JP2023130393A - Water-soluble transmembrane proteins and methods for their preparation and use - Google Patents

Abstract

The present invention provides a water-soluble membrane protein, a method for preparing the same, and a method for using the same.
A computer-implemented method for selecting a water-soluble variant of a G protein-coupled receptor (GPCR), comprising inputting a sequence of the GPCR and obtaining the variant of the GPCR. and then obtaining α-helical secondary structure results for said mutant to confirm maintenance of α-helical secondary structure within said mutant; and obtaining transmembrane region results for said mutant. and confirming the water solubility of the mutant by using the method, thereby selecting a water-soluble mutant of the GPCR.
[Selection diagram] None

Description

(Related application)
This application claims priority to U.S. Patent Application No. 14/669,753 and International Application No. PCT/US 2015/022780, both filed on March 26, 2015, both of which 35U. S. C. 119(e), U.S. Provisional Application No. 62/117,550, filed February 18, 2015, U.S. Provisional Application No. 61/993,783, filed May 15, 2014, and Claims the filing date benefit of U.S. Provisional Application No. 61/971,388, filed March 27, 2014.

Additionally, this application is filed under 35 U.S.C. S. C. 119(e), U.S. Provisional Application No. 62/117,550, filed on February 18, 2015, U.S. Provisional Application No. 61/993,783, filed on May 15, 2014, and We also claim the benefit of the filing date of U.S. Provisional Application No. 61/971,388, filed March 27, 2014.

The entire contents of each of the above referenced applications, including all drawings and sequence listings, are incorporated herein by reference.

Membrane proteins play important roles in all biological systems. Approximately about 30% of all genes in nearly all sequenced genomes encode membrane proteins. However, our detailed understanding of their structure and function lags far behind that of soluble proteins. As of March 2015, over 100,000 structures have been registered in the Protein Structure Data Bank. However, only 945 membrane protein structures have been registered, including 28 types of G protein-coupled receptors and 530 unique structures excluding tetraspanin membrane proteins.

Although membrane receptors are of great importance, there are several obstacles to elucidating their structure and function and their recognition and ligand binding properties. The most important and difficult challenge is that it is extremely difficult to produce milligram quantities of soluble and stable receptors. Inexpensive large-scale production methods are desperately needed and have therefore been the focus of extensive research. Detailed structural studies can only be carried out if these preceding obstacles are overcome.

Zhang et al. (U.S. Pat. No. 8,637,452) (incorporated herein by reference) developed a method for water-soluble GPCRs in which certain hydrophobic amino acids located within the transmembrane region were replaced by polar amino acids. An improved method is described. However, this method requires a large amount of labor. Furthermore, although the modified transmembrane region meets water solubility criteria, improved water solubility and ligand binding are desired. Accordingly, there is a need in the art for improved methods for studying G-protein coupled receptors.

The present invention relates to methods for designing, selecting and/or producing water-soluble membrane proteins and peptides, peptides (and transmembrane domains) designed, selected or produced therefrom, compositions containing said peptides and methods for their use. In particular, the method combines water-insoluble amino acids (leucine, isoleucine, valine and phenylalanine or simple letter code L, I, V, F) with non-ionic water-soluble amino acids (glutamine, threonine and tyrosine or simple letter code Q). , T, Y) using the "QTY principle" to design a library of water-soluble membrane peptides such as GPCR mutants and tetraspanin membrane proteins. Furthermore, two additional nonionic amino acids asparagine (N) and serine (S) may be used for substitution of L, I and V other than F. In embodiments discussed below, asparagine (N) and serine (S) are substituted for Q and T (described as a variant) or L, I or V (described as a native protein). It should be understood that this is assumed to be possible. However, for the sake of brevity, the details of these other embodiments will not be discussed in further detail in this application, as they are known to those skilled in the art from the teachings herein.

The present invention provides modified, synthetic and/or non-naturally occurring one or more alpha-helical domains and water-soluble polypeptides (e.g., "sGPCRs") comprising one or more such modified alpha-helical domains. , wherein the modified one or more α-helical domains include multiple hydrophobic amino acid residues (L, I, V, F) within the α-helical domain of the native membrane protein that are nonionic and hydrophilic. (Q, T, T, Y or "Q, T, Y" respectively) and/or amino acid sequences substituted with N and S. The present invention replaces multiple hydrophobic amino acid residues (L, I, V, F) within one or more α-helical domains of a natural membrane protein with nonionic hydrophilic amino acid residues (Q/N/S, Also included is a method for preparing a water-soluble polypeptide comprising the step of substituting with T/N/S, Y). The present invention replaces multiple hydrophobic amino acid residues (L, I, V, F) within the α-helical domain of natural membrane proteins with nonionic hydrophilic amino acid residues (Q/N/S, T/N, respectively). /S, Y) is further included. The variant can be characterized by the name of the parent or native protein (eg, CXCR4) followed by the abbreviation "QTY" (eg, CXCR4-QTY).

Accordingly, one aspect of the present invention includes (1) inputting the sequence of a membrane protein (e.g., GPCR) for analysis; and (2) transmembrane (TM) domain α-helix of the membrane protein (e.g., GPCR). Obtaining a variant of a membrane protein (e.g., GPCR) in which a plurality of hydrophobic amino acids within a segment (“TM region”) have been substituted, the hydrophobic amino acids being (a) leucine (L), selected from the group consisting of isoleucine (I), valine (V) and phenylalanine (F), (b) said leucine (L) is each independently selected from glutamine (Q), asparagine (N) or serine (S); (c) said isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S), and (d) said phenylalanine is each tyrosine. (Y), followed by (3) obtaining α-helical secondary structure results for the mutant to confirm maintenance of α-helical secondary structure within the mutant; 4) obtaining transmembrane region results for the variant and confirming the water solubility of the variant, thereby designing a water-soluble variant of a membrane protein (e.g., a GPCR). A method of operating a computer program to perform a scripted procedure for designing a water-soluble variant of a membrane protein (e.g., a G protein-coupled receptor (GPCR)) is provided.

In certain embodiments, step (3) is performed before, simultaneously with, or after step (4). Further steps described herein can be incorporated into the above procedure. Preferably, the processing uses predefined calculation steps by the data processing system. The system utilizes automated computational systems and protein variant selection methods.

In certain embodiments, in step (2), substituting a subset of said plurality of hydrophobic amino acids in one and the same TM region of said GPCR to generate one member of a candidate variant library; and substituting one or more different subsets of the plurality of hydrophobic amino acids to create additional members of the library. In certain embodiments, the method may further include ranking all members of said library based on a combined score, where the combined score is determined by the α-helical secondary structure prediction result and the transmembrane region prediction result. is a weighted combination of In certain embodiments, the method further comprises ranking the variants using a ranking function. In certain embodiments, the ranking function may include secondary structure components and water-soluble components. For example, the ranking function may include weighting values for secondary structure components and/or water-soluble components. In certain embodiments, the method further includes performing the method by a data processor, which may further include a memory coupled thereto.

In certain embodiments, the method may further include selecting members of the N species with the highest combination scores to form a first library of variant candidates for the TM region, wherein: where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). be. In certain embodiments, the method may further comprise generating a library of candidate variants for one, two, three, four, five or all six other TM regions of the GPCR. good. In certain embodiments, the method may further include replacing two or more TM regions of the GPCR with corresponding TM regions in a candidate variant library to create a combinatorial variant library. In certain embodiments, the method further comprises producing/expressing said combination variant. In certain embodiments, the method further comprises testing said combination variant for ligand binding (e.g., in a yeast two-hybrid assay), wherein said combination variant exhibits substantially the same ligand binding as compared to said GPCR. Choose what you have. In certain embodiments, the method further comprises testing said combination variant for biological function of said GPCR, wherein said combination variant has substantially the same biological function as compared to said GPCR. Select.

Certain water-soluble polypeptides of the invention have the ability to bind ligands that normally bind to wild-type or native membrane proteins (eg, GPCRs). In certain embodiments, amino acids within the candidate ligand binding site of a natural membrane protein (e.g., a GPCR) are unsubstituted and/or the extracellular and/or intracellular domains of the natural membrane protein (e.g., a GPCR) are unsubstituted. The sequences of are the same.

The (non-ionic) hydrophilic residues (which replace one or more hydrophobic residues in the α-helical domain of natural membrane proteins) include glutamine (Q), threonine (T), tyrosine (Y), selected from the group consisting of asparagine (N) and serine (S) and any combination thereof. In a further aspect, hydrophobic residues selected from leucine (L), isoleucine (I), valine (V) and phenylalanine (F) are substituted. In certain embodiments, a phenylalanine residue in the alpha-helical domain of the protein is replaced with a tyrosine, and each isoleucine and/or valine residue in the alpha-helical domain of the protein is independently replaced with a threonine (or S or N). and/or each leucine residue in the α-helical domain of the protein is independently replaced with glutamine (or S or N).

In certain embodiments, substantially all of the leucine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% substituted with glutamine. In certain embodiments, substantially all of the isoleuci (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are substituted with threonine. In certain embodiments, substantially all of the valine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% substituted with threonine. In certain embodiments, substantially all of the phenylalanine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% substituted with tyrosine. In certain embodiments, one or more (eg, 1, 2 or 3) of the leucines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the isoleucines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the valines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the phenylalanines are unsubstituted.

In certain embodiments, the combinatorial variant library contains less than about 2 million members. In certain embodiments, the sequence of the GPCR includes information regarding the TM region of the GPCR. In certain embodiments, the sequence of the GPCR is obtained from a protein structure database (eg, PDB, UniProt). In certain embodiments, the TM region of the GPCR is predicted based on the sequence of the GPCR. For example, the TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) software module/package can be used to predict the TM region of the GPCR. In certain embodiments, the TMHMM2.0 software module/package utilizes dynamic baselines for peak searching.

In certain embodiments, the method further comprises providing a polynucleotide sequence for each variant of the GPCR. The polynucleotide sequence is codon-optimized for expression in a host (e.g., a bacteria such as E. coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cells, a non-human mammalian cell, or a human cell). There may be.

In certain embodiments, the scripted procedure may include a VBA script. In certain embodiments, the scripted procedure is operable on a Linux system (eg, Ubuntu 12.04 LTS), a Unix system, a Microsoft Windows operating system, an Android operating system, or an Apple iOS operating system. Different programming languages may be used with implementations of the present invention, including C++, JavaScript, MATLAB, and the like. The coded instructions may be stored in a memory device such as a non-transitory computer-readable medium that can be used with computer systems known to those skilled in the art.

In certain embodiments, the alpha helical domain is one of seven transmembrane alpha helical domains within a natural membrane protein that is a G protein coupled receptor (GPCR). In some embodiments, the GPCRs include purinergic receptors (P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ ), M ₁ and M ₃ muscarinic acetylcholine receptors, thrombin receptor (protease activated receptor (PAR )-1, PAR-2), thromboxane (TXA ₂ ), sphingosine 1-phosphate (S1P ₂ , S1P ₃ , S1P ₄ and S1P ₅ ), lysophosphatidic acid (LPA ₁ , LPA ₂ , LPA ₃ ), angiotensin II (AT ₁ ), serotonin (5-HT _2c and 5-HT ₄ ), somatostatin (sst ₅ ), endothelin (ET _A and ET _B ), cholecystokinin (CCK ₁ ), V _1a vasopressin receptor, D ₅ Dopamine receptors, fMLP formyl peptide receptors, GAL ₂ galanin receptors, EP ₃ prostanoid receptors, A ₁ adenosine receptors, α ₁ adrenergic receptors, BB ₂ bombesin receptors, B ₂ bradykinin receptors, calcium Sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK ₁ tachykinin receptor, thyroid stimulating hormone (TSH) receptor, protease activated receptor, neuropeptide receptor, adenosine A2B receptor, P2Y purinergic receptor , a metabotropic glutamate receptor, GRK5, GPCR-30 and CXCR4.

In other embodiments, the native membrane protein or membrane protein is an essential membrane protein. In further embodiments, the native membrane protein is a mammalian protein. The protein of the invention is preferably a human protein. In certain embodiments, reference to a specific GPCR protein (eg, CXCR4) refers to a mammalian GPCR, such as a non-human mammalian GPCR, or a human GPCR.

In some embodiments, the α-helical domain is a G protein-coupled receptor modified, e.g., in an extracellular or intracellular loop, to improve or alter ligand binding, as described elsewhere in the literature. It is one of seven transmembrane α-helical domains within the body (GPCR) variant. For purposes of the present invention, the term "native" or "wild type" shall refer to the protein (or α-helical domain) prior to water solubilization according to the methods described herein.

In certain embodiments, the membrane protein may be a tetraspanin membrane protein characterized by four transmembrane alpha helices. Approximately 54 human tetraspanin membrane proteins have been reviewed and annotated. Many are known to mediate intercellular signaling events that play important roles in controlling cell development, activation, proliferation, and motility. For example, the CD81 receptor plays an important role as a receptor for hepatitis C virus entry and malaria parasite infection. The CD81 gene is located in the tumor suppressor gene region and may be a candidate for mediating cancer malignancy. CD151 is involved in increasing cell motility, invasion and metastasis of cancer cells. CD63 expression correlates with ovarian cancer invasiveness. A characteristic feature of tetraspanin membrane proteins is a cysteine-cysteine-glycine motif within the second or large extracellular loop.

Another aspect of the invention provides that (1) a plurality of hydrophobic amino acids within the transmembrane (TM) domain α-helical segment (“TM region”) of the GPCR are substituted, wherein (a) the hydrophobic (b) said leucine (L) is each independently selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F); (N) or serine (S); (c) said isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S); and (d) each of the phenylalanines is replaced with a tyrosine (Y), after which (2) an α-helical secondary structure is maintained by all seven TM regions of the mutant, and (3) Provide a water-soluble variant of a G protein-coupled receptor (GPCR) characterized by the absence of a predicted transmembrane region.

In certain embodiments, the water-soluble variant is 1 selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64. Contains one or more amino acid sequences. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57. In certain embodiments, the water soluble variant binds CXCR4 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114, and 116-123. Contains the amino acid sequence of It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124. In certain embodiments, the water soluble variant binds CX3CR1 ligand.

In certain embodiments, the water-soluble variants are SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-
one or more amino acid sequences selected from the group consisting of 182. It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183. In certain embodiments, the water soluble variant binds CCR3 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234. Contains the amino acid sequence of It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO: 186, 195, 204, 207, 209, 218, 226 and 235. In certain embodiments, the water soluble variant binds CCR5 ligand.

In certain embodiments, the water-soluble variant is one or more selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281, and 283-290. Contains the amino acid sequence of It may further comprise one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. In certain embodiments, the water soluble variant binds CXCR3 ligand.

In certain embodiments, the water-soluble variant is SEQ ID NO. , 319, 321, 323 or 325. In certain embodiments, the variant is water soluble and binds a homologous natural transmembrane protein ligand.

Another aspect of the invention comprises (a) culturing bacteria in a growth medium under conditions suitable for protein production; and (b) dividing the bacterial lysate into fractions, a soluble fraction and an insoluble pellet fraction. and (c) isolating the protein from the soluble fraction, wherein (1) the protein is a G protein-coupled receptor according to any one of claims 29-46. (2) the yield of the protein is at least 20 mg/L (e.g., 30 mg/L, 40 mg/L, 50 mg/L or more) of the growth medium. A method of producing a protein in bacteria (eg, E. coli) is provided.

In certain embodiments, the bacteria is E. coli BL21 and the growth medium is LB media. In certain embodiments, the protein is encoded by a plasmid within the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter, such as an inducible promoter inducible by IPTG. In certain embodiments, this lysate is produced by sonication. In certain embodiments, the lysate is centrifuged at 14,500 xg or higher to generate the soluble fraction.

Another aspect of the invention is a method for treating a disorder or disease mediated by the activity of a membrane protein in a subject in need thereof, comprising administering to said subject an effective amount of a water-soluble polypeptide as described herein. A method is provided comprising administering a peptide.

In certain embodiments, the water-soluble polypeptide retains the ligand binding activity of the membrane protein. Examples of disorders and diseases that can be treated by administering the water-soluble peptides of the invention include, but are not limited to, cancer (e.g., small cell lung cancer, melanoma, triple negative breast cancer), Parkinson's disease , cardiovascular disease, hypertension and bronchial asthma.

Another aspect of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a water-soluble polypeptide of the invention and a pharmaceutically acceptable carrier or diluent.

In yet another aspect, the invention provides cells transfected with a subject water-soluble peptide comprising a modified alpha-helical domain. In certain embodiments, the cell is an animal cell (eg, a human, non-human mammal, insect, avian, fish, reptile, amphibian, or other cell), yeast, or bacterial cell.

The invention also includes computer-implemented methods executed on a computer system that include one or more of the methods (or steps thereof) described herein. The computer system includes a non-transitory computer-readable medium having computer-executable instructions stored thereon, the computer-executable instructions, when executed by the computer system, cause the computer system to perform the method; When executed by a computer system, it causes the computer system to perform the methods contemplated herein. Further, at least one memory for storing sequence data and quantitative results described herein and a memory connected to the memory configured to perform the methods described herein. A computer system comprising at least one processor is envisioned. A user interface, such as a graphical user interface (GUI), in conjunction with an electronic display device may be used to select processing parameters that operate to control selection methods, including the calculation methods described herein.

Another aspect of the invention provides a non-transitory computer readable medium having a set of instructions stored thereon for performing any of the methods of the invention.

A further aspect of the invention comprises a data processor operative to perform amino acid substitutions similar to any of the methods of the invention, ranking protein variants by a ranking function and A data processing system is provided that is operative to select sex mutants.

It will be appreciated that all embodiments of the invention, including those described in only one aspect of the invention (e.g., screening methods), may be explicitly described, as should be readily understood by those skilled in the art. Unless otherwise waived or otherwise inappropriate, the present invention shall be construed as applicable to all aspects of the invention (e.g., water-soluble proteins or methods of use); should be construed as combinable with any one or more further embodiments of.

The above and other objects, features and advantages of the invention will become apparent from the following more detailed description of exemplary embodiments of the invention, which are illustrated in the accompanying drawings in which like reference numerals refer to the same parts throughout the different drawings. It will be. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIGS. 1A-1D are general illustrations of QTY codes that systematically substitute hydrophobic amino acids L, I, V, and F with Q, T, T, Y, respectively. (FIG. 1A) The amino acids leucine and glutamine have similar molecular shapes, similarly, isoleucine and valine have similar molecular shapes to threonine, and phenylalanine and tyrosine have similar molecular shapes. Leucine, isoleucine, valine and phenylalanine are hydrophobic and cannot bind water molecules. In contrast, glutamine can bind four water molecules, i.e. two hydrogen donors and two hydrogen acceptors, and the -OH groups on threonine and tyrosine can bind three water molecules, i.e. one hydrogen donor and two hydrogen acceptors. Can be bound to an acceptor. FIG. 1B is a side view of the alpha helix. After applying the QTY code of systematic amino acid changes, the α-helix becomes water-soluble. Figure 1C is a top view of the α-helix before and after QTY code substitution. The helix on the left is the native membrane helix with primarily hydrophobic amino acids, and the helix on the right is the same helix after applying QTY code substitutions. This helix has the most hydrophilic amino acids at this point. (FIG. 1D) Before the QTY code, the GPCR membrane proteins are surrounded by hydrophobic lipid molecules such that they are embedded within a lipid membrane (left part of FIG. 1D). After applying the QTY code, the GPCR membrane protein becomes water-soluble and no longer requires surfactant surrounding it for stabilization (right part of Figure 1D). TMHMM prediction of the transmembrane domain region of CXCR4. This prediction shows seven distinguishable hydrophobic transmembrane segments. In contrast, in the TMHMM prediction of the variant of CXCR4 (CXCR4-QTY) subjected to the QTY replacement method of the present invention, there are no longer any visible and distinguishable seven hydrophobic transmembrane segments. The predicted α-helical wheel structure of the fully QTY-coded modified TM1 domain of CXCR4 is shown. Figure 2 is an illustration of candidate variants in each of the seven TM regions of GPCR CXCR4. Sequence alignment of the wild type protein and each QTY variant of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. Sequence alignment of the wild type protein and each QTY variant of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. Sequence alignment of the wild type protein and each QTY variant of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. Sequence alignment of the wild type protein and each QTY variant of CXCR4, CXCR3, CCR3 and CCR5. The QTY code applies only to the seven hydrophobic transmembrane segments and not to the extracellular and intracellular segments. 3 is a flowchart of an exemplary embodiment of the method. 2 is another flowchart of an exemplary embodiment of the method. 1 is an illustration of a computer system of the present invention. 1 is a schematic diagram of a flowchart describing the process steps of certain preferred embodiments of the invention; FIG. 1 is a schematic diagram of a flowchart describing the process steps of certain preferred embodiments of the invention; FIG.

A description of preferred embodiments of the invention follows. The words "a species" or "an" are intended to include one or more species, unless otherwise specified.

In some aspects, the present invention replaces the hydrophobic residues of the seven transmembrane alpha helices of natural proteins, leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with hydrophilic residues. relates to the use of the QTY (glutamine, threonine and tyrosine) substitution (or "QTY code") method (or "principle") to convert glutamine (Q), threonine (T) and tyrosine (Y) to glutamine (Q), threonine (T) and tyrosine (Y). In certain embodiments, asparagine (N) and serine (S) can also be used as replacement residues for L, I and/or V other than F, as described above. The present invention can convert water-insoluble native membrane proteins into more water-soluble counterparts that still retain some or substantially all of the functions of the native protein.

The present invention includes methods for designing water-soluble peptides. The method is first described with respect to GPCR proteins, using human CCR3, CCR5, CXCR4 and CX3CR1 as specific examples. However, the general principles of the invention also apply to other proteins that have transmembrane (α-helical) regions.

GPCRs typically have seven transmembrane α-helices (seven TMs) and eight loops (eight NTMs) connected by seven TM regions. These transmembrane segments may be referred to as TM1, TM2, TM3, TM4, TM5, TM6 and TM7. The eight non-transmembrane loops include four extracellular loops EL1, EL2, EL3 and EL4 and four intracellular loops IL1, IL2, IL3 and IL4, thus a total of eight loops (each connected to only one TM region). It is also divided into N-terminal and C-terminal loops, each with a free end. Thus, the 7TM-GPCR protein can be divided into 15 fragments based on transmembrane and non-membrane characteristics.

One aspect of the invention is a method of causing a computer program to perform scripted procedures to select or prepare a water-soluble variant of a membrane protein (e.g., a G protein-coupled receptor (GPCR)), the method comprising:
(1) inputting the sequence of the membrane protein (for example, GPCR) for analysis;
(2) A variant of the membrane protein (e.g., GPCR) in which multiple hydrophobic amino acids in the transmembrane (TM) domain α-helical segment (“TM region”) of the membrane protein (e.g., GPCR) are substituted. A process of obtaining
(a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) the leucine (L) is each independently substituted with glutamine (Q), asparagine (N) or serine (S),
(c) said isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N), or serine (S), and (d) said phenylalanine is each substituted with tyrosine (Y). followed by (3) obtaining α-helical secondary structure results for the mutant to confirm maintenance of α-helical secondary structure within the mutant;
(4) obtaining transmembrane region results for the variant and confirming the water solubility of the variant, thereby selecting a water-soluble variant of the membrane protein (e.g., GPCR). Provided is a method characterized by:

As used herein, "water-soluble variant of a membrane (spanning) protein" or "water-soluble membrane (spanning) variant" can be used interchangeably.

The exact order in which the steps of the invention are performed may vary. For example, in certain embodiments, step (3) is performed before step (4). In certain embodiments, step (3) is performed simultaneously with step (4). In certain embodiments, step (4) is followed by step (3).

In certain embodiments, the plurality of hydrophobic amino acids are randomly selected from all hydrophobic amino acid candidates L, I, V and F located in all TM regions of the protein. In certain embodiments, the plurality of hydrophobic amino acids comprises about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% , 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42 %, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In certain embodiments, the plurality of hydrophobic amino acids comprises at least about 10%, 15%, 20%, 25% of all candidate hydrophobic amino acids L, I, V and F located in all TM regions of the protein. , 30%, 35%, 40%, 45%, 50%. In certain embodiments, the plurality of hydrophobic amino acids comprises about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60% or 50% or less. In certain embodiments, randomly selected hydrophobic amino acids L, I, V, and F may be approximately evenly distributed across all TM regions, or alternatively 1, 2, 3, 4, 5, or 6 TM regions. It may be preferentially or exclusively distributed in a region.

In certain embodiments, all candidate hydrophobic amino acids L, I, V and F on all TM regions of the protein are substituted. For example, every L is independently substituted with Q (or S or N), and/or every I and V is independently substituted with T (or S or N), and/or All F's are replaced with Y. In certain embodiments, all L's are replaced with Q, all I and V's are replaced with T, and all F's are replaced with Y.

In certain embodiments, instead of randomly substituting selected hydrophobic amino acids L, I, V, and F in all TM regions, all substitutions are made first in any one of the TM regions (e.g., most (N-terminal or C-terminal TM region), and only desired substitution mutants are selected as members of the mutant candidate library. the position being substituted (e.g., the 3rd residue is substituted for the 10th residue in the TM region), or the identity of the substituted residue (e.g., T for an I or V substitution). and/or S), all members of the library differ in substitutions within the selected TM region. Desired substitutional variants are selected based on predetermined criteria, such as a scoring system that takes into account α-helical secondary structure predictions and/or transmembrane region predictions.

This method can be repeated for 1, 2, 3, 4, 5, 6 additional TM regions of the protein or all remaining TM regions of the protein, with each iteration storing in an electronic memory or database. Create a mutant candidate library that can Within the same library, all variants differ in substitutions within the selected TM region (see above) but are otherwise identical within the remaining TM and non-TM regions.

Domain swapping or domain shuffling using sequences in two or more such libraries produces combinatorial variants with hydrophobic amino acid L, I, V, F substitutions in two or more TM regions. Depending on the number of members in each library, the total number of possible combinations of combinatorial variants can approach millions with only a few members in each library. For example, for a GPCR with seven TM regions, if there are eight members in each of the seven libraries, the total number of combined variants based on the libraries is ⁸⁷ or about 2.1 million. In certain embodiments, the combinatorial variant library contains less than about 5 million, 4 million, 3 million, 2 million, 1 million, or 500,000 members.

Accordingly, in certain embodiments, in step (2), one subset of said plurality of hydrophobic amino acids in one and the same TM region of said protein (e.g., a GPCR) is substituted to Members of the species are created and one or more different subsets of the plurality of hydrophobic amino acids are substituted to create further members of the library.

In certain embodiments, the method further comprises ranking all members of the library based on a combined score, the combined score being a weighting of the α-helical secondary structure prediction results and the transmembrane region prediction results. This is a combination of

As one skilled in the art will appreciate, domains with different sequences likely predict different water solubility and propensity for α-helix formation. A "score" is assigned for a particular predicted water solubility or range of water solubility, tendency or range of propensity to form alpha helical structures. This score may be qualitative (0, 1), where 0 may represent a domain with e.g. unacceptable predicted water solubility and 1 may represent e.g. an acceptable predicted water solubility. can represent a domain with . This score can be based on a threshold value, for example. Alternatively, the score can be evaluated, for example, on a scale of 1 to 10 established as characterized by an increasing degree of water solubility. Alternatively, this score may be quantitative, such as describing predicted solubility in units of mg/mL. Once scores have been assessed for each domain, domain variants can be easily compared (or ranked) by one or preferably both of those scores, both being water soluble and forming an alpha helix. domain variants can be selected. Accordingly, preferred embodiments may utilize ranking functions that may be used to calculate ranking data. Analyze and characterize water-soluble proteins prepared based on the system currently described and constrain computational models using those substitution combinations that are not effective in achieving a given biological function. It should also be noted that inputs to the system can thus be obtained that allow for more efficient information processing.

For example, using the methods of the invention, one or more variants can be designed and produced in vitro and/or in vivo, based on any of a number of art-recognized methods. one or more biological functions of the variant can be determined. For GPCRs, for example, ligand binding and/or downstream signaling by the mutant in question can be compared to that of a wild-type GPCR, and the QTY substitution pattern used to produce a particular mutant can be compared to biological It can be associated with increasing, maintaining or decreasing activity. Such structural/functional relationship information obtained based on one or more variants can be used for machine learning or by imposing further constraints on the computational model of the invention to improve the method of the invention. It is possible to more efficiently rank the mutants produced by In this way, new mutant candidates with substitution patterns that more closely match the substitution patterns of known successful mutants will be considered candidates for new mutants with substitution patterns that more closely match the substitution patterns of known successful mutants or with substitution patterns that more closely match the substitution patterns of known successful mutants. can be ranked higher than another variant candidate whose substitution pattern more closely matches that of the unsuccessful variant.

When the TMHMM program is run as a standalone software module/package (e.g. for Linux systems), it can be used to predict the propensity to form transmembrane regions/proteins. Generate a score. This score can be used as a quantitative prediction of water solubility in the methods of the invention.

Thus, in certain embodiments, the α-helical secondary structure component of the ranking function is 0.5 or 1 with no predicted α-helical secondary structure, and 0.5 or 1 with no predicted α-helical secondary structure. It may also be a quantitative score such as 0 when the In certain embodiments, a TM region prediction program, such as TMHMM2.0, provides a number between 0 and 1, where 0 is no predicted TM region and 1 is the strongest tendency to form one or more TM regions. Transmembrane region results can be obtained by: Therefore, the two scores can be combined directly or with weights such that the combined score represents an overall assessment of preserved secondary structure as well as predicted water solubility (as measured by the tendency to form TM regions). . For example, a combination score of 0 indicates that the variant does not have the predicted TM region but maintains the predicted α-helical secondary structure and is therefore a desired variant. On the other hand, mutants that have a strong tendency to form TM regions (e.g. due to the presence of many hydrophobic residues) tend to have larger combinatorial scores and are therefore less desirable under this scoring scheme. .

In certain embodiments, the method includes removing mutants that have α-helical secondary structure predictions that tend to indicate that the α-helical secondary structure is disrupted or disrupted. In certain embodiments, the method includes removing variants with transmembrane region predictions that tend to exhibit a strong tendency to form TM regions. Therefore, the system can be equipped with a BEAMing module that allows variants to be excluded by further selection processing.

In certain embodiments, a weight of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% is used for α-helical secondary structure prediction. The ranking function can be selected to include a weighting scheme that assigns the results to the results and the remainder to the transmembrane region prediction results. Depending on the desired characteristic, such as biological function, the weighting features can be selected manually by the user, or the weighting features can be automatically selected by the software.

In certain embodiments, the method further comprises selecting members of the N species with the highest combination scores to form a first library of variant candidates for the TM region, wherein: N is a predetermined integer (eg, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).

In certain embodiments, the method involves generating a library of variant candidates for one, two, three, four, five, six or all remaining TM regions of the protein (e.g., GPCR). The method further includes the step of: Each entry in the library may include fields used to define attributes of the entry, including ranking data generated by one or more ranking functions.

In certain embodiments, the method involves replacing two or more (e.g., all) TM regions of the protein (e.g., GPCR) with corresponding TM regions in a candidate variant library to create a combinatorial variant library. The method further includes the step of: As used herein, "corresponding TM region" refers to a TM region within a candidate variant library that is the same or homologous to the TM region of the protein (eg, GPCR) with which it is being combined. For example, if the second and third TM regions from the N-terminus of a GPCR are substituted, TM region sequences in the library that have substitutions only in the second TM region and substitutions only in the third TM region The TM region sequences in the library containing the GPCR are imported/paste/transferred into the second and third TM regions of the GPCR to generate combinatorial variants.

In certain embodiments, substantially all of the leucine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are substituted with glutamine. In certain embodiments, substantially all of the isoleucine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are substituted with threonine. In certain embodiments, substantially all of the valine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are substituted with threonine. In certain embodiments, substantially all of the phenylalanine (e.g., 96%, 97%, 98%, 99% or 100%) or 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% are substituted with tyrosine. In certain embodiments, one or more (eg, 1, 2 or 3) of the leucines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the isoleucines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the valines are unsubstituted. In certain embodiments, one or more (eg, 1, 2 or 3) of the phenylalanines are unsubstituted.

In certain embodiments, the method further comprises producing/expressing said combination variant. In certain embodiments, the method further comprises testing said combination variant for ligand binding (e.g., in vitro or in a biological system such as a yeast two-hybrid assay), wherein said combination variant is tested for ligand binding of said GPCR. Compare and select those with substantially the same ligand binding. In certain embodiments, the method further comprises testing the combination variant for a biological function of the GPCR, wherein the combination variant has substantially the same biological function as compared to the biological function of the GPCR. Choose one that has the desired function.

In certain embodiments, the sequence of a TM protein (e.g., a GPCR) includes information about the TM regions of the protein, e.g., the location of one or more transmembrane regions of the TM protein, such as the location of all TM regions. . Such a sequence may belong to a protein that has a resolved crystal structure with a defined TM region. Such sequences may also belong to proteins with TM region information annotated based on previous studies, and such information can be found in public sources such as PDB, UniProt, Genbank, EMBL, DBJ, etc. or readily available from a proprietary database

The Protein Structure Data Bank (PDB) is a weekly updated repository for three-dimensional structural data of large biomolecules such as proteins and nucleic acids. Data, typically obtained by X-ray crystallography or NMR spectroscopy, and submitted by biologists and biochemists around the world, are made available on the Internet via the websites of its member organizations (PDBe, PDBj and RCSB). Freely accessible. The PDB is supervised by the World Protein Structural Data Bank or wwPDB. The PDB is an important resource in areas of structural biology such as structural genomics, and most major scientific journals and several funding agencies are currently seeking scientists to submit their structural data to the PDB.

If we consider the contents of the PDB as primary data, there are hundreds of derivative (ie, secondary) databases that categorize that data differently. For example, while SCOP and CATH both classify structures according to structure type and assumed evolutionary relationships, and GO classifies structures based on genes, crystallographic databases classify structures according to the 3D structure of proteins. remembers information about. All such publicly available databases may be used to obtain input sequence information, including information regarding the presence and location of transmembrane regions.

Another publicly available database that can provide sequence information used in the methods of the invention is UniProt. UniProt is a comprehensive, high-quality, freely accessible database of protein sequence and functional information, with many entries derived from genome sequencing projects. This includes a lot of information about the biological function of proteins derived from the research literature. UniProt provides four core databases: UniProtKB (including subdivisions Swiss-Prot and TrEMBL), UniParc, UniRef and UniMes. Among these, UniProtKB/Swiss-Prot is a manually annotated, non-redundant protein sequence database that combines information extracted from the scientific literature and computer analysis evaluated by biocurators. The purpose of UniProtKB/Swiss-Prot is to provide all known relevant information about a particular protein. The annotations are regularly reviewed to keep abreast of current scientific findings. Manual annotation of entries includes detailed analysis of protein sequences and scientific literature. Sequences from the same gene and the same species are merged into the same database entry. Identify differences between sequences and document their causes (e.g., alternative splicing, natural mutations, etc.). Manually evaluate computer predictions and select relevant results for inclusion in the entry. These predictions include post-translational modifications, transmembrane domains and topology, signal peptides, domain identification and protein family classification, all of which can be used to generate useful sequences related to TM regions used in the methods of the invention. You can get information.

In certain embodiments, the sequence of the TM protein (eg, GPCR) does not include information regarding the location of one or more (eg, any) transmembrane regions. However, the one or more TM regions can be predicted based on sequence homology with related proteins having known TM regions. For example, related proteins may be homologous proteins in different species.

In certain embodiments, the sequence of the TM protein (e.g., GPCR) does not include information regarding the location of one or more (e.g., any) transmembrane regions, and such information is readily available based on known information. is not available. In this embodiment, the invention utilizes art-approved methods such as the TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) program developed by the Center for Biological Sequence Analysis. The TM area is calculated using See further details regarding this below.

In certain embodiments, the method further comprises providing a polynucleotide sequence for each variant of the protein (eg, GPCR). Such polynucleotide sequences can be easily generated based on the protein sequence of the protein in question (eg, GPCR) and the known genetic code. In certain embodiments, the polynucleotide sequence is codon optimized for expression in the host. The host may be a bacterium such as E. coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as an Sf9 cell, a non-human mammalian cell or a human cell.

In certain embodiments, the protein is a GPCR, such as the purinergic receptors (P2Y ₁ , P2Y ₂ , P2Y ₄ , P2Y ₆ ), M ₁ and M ₃ muscarinic acetylcholine receptors, thrombin receptor (protease activated receptor (PAR)-1, PAR-2), thromboxane (TXA ₂ ), sphingosine 1-phosphate (S1P ₂ , S1P ₃ , S1P ₄ and S1P ₅ ), lysophosphatidic acid (LPA ₁ , LPA ₂ , LPA ₃ ), angiotensin II (AT ₁ ), serotonin (5-HT _2c and 5-HT ₄ ), somatostatin (sst ₅ ), endothelin (ET _A and ET _B ), cholecystokinin (CCK ₁ ), V _1a vasopressin receptor , _D5 dopamine receptors, fMLP formyl peptide receptors, GAL ₂ galanin receptors, EP ₃ prostanoid receptors, A ₁ adenosine receptors, α ₁ adrenergic receptors, BB ₂ bombesin receptors, B ₂ bradykinin receptors body, calcium sensing receptor, chemokine receptor, KSHV-ORF74 chemokine receptor, NK ₁ tachykinin receptor, thyroid stimulating hormone (TSH) receptor, protease activated receptor, neuropeptide receptor, adenosine A2B receptor, P2Y A GPCR selected from the group consisting of purinergic receptors, metabotropic glutamate receptors, GRK5, GPCR-30 and CXCR4.

In certain embodiments, the scripting steps of the method include VBA scripts.

In certain embodiments, the scripted procedure is operable on a Linux system (eg, Ubuntu 12.04 LTS), a Microsoft Windows operating system, or an Apple iOS operating system.

In certain embodiments, the method comprises:
(1) If necessary, predicting the α-helical structure of the protein (e.g., GPCR) and identifying the first transmembrane region of the transmembrane (spanning) protein;
(2) modifying multiple hydrophobic amino acids with the QTY code defined herein to obtain a modified first transmembrane sequence;
(3) the tendency of the α-helical structure of the first modified transmembrane sequence in (2) (e.g., in the context of a modified membrane (spanning) protein with a first modified transmembrane sequence); scoring to obtain a structural score;
(4) scoring the water solubility prediction of the first modified transmembrane sequence of (2) (e.g., in the context of a modified transmembrane protein having the first modified transmembrane sequence); obtaining a solubility score;
(5) repeating (2) to (4) to obtain a first library of first modified transmembrane variants that are putatively water-soluble;
(6) comparing the structure and solubility scores of each of the first putatively water-soluble first modified transmembrane variants in the first library, preferably using said structure and solubility scores; ranking the first modified transmembrane variants that are putatively water soluble;
(7) a plurality of putatively water-soluble first modified transmembrane variants, where the plurality is the integer H or preferably less than 10, 9, 8, 7, 6, 5 or 4; selecting a second library of modified transmembrane variants of the first putatively water-soluble variants;
(8) repeating steps (1) to (7) for the second, third, fourth, fifth, sixth, seventh or preferably all transmembrane regions of the protein (modified by the present method); the total number of transmembrane regions is an integer n),
(9) Identifying the amino acid sequence of a protein that is not included in any of the transmembrane regions modified in steps (1) to (8) and that includes any extracellular or intracellular domain of the protein. ,
(10) producing combination variants of modified transmembrane proteins that are putatively water-soluble (see above); and (11) optionally, each of the modified transmembrane variants that are putatively water-soluble. all or substantially all of the steps of identifying a nucleic acid sequence.

The nucleic acid sequences identified in the above method are used to generate nucleic acid sequences for each of the putatively water-soluble modified transmembrane variants and for each of the non-transmembrane domains (including extracellular and intracellular domains). , and can be expressed combinatorially to generate a library of up to H ⁿ putatively water-soluble transmembrane protein variants. For example, if H is 8 and n is 7, a library of approximately 2 million water-soluble protein variants can be designed.

Another aspect of the invention relates to the expression of water-soluble variant proteins (eg, GPCRs) designed based on the methods of the invention. This aspect of the invention is achieved, in part, by water-soluble mutant proteins (e.g., GPCRs) designed based on the methods of the invention, and expression in vitro and in commonly used cell-free expression systems such as E. coli. It is based on the surprising discovery that high levels of expression can be achieved both in cell-based expression systems and in cell-based expression systems. Also, the expressed protein is highly soluble, in contrast to the insoluble aggregates or pellets in which most membrane proteins typically reside, such as in the soluble fraction of a lysate of an E. coli culture. It can be easily purified from the soluble fraction of the expression system.

Therefore, one aspect of the present invention is
(a) culturing the bacteria in a growth medium under conditions suitable for protein production;
(b) fractionating the bacterial lysate to produce a soluble fraction and an insoluble pellet fraction;
(c) isolating the protein from the soluble fraction, comprising:
(1) the protein is a variant protein of the subject matter of the invention (e.g., a G protein-coupled receptor (GPCR)), and (2) the yield of the protein is at least 20 mg/L (e.g., 30 mg/L) of the growth medium. 40 mg/L, 40 mg/L, 50 mg/L or more).

In certain embodiments, the bacteria is E. coli BL21 and the growth medium is LB media. In certain embodiments, the protein is encoded by a plasmid within the bacterium. In certain embodiments, expression of the protein is under the control of an inducible promoter. For example, the inducible promoter may be inducible by IPTG. In certain embodiments, the lysate is produced by sonication. In certain embodiments, the lysate is centrifuged at 14,500 xg or higher to generate the soluble fraction.

Using the general aspects of the invention described above, specific features or specific embodiments of the invention are further described below.

Transmembrane Region Prediction Certain methods of the invention include predicting transmembrane regions of proteins such as GPCRs. There are many programs and software known in the art relating to TM regions, any of which may be used individually or in combination in the methods of the present invention requiring a TM region prediction step. These programs typically have very simple user interfaces that typically require the user to provide an input sequence in a specified format (such as FASTA or text format), and may contain text and/or graphics. gives the prediction result using There are also programs that provide more advanced functionality, such as allowing users to specify specific parameters to fine-tune prediction results. All such programs can be used in the method of the invention.

One exemplary TM region prediction program is TMHMM (provided by the Biological Sequence Center at the Technical University of Denmark), which accurately predicts 97-98% of TM region helices. It uses hidden Markov models to predict transmembrane helices within proteins. The input protein sequence may be in FASTA format and the output may be presented as an html page containing an image of the predicted location of the TM region. In a study by Moller et al. entitled “Evaluation of Methods for the Prediction of Membrane Spanning Regions” (Bioinformatics 17(7):646-653, 2001), TMHMM was It was judged to be the best program to perform a transmembrane prediction program.

The following programs were compared in the study: TMHMM 1.0, 2.0 and 2.0 retraining versions (Sonnhammer et al., Int. Conf. Intell. Syst. Mol. Biol. AAAI Press, Montreal , Canada, pp.176-182, 1998; Krogh et al., J Mol Biol. 305(3):567-80, 2001), MEMSAT1.5 (Jones et al., Biochemistry 33:3038-3049, 1994) , Eisenberg (Eisenberg et al., Nature 299:371-374, 1982), Kyte/Doolittle (Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982), TMAP (Persson and Argos, J. Protein Chem. 16:453-457, 1997), DAS (Cserzo et al., Protein Eng. 10:673-676, 1997), HMMTOP (Tusnady and Simon, J. Mol. Biol. 283:489-506, 1998) , SOSUI (Hirokawa et al., Bioinformatics 14:378-379, 1998), PHD (Rost et al., Int. Conf. Intel. Syst. Mol. Biol. AAAI Press, St. Louis, USA, pp.192- 200, 1996), TMpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166, 1993), KKD (Klein et al., Biochim. Biophys. Acta. 815:468-476, 1985), ALOM2 (Nakai and Kanehisa, Genomics 14:489-911, 1992) and Toppred2 (Claros and Heijne, Comput. Appl. Biosci. 10:685-686, 1994), all of which are used to predict the TM region in the method of the present invention. can be used. All cited references are incorporated herein by reference.

The principle of TMHMM is based on Krogh et al., Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. Journal of Molecular Biology, 305(3):567-580, January 2001 (incorporated by reference) and Sonnhammer et al., A hidden Markov model for predicting transmembrane helices in protein sequences. In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, editors, Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology, pages 175-182, Menlo Park , CA, 1998, AAAI Press (incorporated by reference).

DAS (Dense Alignment Surface, Cserzo et al., “Prediction of transmembrane alpha-helices in procariotic membrane proteins: the Dense Alignment Surface method,” Prot Eng. 10(6): 673-676, 1997, Stockholm University, Sweden) predict transmembrane regions using a dense alignment surface method. DAS is based on a low stringency dot plot of query sequences against a set of library sequences (heterologous membrane proteins) using a special scoring matrix obtained previously. The method provides a high precision hydrophobicity profile of the query from which the location of candidate transmembrane segments can be obtained. The novelty of the DAS-TMfilter algorithm is the second prediction cycle to predict TM segments within the sequences of the TM library. To use the DAS server, the user must visit www. sbc. su. Input a protein sequence at se/~miklos/DAS/, and the DA server predicts the TM region of the input sequence.

HMMTOP (Hungarian Academy of Sciences, Budapest) is a G.I. E. An automated server for predicting transmembrane helices and protein topology using hidden Markov models developed by Tusnady. The method used by this prediction server is based on G.E Tusnady and I. Simon (1998) “Principles Governing Amino Acid Composition of Integral Membrane Proteins: Applications to Topology Prediction”. 283: 489-506 (incorporated by reference). New features of HMMTOP 2.0 version are described in G.E Tusnady and I. Simon (2001) “The HMMTOP transmembrane topology prediction server,” Bioinformatics 17: 849-850 (incorporated by reference). ing.

The MEMSAT2 transmembrane prediction page (www.sacs.ucsf.edu/cgi-bin/memsat.py) predicts transmembrane segments within proteins using FASTA or text format as input. The associated program, MEMSAT (1.5) software, is copyrighted by Dr. David Jones (Jones et al., Biochemistry 33:3038-3049, 1994). MEMSAT V3, the latest version of MEMSAT, is the widely used whole-helical membrane protein prediction method MEMSAT. The method was benchmarked on a test set of transmembrane proteins of known topology. MEMSAT was estimated to have greater than 78% accuracy in predicting the structure of all-helical transmembrane proteins and the location of their constituent helical elements within the membrane from sequence data. MEMSAT-SVM is a highly accurate prediction method of transmembrane helix topology. It can distinguish between signal peptides and identify cytoplasmic and extracellular loops. Both MEMSAT3 and MEMSAT-SVM are part of the PSIPRED Protein Sequence Analysis Workbench, which brings together several structure prediction methods in one place at the University of London.

The Phobius server (phobius.sbc.su.se) is for predicting transmembrane topology and signal peptides from protein amino acid sequences in FASTA format. For Phobius, see Lukas et al., “A Combined Transmembrane Topology and Signal Peptide Prediction Method,” Journal of Molecular Biology 338(5):1027-1036, 2004). Are listed. For PoyPhobius, see Lukas et al., “An HMM posterior decoder for sequence feature prediction that includes homology information,” Bioinformatics, 21 (Suppl 1):i251- i257, 2005. And for the Phobius web server, see Lukas et al., “Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server,” Nucleic Acids Res. 35:W429-32, 2007 (all cited technical content is incorporated by reference).

SOSUI is for distinguishing between membrane proteins and soluble proteins, along with prediction of transmembrane helices. SOSUI predicts transmembrane regions using Hydrophobicity Analysis for Topology and Probe Helix Method for Tertial Structure. The accuracy of protein classification is said to be as high as 99%, and the corresponding value for transmembrane helix prediction is said to be about 97%. This SOSUI system provides Internet access www. tuat. ac. jp/mitaku/sosui.

TMPred (Node of the European Molecular Biology Network, Switzerland) predicts the orientation of transmembrane regions and proteins in a query sequence. Specifically, the TMPred algorithm is based on statistical analysis of TMbase, a database of naturally occurring transmembrane proteins. The prediction is made using a combination of several weight matrices for scoring. See Hofmann & Stoffel (1993) “TMbase - A database of membrane spanning protein segments,” Biol. Chem. Hoppe-Seyler, 374:166.

The SPLIT4.0 server is a membrane protein secondary structure prediction server (split.pmfst.hr/split/4) that predicts the transmembrane (TM) secondary structure of membrane proteins in SWISS-PROT format using the preference function method. It is. Juretic et al., “Basic charge clusters and predictions of membrane protein topology,” J. Chem. Inf. Comput. Sci., 42:620-632, 2002 (incorporated by reference). Please refer to

PRED-TMR predicts transmembrane domains within a protein simply using the protein sequence itself. This algorithm refines standard hydrophobicity analysis by detecting candidate termini ("ends", start and end) of transmembrane regions. This allows us to discard regions of high hydrophobicity that are not delimited by clear start and end configurations and to identify putative transmembrane segments that are not distinguishable by their hydrophobic composition. The accuracy obtained for a test set of 101 heterologous transmembrane proteins with reliable topology compares well with the accuracy of other common existing methods. Only a slight decrease in prediction accuracy was observed when this algorithm was applied to all transmembrane proteins in the SwissProt database (Release 35). Pasquier et al., “A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm. Novel Method: PRED-TMR Algorithm),” Protein Eng., 12(5):381-385, 1999 (incorporated by reference).

In the related PRED-TMR2, its application is extended by a preprocessing step represented by an artificial neural network that can distinguish transmembrane proteins from soluble or fibrillar proteins with high accuracy. When applied to several test sets of transmembrane proteins, this system gives a 100% perfect prediction rating by classifying all sequences within the transmembrane class. When applied to 995 non-transmembrane proteins extracted from the PDBselect database, the neural network incorrectly predicts 23 of them to be transmembrane (97.7% correct assignment). Pasquier and Hamodrakas, “An hierarchical artificial neural network system for the classification of transmembrane proteins,” Protein Eng., 12(8):631-634, 1999 (incorporated by reference).

Prediction of Protein α-Helix Secondary Structure Certain methods of the invention include predicting the α-helix secondary structure of a protein, such as a GPCR. Many such programs and software are known in the art, any of which may be used individually or in combination in the methods of the present invention involving an α-helix secondary structure prediction step. Good too. All such programs can be used in the method of the invention.

Early methods of secondary structure prediction were limited to predicting three predominant states: helix, sheet, or random coil. These methods were based on the tendency of individual amino acids to form helices or sheets, sometimes coupled with rules for estimating the free energy of forming secondary structure elements. Such methods were typically about 60% accurate in predicting which of the three states (helix/sheet/coil) a residue would assume. The first widely used technique to predict protein secondary structure from amino acid sequences was the Chou-Fasman method.

Significant increases in accuracy (to almost 80%) are made by exploiting the information provided by multiple sequence alignments, and can be found throughout evolution at a given position (and its vicinity, typically about 7 on one side). Knowing the complete distribution of amino acids occurring at a residue) provides a very good picture of the structural trends near that position. For example, a given protein may have a glycine at a given position, which itself may suggest a random coil. However, multiple sequence alignments may reveal that helix-loving amino acids occur at that position (and nearby positions) in 95% of homologous proteins through evolution. Furthermore, by examining the average hydrophobicity at that position and nearby positions, the same alignment may also suggest a residue solvent exposure pattern consistent with an α-helix. Taken together, these factors seem to suggest that the glycine in the original protein adopts an α-helical structure rather than a random coil. Thus, in the method of the present invention, an α-helix secondary structure prediction program including a neural network, hidden Markov model and support vector machine may combine all available data to make a three-state prediction. Such prediction methods also provide confidence scores for their predictions at all locations.

Secondary structure prediction methods are continuously benchmarked (e.g. EVA (benchmark)). EVA continuously runs benchmark projects to evaluate the quality of protein structure prediction and secondary structure prediction methods. Compare methods for predicting both secondary and tertiary structure, such as homology modeling, protein threading and contact order prediction, with results from weekly newly solved protein structures deposited in the Protein Structure Data Bank (PDB) . This project aims to determine the expected predictive accuracy for non-expert users of common publicly available predictive web servers.

According to these tests, the most accurate method at present is Psipred, SAM (Karplus, "SAM-T08, HMM-based protein structure prediction," Nucleic Acids Res. (2009) 37 (Web Server issue): W492-497. doi:10.1093/nar/gkp403), PORTER (Pollastri & McLysaght, "Porter: a new, accurate server for protein secondary prediction structure" Server)," Bioinformatics 21 (8):1719-1720, 2005), PROF (Yachdav et al. (2014). Nucleic Acids Res. 42 (Web Server issue): W337-343. doi:10.1093/nar/gku366) and SABLE (Adamczak et al. (2005) "Combining prediction of "Combined prediction of secondary structure and solvent accessibility in proteins," Proteins 59 (3): 467-475. doi:10.1002/prot.20441). Additionally, the standard method for assigning a secondary structure class (helix/strand/coil) to a PDB structure is DSSP (Kabsch W and Sander (1983) "Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features (Dictionary of Protein Secondary Structure: Pattern Recognition of Hydrogen Bonds and Geometric Features)," Biopolymers 22 (12): 2577-2637. doi:10.1002/bip.360221211), and benchmark against predictions for these will be held. All are incorporated by reference and all may be used in the methods of the invention.

The DSSP algorithm is a standard method for assigning secondary structure to the amino acids of a protein considering the protein's atomic resolution coordinates. DSSP begins by simply identifying the intrabackbone hydrogen bonds of a protein using electrostatic definitions, assuming partial charges of -0.42e and +0.20e for the carbonyl oxygen and amide hydrogen, respectively, and their Assign the inverse to the carbonyl carbon and amide nitrogen. A hydrogen bond is identified if E in the equation below is less than -0.5 kcal/mol.

と

との角度は７０°未満である）、空白（すなわちスペース）は他の規則が適用されない場合に使用され、ループを指す。これらの８種類は通常、３つのより大きなクラスすなわちヘリックス（Ｇ、ＨおよびＩ）、ストランド（ＥおよびＢ）ならびにループ（それ以外の全て）にグループ化される。 Based on this, eight types of secondary structures are assigned. 3 ₁₀ helices, α helices and π helices have the symbols G, H and I and are recognized by their residues having repeating sequences of hydrogen bonds separated by 3, 4 or 5 residues, respectively. There are two types of β-sheet structures, β-bridges have the symbol B and longer sets of hydrogen bonds and β-bulges have the symbol E. T is used for rotations characterized by hydrogen bonds typical of helices, and S is used for regions of high curvature (where

and

is less than 70°), white space (i.e., a space) is used when no other rules apply, and refers to a loop. These eight types are usually grouped into three larger classes: helices (G, H and I), strands (E and B) and loops (all else).

PSIPRED (PSI-BLAST Secondary Structure Prediction) is a technique used to investigate protein structure. It uses neural networks or machine learning methods in its algorithms. This is a server-side program featuring a website that serves as a front-end interface, allowing prediction of protein secondary structure (β-sheets, α-helices and coils) from the primary sequence. bioinf. cs. ucl. ac. See uk/psipred. The idea of this method is a machine learning method that uses information from evolutionarily related proteins to predict the secondary structure of new amino acid sequences. Specifically, PSI-BLAST is used to find related sequences and build a position-specific scoring matrix. This matrix is processed by a neural network built and trained to predict the secondary structure of the input array. This prediction method or algorithm is divided into three stages: sequence profile creation, initial secondary structure prediction and filtering of the predicted structure. PSIPRED operates to normalize sequence profiles created by PSI-BLAST. The initial secondary structure is then predicted by using neural networking. For each amino acid in the sequence, give the neural network a window of 15 acids. Additional information is attached indicating whether these windows span the N or C terminus of the strand. This results in a final input layer consisting of 315 input units divided into 15 groups of 21 units. This network has a single hidden layer of 75 units and three output nodes (one for each secondary structure element: helix, sheet, coil). A second neural network is used to filter the predicted structure of the first network. Give this network a window of 15 locations. An indication of the possible position of this window at the chain end is also transferred. This results in 60 input units divided into 15 groups of 4. This network has a single hidden layer of 60 units and results at three output nodes (one for each secondary structure element: helix, sheet, coil). Three final output nodes send the score of each secondary structure element for the center position of this window. Using the secondary structure with the highest score, PSIPRED generates the protein prediction. The Q3 value is the percentage of residues correctly predicted in the secondary structural states ie helix, strand and coil.

Step-by-Step Description of Exemplary Embodiments Using the invention as outlined above, certain non-limiting exemplary embodiments will now be described with reference to the representative flowcharts in the figures. This will be explained below.

FIG. 9A depicts one non-limiting embodiment of the invention. This figure generally depicts a method 200 of the present invention, in which selected hydrophobic amino acids L, I, V and F within the TM region of a protein of interest (e.g., a GPCR) are added to any particular TM region/domain Substitutions are made according to the "QTY code" of the present invention without limitation.

In a specific embodiment thereof, the method begins (202) by obtaining or reading (204) input of a protein sequence, which may or may not be a transmembrane protein. This protein sequence is then combined with TM region prediction (206) (if such information is not already available from the input protein sequence) and α-helical secondary prediction based on any of the art-recognized methods. It can be used for structural prediction. For example, TM region prediction can be performed using a program such as a TMHMM program (240). If this prediction does not result in any TM region at 242, one or more different TM region prediction programs, such as SOSUI, may be used (250) to enable prediction of the presence/absence of a TM region. If no TM region is predicted at 252 based on such a program, there may be no TM region in the protein (254) and the method ends (260).

On the other hand, if one or more TM regions are predicted by any of the suitable programs at 242, a TM region protein sequence is obtained (244) and within such one or more TM regions the QTY code of the invention is can be applied to the hydrophobic amino acids L, I, V and F. More specifically, according to the QTY code, each leucine in the TM region can be independently substituted with glutamine (Q), serine (S) or asparagine (N) (212) or left unsubstituted; Isoleucine and valine in the TM region can each be independently substituted with threonine (T), serine (S) or asparagine (N) or left unsubstituted, and each phenylalanine in the TM region can be replaced with tyrosine (Y). ) or leave it unreplaced. The result of such QTY substitutions is to produce one or more putatively water-soluble variants of the original transmembrane protein. Note that the number of substitutions made for each amino acid within a certain region can be selected as a parameter.

The α-helical secondary structure in each of the putative water-soluble variants can then be predicted using any art-recognized program such as PORTER (210). The results can be compared to the original protein results, preferably predicted using the same program (eg PORTER) (208). Note that, before, simultaneously with, or after the step of predicting the TM region of the original protein, the α-helical secondary structure of the original protein can be predicted using any program approved in the technical field.

If the α-helical secondary structure prediction results indicate that the water-soluble variant candidate at 214 maintains the same or most of the α-helical secondary structure as the original protein, it indicates that the This suggests that the specific pattern of QTY substitutions does not affect or significantly affect the α-helical secondary structure in the original protein. This TM region can then be predicted (220), confirmed (222), and variant sequences generated (224). Optionally, if this result indicates that one or more of the one or more α-helical secondary structures in the original protein is disrupted at 214, the variant is discarded as undesirable in this step. , thus the method can be ended.

On the other hand, the method of the invention also requires showing that the predicted QTY variant has a reduced or no tendency to form a TM region compared to the original protein. Thus, putatively water-soluble variants can be subjected to TM region prediction, such as using the same TM region prediction program used for the initial TM region prediction in the original protein (if desired). If the results indicate that significant TM regions are still present, the variant may be discarded. On the other hand, if the results indicate the absence of a TM region or a reduced tendency to form a TM region, the mutant can be modified to have higher water solubility than the original protein but maintain an α-helical secondary structure; and therefore can be selected as a desired variant that presumably retains the function of the original protein.

If desired, further steps can be performed to further characterize the resulting water-soluble variants. Such further characterization may include calculating the pI of the variant (226) and comparing this pI to the pI of the original protein. The pI should remain unchanged or change very slightly (ie less than 30% or preferably less than 20% or more preferably less than 10%). Other further characterizations may include creating a helical wheel model (eg, as shown in FIG. 3) 246 to indicate the location and any clustering of QTY substitutions in any particular TM region.

Another exemplary embodiment of the present invention for designing transmembrane regions of proteins (e.g., GPCRs) according to the QTY codes of the present invention is performed using a computer system using the exemplary method 10 described in FIG. 9B. Some of the detailed steps that can be performed above are further described below. Many of the steps are optional or can be combined according to the method of the invention.

1: In step 1, a computer interface of a computer system receives a protein sequence, selects it for analysis, and transmits data describing the input protein (e.g., its sequence) through the computer interface of the computer system. (12). The data entered may be a protein name, database reference or protein sequence. For example, the protein sequence can be uploaded via a computer interface.

2: In step 2, further data regarding the protein may be identified, determined, obtained and/or entered, including its name or sequence, and may be entered via a computer interface. One source for obtaining protein data (20) is a database named UniProt (www.uniprot.org). Alternatively, the method of the invention may store data regarding the protein or sequences related to the protein for later retrieval by the user during this step. In embodiments, the program may prompt the user to select a database or file to search for additional data (eg, sequence data) regarding the protein selected for analysis.

3: In step 3, the user can enter, upload or obtain data identifying the transmembrane region. For example, users may be prompted to obtain data from public sources such as UniProt. That information can be verified (30) and collected from the database for use in step 5.

4: Alternatively or additionally, even if TM region information is not readily available from the input protein sequence, the transmembrane region can be established by any art-recognized method ( 40). Transmembrane regions are generally characterized by an α-helical conformation. For example, a software module/package named TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) developed by the Center for Biological Sequencing (www.cbs.dtu.dk/services/TMHMM) was used to predict transmembrane penetration. Helix predictions can be made. This software version may have problems with peak search and may fail to find the 7 TM regions for GPCRs. Accordingly, a modified version of this program in which a dynamic baseline is introduced into the peak search method executed by the computer system may be used if desired. Here, for example in the case of GPCR, if all seven TM regions are not found using the initial baseline value, the baseline can be changed to a lower value. For example, the default baseline may be set to 0.2. To identify the missing seventh transmembrane region, the baseline value can be set to 0.1. If more than 8 TM regions are found, the baseline can be changed to a higher value, such as 0.15, to filter out off-the-mark TM predictions. For example, when the amino acid sequence of CCR-2 was submitted to TMHMM2.0 software, only six transmembrane regions were initially identified. However, when the baseline value of TMHMM2.0 was set to 0.07, a total of seven correct transmembrane regions were identified. The results of the TM region prediction are then provided to step 5.

5: In step 5, after identifying the TM data by new predictions or by obtaining such information from the initial sequence input, the sequence of the GPCR is divided into a total of 15 fragments (i.e., 7 fragments) according to the TM region information. (50) into transmembrane segments (7 TMs) (52) and eight non-membrane segments (8 NTMs) (54); i.e., seven TMs and eight NTMs for each of the typical GPCRs. NTM fragments must be present.

Of course, the system may perform one or more, eg, all, of the steps described above using a computer interface for user input. Also, of course, the present system can omit one or more of the above steps or combine two or more steps.

6: In step 6, partial QTY substitutions are made to a selected subset of hydrophobic amino acids L, I, V and F within a given TM region of the protein (60). Specifically, the first transmembrane region (typically, but not necessarily, the transmembrane region most proximal to the N-terminus of the protein) is first selected for mutation. Some or all of the hydrophobic amino acids (L, I, V and F) in the first transmembrane region are then replaced with the corresponding nonionic hydrophilic amino acids (Q/S/N, T/S/N , T/S/N or Y). Of course, in this case this amino acid is not actually substituted into the protein. Rather, this amino acid designation is substituted in the modeling sequence. Accordingly, the term "sequence" shall include "sequence data." Typically, most or all of the hydrophobic amino acids are selected for substitution. If less than all amino acids are selected, it may be desirable to leave one or more N- and/or C-terminal amino acids of the transmembrane region hydrophobic and select internal hydrophobic amino acids. Additionally or alternatively, it may be desirable to choose to replace all of the leucines (L) within the transmembrane region. Additionally or alternatively, it may be desirable to selectively replace all isoleucines (I) within the transmembrane region. Additionally or alternatively, it may be desirable to selectively replace all valines (V) within the transmembrane region. Additionally or alternatively, it may be desirable to selectively replace all phenylalanines (F) within the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more phenylalanines within the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more valines within the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more leucines within the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more isoleucines within the transmembrane region. Additionally or alternatively, it may be advantageous to retain one or more hydrophobic amino acids within a transmembrane region whose wild-type sequence is characterized by three or more consecutive hydrophobic amino acids.

7: Step 7 places the so designed transmembrane region back into the original protein sequence context. That is, each set of substitutions generates one specific putative variant for that TM region, so that a mutated or redesigned TM region (62) with a QTY substitution can be compared to its original protein counterpart. transmembrane mutants or “putative mutants” (70). Together, these related putative variants form a first library of putative variants.

8: Steps 82 and 84 then subject each putative variant to the transmembrane region prediction method (84) described herein (eg, loss of predicted TM region). The mutants are also evaluated for their sequence's propensity to form alpha helices (82). The mutants are also subjected to the water solubility prediction method described herein. For example, the variant is evaluated for its sequence's propensity to be water soluble score. Such scores may be based on the predicted tendency to form TM regions, where a strong tendency to form TM regions is associated with low water solubility, and a low tendency to form TM regions or its The lack of tendency is associated with high water solubility. Of course, complete water solubility at all concentrations is not necessary for most commercial purposes. Aqueous solubility is preferably determined to be that necessary for functionality in the expected conditions of use (eg, ligand binding assays).

9: In step 9, putative variants that predict loss of alpha helical structure and/or "water insolubility" (as predicted under expected conditions of use) are discarded. For example, a combination score or rank (90), which is a weighted combination based on a ranking function of α-helix secondary structure prediction results and TM region/water solubility prediction results, is used to predict α-helix structure and water solubility. putative mutants can be selected. For example, if it is possible to predict that the α-helical structure may be compromised, we can assign highly water-soluble or 0, 1, 2, or 3 hydrophobic amino acids (e.g., a higher weight for the predicted water solubility). Characterized transmembrane mutants can be selected. Alternatively or additionally, advanced α-helical structures characterized by 3, 4, 5 or 6 hydrophobic amino acids (eg, higher weight for α-helical secondary structure prediction results) can be selected.

10: In step 10, putative variants within the same library can be sorted or ranked (100) based on the scoring scheme (94) outlined above. A predetermined number of putative variants can then be selected as final members of the first putative variant library. For example, in the above combination scores, a score of 0 means no tendency to form TM regions and complete preservation of the original α-helical secondary structure, thus being the most desired putative variant. . A slightly higher score may indicate a slighter tendency to form TM regions (or a lower tendency to be water soluble). Therefore, although this putative variant is less desirable, it can still be selected based on its superior combination score compared to other putative variants in the library.

In certain embodiments, a predetermined number of desired putative variants can be selected, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

Repeat these steps (e.g., steps 6-10) for the second, third, fourth, fifth, sixth and/or seventh (or more) transmembrane regions or domains to A library of putative variants can be generated, one for each of the TM regions or domains.

11: In step 11, a combination of a TM region or domain with a putative variant and an unsubstituted non-TM region can be selected (110). For example, one, two, three or four domains containing a putative variant with a high α-helical structure score and one, two, three domains containing a putative variant with a high water solubility score. One, four, five or six domains can be combined. In another example, in the selection of multiple variants, a domain/TM region characterized in that all hydrophobic amino acids are replaced with hydrophilic amino acids, thus maximizing its water solubility score; , a second domain/TM region carrying 4 or 5 hydrophobic amino acids. As is known in the art, such selected putative variants are "recombined" with extracellular and intracellular domains to create the first combinatorial library of putatively water-soluble protein variants. It can be made.

In certain embodiments, all or fragments of putatively water-soluble protein variants of an initial combinatorial library designed as described herein are prepared (produced or expressed in vitro or in host cells). ) and preferably screened for water solubility and/or ligand binding in high throughput screening. For example, amplification of the library can express protein combination variants that are putatively less than 100% water soluble. Reporter systems can be used to screen for ligand binding, as is well known in the art. Using the methods of the invention, libraries of putatively water-soluble modified transmembrane combinatorial variants containing functionally combined extracellular and intracellular domains can be rapidly identified, and the appropriate Water-soluble protein variants can be produced that have three-dimensional structure and retain ligand binding function (including binding affinity) or other functions. The software can include a learning module that uses the confirmed functionality of protein variants to exclude certain variants or rank them differently.

In certain embodiments, to be experimentally practical, the initial combinatorial library has approximately 2 million potentially water-soluble GPCR or CXCR4 variants. Of course, libraries with more or fewer variants can also be designed. In certain embodiments, smaller libraries may be preferred as they can be optimized based on analysis of the study results described herein. Analysis of the study results will likely establish trends for optimizing the number of domain variants to be recombined and assumptions for selecting domain variants.

In certain embodiments, the TM of transmembrane proteins is modified for modification based on helix formation propensity, also known as "helix prediction score" (see www.proteopedia.org/wiki/index.php/Main_Page). Select specific hydrophobic amino acids within the region. The various fragments are randomly combined to form approximately 2 million ( ⁸⁷ ) variants of the full-length GPCR gene. The number of predicted variants is generally determined by the formula H ⁿ , where n = number of transmembrane regions modified and/or altered by the method (in the GPCR example, n = 7) and H = combinatorial variants. (the number of putative mutants in each transmembrane region available to produce).

Once an initial combinatorial library, or set of domain variants to be recombined, has been selected, nucleic acid molecules, DNA or cDNA molecules, can be designed that encode the proteins within the initial combinatorial library. Preferably, these nucleic acid molecules are designed to perform codon optimization and intron deletion for the selected expression system to generate a library of coding sequences. For example, if the expression system is E. coli, codons optimized for E. coli expression can be selected. www. dna20. com/resources/genedesigner. Also, a promoter region, such as a promoter suitable for expression in an expression system (eg, E. coli), is selected and operably linked to a coding sequence within a library of coding sequences.

The initial library of coding sequences, or a portion thereof, is then expressed to generate a library of putatively water-soluble GPCRs. This library is then subjected to a ligand binding assay. In binding assays, a putatively water-soluble GPCR is contacted with a ligand, preferably in an aqueous medium, and ligand binding is detected.

The present invention includes transmembrane domain variants and nucleic acid molecules encoding the same obtained or obtainable from the methods described herein.

The invention provides that at least 50%, preferably at least about 60%, more preferably at least about 70% or 80% of naturally occurring transmembrane proteins (e.g. GPCRs) are replaced by Q, T, T or Y, respectively, e.g. Water-soluble GPCR variants (“sGPCRs”) characterized by multiple transmembrane domains independently characterized by at least about 90% hydrophobic amino acid residues (L, I, V and F) are also envisioned. . The sGPCRs of the invention are characterized by water solubility and ligand binding. In particular, sGPCRs bind the same natural ligands as the corresponding natural GPCRs.

The present invention is a method of treating disorders and diseases mediated by the activity of membrane proteins, comprising the use of water-soluble polypeptides to treat disorders and diseases mediated by the activity of membrane proteins, wherein said water-soluble polypeptides have a modified alpha-helical structure. and the water-soluble polypeptide retains the ligand-binding activity of the native membrane protein. Examples of such disorders and diseases include, but are not limited to, cancer, small cell lung cancer, melanoma, breast cancer, Parkinson's disease, cardiovascular disease, hypertension, and asthma.

As described herein, the water-soluble peptides described herein can be used for the treatment of diseases or diseases mediated by the activity of membrane proteins. In certain embodiments, the water-soluble peptides can function as "decoys" for membrane receptors and bind ligands that would otherwise activate membrane receptors. Accordingly, the water-soluble peptides described herein can be used to reduce the activity of membrane proteins. These water-soluble peptides remain in the circulation and can competitively bind specific ligands, thereby reducing the activity of membrane-bound receptors. For example, the GPCR CXCR4 is overexpressed in small cell lung cancer and promotes tumor cell metastasis. Coupling to this ligand with water-soluble peptides as described herein can significantly reduce metastasis.

The chemokine receptor CXCR4 is known in virus research as the major co-receptor for T-cell line-tropic HIV entry (Feng et al. (1996) Science 272: 872-877; Davis et al. 1997) J Exp Med 186: 1793-1798; Zaitseva et al. (1997) Nat Med 3: 1369-1375; Sanchez et al. (1997) J Biol Chem 272: 27529-27531). Stromal cell-derived factor 1 (SDF-1) is a chemokine that specifically interacts with CXCR4. When SDF-1 binds to CXCR4, CXCR4 interacts with downstream kinase pathways such as Ras/MAP kinase and phosphatidylinositol 3-kinase (PI3K)/Akt in lymphocytes, megakaryocytes, and hematopoietic stem cells (Bleul et al. (1996) ) Nature 382: 829-833; Deng et al. (1997) Nature 388: 296-300; Kijowski et al. (2001) Stem Cells 19: 453-466; Majka et al. (2001) Folia. Histochem. Cytobiol. 39: 235-244; Sotsios et al. (1999) J. Immunol. 163: 5954-5963; Vlahakis et al. (Chen et al. (1998) Mol Pharmacol 53: 177-181). In mice transplanted with human lymph nodes, SDF-1 induces CXCR4-positive cell migration to the transplanted lymph nodes (Blades et al. (2002) J. Immunol. 168: 4308-4317).

Recent studies have shown that CXCR4 interaction can control the migration of metastatic cells. Hypoxia, or a decrease in oxygen tension, is a microenvironmental change that occurs in most solid tumors and is a major inducer of tumor angiogenesis and treatment resistance. Hypoxia increases CXCR4 levels (Staller et al. (2003) Nature 425: 307-311). Microarray analysis of a subpopulation of cells from a bone metastasis model with increased metastatic activity revealed that one of the genes increased in the metastatic phenotype was CXCR4. Furthermore, overexpression of CXCR4 in isolated cells significantly increased metastatic activity (Kang et al. (2003) Cancer Cell 3: 537-549). In samples taken from various breast cancer patients, Muller et al. (2001) Nature 410: 50-56 found that CXCR4 expression levels were higher in primary tumors versus normal mammary gland or epithelial cells. I found it. Furthermore, CXCR4 antibody treatment was found to inhibit regional lymph node metastasis compared to the control isotype, which all metastasized to lymph nodes and lungs (Muller et al. (2001)). Therefore, the decoy therapy model is suitable for treating CXCR4-mediated diseases and disorders.

In another embodiment, the invention relates to the treatment of diseases or disorders associated with CXCR4-dependent chemotaxis with abnormal leukocyte recruitment or activation. The diseases include arthritis, psoriasis, multiple sclerosis, ulcerative colitis, Crohn's disease, allergies, asthma, AIDS-related encephalitis, AIDS-related maculopapular eruption, AIDS-related interstitial pneumonia, AIDS-related enteropathy, and AIDS-related diseases. selected from the group consisting of periportal pneumonia and AIDS-associated glomerulonephritis.

In another aspect, the invention provides for arthritis, lymphoma, non-small cell lung cancer, lung cancer, breast cancer, prostate cancer, multiple sclerosis, central nervous system developmental disorders, dementia, Parkinson's disease, Alzheimer's disease, tumors, fibroids, It relates to the treatment of a disease or disorder selected from astrocytoma, myeloma, glioblastoma, inflammatory diseases, organ transplant rejection, AIDS, HIV infection or angiogenesis.

The present invention also encompasses pharmaceutical compositions comprising the water-soluble polypeptide and a pharmaceutically acceptable carrier or diluent.

The present compositions may contain pharmaceutically acceptable non-toxic carriers, defined as excipients commonly used to formulate pharmaceutical compositions for administration to animals or humans, depending on the desired formulation. Alternatively, a diluent may also be included. The diluent is selected so as not to affect the biological activity of the drug or pharmaceutical composition. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. The pharmaceutical composition or formulation may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like. Pharmaceutical compositions include proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex functionalized SEPHAROSE™, agarose, cellulose, etc.), polymerized amino acids, amino acid copolymers and lipids. Large slowly metabolized macromolecules such as aggregates (eg, oil droplets or liposomes) can also be included.

The composition can be administered parenterally, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the composition into a solution or suspension. Such solutions or suspensions may also include sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Parenteral formulations may also contain antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, and chelating agents such as EDTA. Buffers such as acetate, citrate or phosphate and tonicity agents such as sodium chloride or dextrose may also be added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Furthermore, auxiliary substances such as wetting agents, emulsifiers, surfactants, pH buffering substances, etc. may be present in the composition. Other components of the pharmaceutical composition are oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions.

Injectable preparations can be prepared either as liquid solutions or suspensions, and solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared. The formulation may also be emulsified or encapsulated in liposomes or microparticles such as polylactic acid, polyglycolide or copolymers to enhance the adjuvant effect as described above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997). The compositions and medicaments described herein can be administered in the form of depot injection or implant preparations that can be formulated to provide sustained or pulsatile release of the active ingredient.

Transdermal administration includes transdermal absorption of the composition through the skin. Transdermal preparations include patches, ointments, creams, gels, salves, and the like. Transdermal delivery can be achieved using skin patches or transferosomes. See Paul et al., Eur. J. Immunol. 25: 3521-24, 1995 and Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

"Treat" or "treatment" means to prevent or delay the onset of symptoms, complications or biochemical signs of a disease, to reduce or ameliorate its symptoms, or to prevent or prevent further progression of a disease, disease or disorder. Including inhibiting. A "patient" is a human subject in need of treatment.

"Effective amount" means sufficient amount of therapeutic agent to ameliorate one or more symptoms of a disease, and/or prevent progression of the disease, cause resolution of the disease, and/or achieve the desired effect. Refers to quantity.

Computer Systems Various aspects and functionality described herein may be implemented as dedicated hardware or software components running on one or more computer systems. There are many examples of computer systems currently in use. Examples of these include net appliances, personal computers, workstations, mainframes, networked clients, servers, media servers, application servers, database servers, and web servers, among others. Other examples of computer systems can include mobile computing devices such as cell phones and personal digital assistants, network equipment such as load balancers, routers and switches. Furthermore, aspects may be located on a single computer system or distributed among multiple computer systems connected by one or more communication networks.

For example, the various aspects, functions, and methods may be distributed among one or more computer systems configured to provide services to one or more client computers or to perform tasks entirely as part of a distributed system. Good too. Additionally, aspects may be performed on a client-server or multi-tier system with components distributed among one or more server systems that perform various functions. Accordingly, embodiments are not limited to performance on any particular system or systems. Further, the aspects, functions and methods may be implemented in software, hardware or firmware or any combination thereof. Accordingly, the aspects, features and methods may be implemented in methods, operations, systems, system elements and components using a variety of hardware and software configurations; these examples include any particular distributed Not limited to architecture, network or communication protocols.

Referring to FIG. 10, a block diagram of a distributed computer system 300 in which various aspects and functions are implemented is shown. As illustrated, distributed computer system 300 includes one or more computer systems that exchange information. More specifically, distributed computer system 300 includes computer systems 302, 304, and 306. As illustrated, computer systems 302 , 304 , and 306 are interconnected via a communications network 308 and may exchange data via communications network 308 . Network 308 may include any communications network through which computer systems can exchange data. To exchange data using network 308, computer systems 302, 304, and 306 and network 308 may use various methods, protocols, and standards. Examples of these protocols and standards include NAS, Web, storage and other data movement protocols suitable for use in big data environments. To ensure that data transmissions are secure, computer systems 302, 304, and 306 may transmit data over network 308 using various security measures, such as, for example, SSL or VPN technology. Although distributed computer system 300 depicts three networked computer systems, distributed computer system 300 is not so limited and can include any number of networked computer systems using any medium and communication protocol. It may include a computer system and a computing device.

As shown in FIG. 10, computer system 302 includes a processor 310, a memory 312, an interconnect element 314, an interface 316, and a data storage element 318. To implement at least some of the aspects, features, and methods disclosed herein, processor 310 performs a series of instructions that result in processed data. Processor 310 may be any type of processor, multiprocessor, or controller. Examples of processors include commercially available processors such as Intel Xeon, Itanium, Core, Celeron or Pentium processors, AMD Opteron processors, Apple A4 or A5 processors, Sun UltraSPARC processors, IBM Power5+ processors, IBM mainframe chips or quantum computers. can be mentioned. Processor 310 is connected to other system components, including one or more memory devices 312, by interconnect elements 314.

Memory 312 stores programs (eg, sequences of instructions coded for execution by processor 310) and data during operation of computer system 302. Accordingly, memory 312 may be relatively high performance volatile RAM, such as dynamic RAM ("DRAM") or static memory ("SRAM"). However, memory 312 may include any device for storing data, such as a disk drive or other non-volatile storage device. In various examples, memory 312 may be organized into individualized and possibly unique structures to perform the functions disclosed herein. These data structures may be sized and organized to store specific data values and data types.

Components of computer system 302 are connected by interconnect elements, such as interconnect element 314. Interconnection element 314 may include any communication coupling between system components such as one or more physical buses following proprietary or standard computing bus technologies such as IDE, SCSI, PCI and InfiniBand. Interconnection element 314 allows communications, including instructions and data, to be exchanged between system components of computer system 302.

Computer system 302 also includes one or more interface devices 316, such as input devices, output devices, and combinations of input/output devices. An interface device may receive input or provide output. More particularly, the output device may provide information for external presentation. The input device may receive information from an external source. Examples of interface devices include keyboards, mouse devices, trackballs, microphones, touch screens, printing devices, display screens, speakers, network interface cards, and the like. Interface devices allow computer system 302 to exchange information and communicate with external entities such as users and other systems.

Data storage element 318 comprises a computer readable and writable non-volatile or non-transitory data storage medium on which instructions defining a program or other object to be executed by processor 310 are stored. Data storage element 318 may also contain information recorded on or in a medium and processed by processor 310 during execution of the program. More specifically, the information may be stored in one or more data structures specifically configured to save storage space or enhance data exchange performance. The instructions may be permanently stored as encoded signals and may cause processor 310 to perform the functions described herein. The medium may be, for example, an optical disk, a magnetic disk or a flash memory, among others. In operation, processor 310 or some other controller stores data from a non-volatile storage medium to a memory that allows faster access to the information by processor 310 than the storage medium contained in data storage element 318. 312 or other memory. However, the memory may be located within data storage element 318 or memory 312, and processor 310 processes the data in memory and then transfers the data to a storage medium associated with data storage element 318 after the processing is complete. Copy to. Various components may manage data movement between storage media and other memory elements, and these examples are not limited to particular data management components. Furthermore, these examples are not limited to particular memory or data storage systems.

Although computer system 302 is illustrated as one type of computer system capable of implementing various aspects and functions, the aspects and functions are not limited to implementation on computer system 302 shown in FIG. 10. Various aspects and functions may be implemented on one or more computers having a different architecture or components than that shown in FIG. For example, computer system 302 may include specially programmed, special-purpose hardware, such as an application-specific integrated circuit ("ASIC"), designed to perform the specific operations disclosed herein. You can leave it there. On the other hand, another example is with a grid of some general purpose computing devices running MAC OS System X with Motorola PowerPC processors and some specialized computing devices running their own hardware and operating systems. can perform the same function.

Computer system 302 may be a computer system that includes an operating system that manages at least some of the hardware elements included in computer system 302. In some examples, a processor or controller, such as processor 310, executes an operating system. Examples of specific operating systems that may run include Windows-based operating systems such as Windows NT, Windows 2000 (Windows ME), Windows XP, Windows Vista, or Windows 7 operating systems available from Microsoft; the available MAC OS System Examples include the Solaris operating system available from Oracle Corporation or the UNIX operating system available from various sources. Many other operating systems may be used, and these examples are not limited to any particular operating system.

Processor 310 and operating system together define a computer platform on which application programs are written in high-level programming languages. These component applications may be executable intermediate bytecodes or interpreted code that communicate over a communication network, eg the Internet, using a communication protocol, eg TCP/IP. Similarly, the aspect is . NET, SmallTalk, Java(R), C++, Ada, C# (C Sharp), Python, or JavaScript(R). Other object oriented programming languages may also be used. Alternatively, functions, scripts or logic programming languages may be used.

Furthermore, various aspects and functionality may be implemented in an unprogrammed environment. For example, a document created in HTML, XML, or other formats may provide aspects of a graphical user interface or perform other functions when viewed in a browser program window. Furthermore, various examples may be implemented as programmed or unprogrammed elements or any combination thereof. For example, a web page may be implemented using HTML, but data objects required within the web page may be written in C++. Therefore, the example is not limited to a particular programming language, but any suitable programming language may be used. Accordingly, functional components disclosed herein include a wide variety of elements configured to perform the functions described herein (e.g., specialized hardware, executable code, data structures, etc.). or objects).

In some examples, components disclosed herein may read parameters that affect the functions performed by the component. These parameters may be physically stored in any form of suitable memory, including volatile memory (such as RAM) or non-volatile memory (such as a magnetic hard drive). Additionally, such parameters may be logically stored in proprietary data structures (such as a database or file defined by a user-space application) or commonly shared data structures (such as an application registry defined by an operating system). Good too. Additionally, some examples provide both a system and a user interface that allow external entities to modify parameters and thereby configure the behavior of the component.

The software for performing the computational method is shown generally in FIG. 11A, where the user selects operating parameters for performing the procedure on the computer ( 402), where one or more sequences are input (404) and substitutions are made (408). The system is operable to confirm 408 secondary structure and confirm water solubility of the one or more variants. As shown in FIG. 11B, the program can include further processing options in addition to those described above, where one or more ranking functions can be stored (442) and used by the user. The system may automatically select a ranking function to perform (444). The system then generates a rank (446) as described herein, and the user then generates (448) the selected variant and measures function (448), after which Functional data may be entered and the procedure modified 450 based thereon.

The invention will be better understood in connection with the following examples, which are intended by way of illustration only and are not intended to limit the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit of the invention and the scope of the appended claims.

である。 Example 1: CXC chemokine receptor type 4 isoform a (CXCR4)
CXCR4 is a 356 amino acid long chemokine receptor. It has a pI of approximately 8.61 and a molecular weight of 40221.19 Da. The sequence of CXCR4 published in the literature is

It is.

This sequence is subjected to TMHMM to identify the transmembrane domain shown in FIG.

All or substantially all of the hydrophobic amino acids L, I, V and F are replaced with Q, T and Y (respectively) to yield the following sequence.

The predicted pI of this protein is 8.54 and the molecular weight is 40551.64 Da. Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain (TM1) comprising amino acids 47-70 of SEQ ID NO: 2 and a protein comprising the same. As an example, Figure 3 represents an α-helix prediction of the TM1 sequence. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of the extracellular and intracellular loop sequences (sequences not underlined) of SEQ ID NO: 2. Additionally or alternatively, proteins comprising TM1 herein include SEQ ID NO: 2 or one, two, three or optionally of the natural L, I, V and F amino acids set forth in SEQ ID NO: 1. Contains one or more additional transmembrane regions (sequences underlined) within homologous sequences retaining four or more.

The native protein sequence of CXCR4 (differing in the N-terminal amino acid) is again subjected to the method. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain variants for each transmembrane domain. The results are shown in FIG. 4 and the table below.

MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNK (SEQ ID NO: 3; EC1)

TM1 mutant:
IFLPTTYSTTFQTGTTGNGQVTQVM (SEQ ID NO: 4)
IFQPTTYSTTFQTGTTGNGQVTQVM (array number 5)
IFQPTTYSTTFQTGTTGNGQVTQTM (Sequence number 6)
IFQPTTYSTTYQTGTTGNGQVTQTM (Sequence number 7)
IFQPTTYSTTYQTGTTGNGQTTQVM (Sequence number 8)
IFQPTTYSTTYQTGTTGNGQTIQTM (SEQ ID NO: 9)
IFQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO: 10)
TYQPTTYSTTYQTGTTGNGQTTQTM (SEQ ID NO: 11)

GYQKKLRSMTDKYR (SEQ ID NO: 12; IC1)

TM2 variant:
LHLSTADQQFTTTQPFWAVDAV (SEQ ID NO: 13)
LHLSVADQQYTTTQPFWATDAV (SEQ ID NO: 14)
LHQSVADQQYVTTQPFWATDAT (SEQ ID NO: 15)
QHQSVADQQFTTTQPFWATDAT (SEQ ID NO: 16)
LHQSVADQQYTITQPYWATDAT (SEQ ID NO: 17)
QHLSVADQQYTITQPYWATDAT (SEQ ID NO: 18)
QHLSTADQQYVTTQPYWATDAT (SEQ ID NO: 19)
QHQSTADQQYTTTQPYWATDAT (SEQ ID NO: 20)

ANWYFGNFLCK (SEQ ID NO: 21; EC2)

TM3 mutant:
AVHVTYTVNQYSSVQIQAFT (SEQ ID NO: 22)
AVHTTYTVNQYSSVQIQAFT (Sequence number 23)
AVHTTYTVNQYSSVQTQAFT (Sequence number 24)
ATHTTYTVNQYSSVQTQAFT (SEQ ID NO: 25)
ATHTIYTTNQYSSVQTQAFT (SEQ ID NO: 26)
AVHTTYTTNQYSSVQTQAFT (SEQ ID NO: 27)
ATHTTYTTNQYSSVQTQAFT (SEQ ID NO: 28)
ATHTTYTTNQYSSTQTQAYT (SEQ ID NO: 29)

SLDRYLAIVHATNSQRPRKLLAEK (SEQ ID NO: 30; IC2)

TM4 mutant:
VTYTGVWTPAQQQTIPDFIF (SEQ ID NO: 31)
TTYTGTWIPAQQQTIPDFIF (SEQ ID NO: 32)
TTYTGTWTPAQQQTIPDFIF (SEQ ID NO: 33)
TTYTGTWTPAQQQTIPDFIY (SEQ ID NO: 34)
TTYVGTWTPAQQQTTPDYIF (SEQ ID NO: 35)
TTYVGTWTPAQQQTTPDFIY (SEQ ID NO: 36)
TTYTGVWTPAQQQTTPDYTF (SEQ ID NO: 37)
TTYTGTWTPAQQQTTPDYTY (SEQ ID NO: 38)

ANVSEADDRYICDRFYPNDLW (SEQ ID NO: 39; EC3)

TM5 mutant:
VVVFQFQHTMVGQTQPGTTTQ (SEQ ID NO: 40)
VVVFQFQHTMTGQTQPGTTTQ (SEQ ID NO: 41)
VVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 42)
VVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO: 43)
TVVFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 44)
VVTFQYQHTMTGQTQPGTTTQ (SEQ ID NO: 45)
TVVYQYQHTMTGQTQPGTTTQ (SEQ ID NO: 46)
TTTYQYQHTMTGQTQPGTTTQ (Sequence number 47)

SCYCIIISKLSHSKGHQKRKALKTT (SEQ ID NO: 48; IC3)

TM6 mutant:
VTQIQAFFACWQPYYTGTST (array number 49)
VIQIQAYFACWQPYYTGTST (SEQ ID NO: 50)
VIQIQAYYACWQPYYTGTST (SEQ ID NO: 51)
VIQTQAFYACWQPYYTGTST (SEQ ID NO: 52)
VIQTQAYFACWQPYYTGTST (SEQ ID NO: 53)
VTQIQAFYACWQPYYTGTST (Sequence number 54)
VIQTQAYYACWQPYYTGTST (SEQ ID NO: 55)
TTQTQAYYACWQPYYTGTST (SEQ ID NO: 56)

DSFILLEIIKQGCEFENTVHK (SEQ ID NO: 57; EC4)

TM7 mutant
WISITEAQAFFHCCLNPIQY (SEQ ID NO: 58)
WISITEAQAFYHCCLNPIQY (SEQ ID NO: 59)
WISITEAQAYFHCCQNPTLY (SEQ ID NO: 60)
WISTTEALAFYHCCQNPTQY (SEQ ID NO: 61)
WISTTEALAYFHCCQNPTQY (SEQ ID NO: 62)
WISITEALAYYHCCQNPTQY (SEQ ID NO: 63)
WISTTEALAYYHCCQNPTQY (SEQ ID NO: 64)
WTSTTEAQAYYHCCQNPTQY

AFLGAKFKTSAQHALTSVSRGSLKILSKGKRGGHSSVSTESESSSFHSS (SEQ ID NO: 65; IC4)

The sequences before, between and after each list of transmembrane domain variants (SEQ ID NOs: 3, 12, 21, 30, 39, 48, 57 and 65) are N', middle and C' extracellular and intracellular, respectively. It seems clear from the above that this is an area.

The above sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as is known in the art. This coding sequence is then recombined and expressed to produce SEQ ID NOs: 3, 12, 21, 30, each containing one transmembrane domain variant within each variant list between the respective intracellular and extracellular domains. , 39, 48, 57, and 65.

The library so produced was then assayed for the CXCR4 cognate ligand, SDF1a (or CCL12), on a plasmid expressed in yeast that binds in living yeast cells. Ligand binding was detected by gene activation with a yeast two-hybrid system, and samples were then sequenced. Nineteen CXCR4 variants were sequenced. The results are shown in FIG.

Example 2: CXC chemokine receptor type 3 isoform b (CX3CR1)
CX3CR1 is a 355 amino acid long chemokine receptor. It has a pI of approximately 6.74 and a molecular weight of 40396.4 Da. This sequence is subjected to TMHMM to identify its transmembrane domain. All or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain were replaced with Q, T and Y (respectively) to align with the wild type (top line). Get the array (bottom line).

The predicted pI of this protein variant is 6.74 and the molecular weight is 41027.17 Da. Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain comprising the underlined amino acids of SEQ ID NO:67. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of the extracellular and intracellular loop sequences (sequences not underlined) of SEQ ID NO: 66. Additionally or alternatively, proteins comprising TM1 herein include SEQ ID NO: 67 or one, two, three of the naturally occurring V, L, I and F amino acids set forth in SEQ ID NO: 66, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more.

The native protein sequence of CX3CR1 is again subjected to this method. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain variants for each transmembrane domain. The results are shown in the table below.

MDQFPESVTENFEYDDLAEACYIGDIVVFGT (SEQ ID NO: 68)

TM1 mutant:
TYQSTYYSTTFATGQVGNQQVVFALTNS (SEQ ID NO: 69)
TYQSTYYSTTYATGQVGNQQVVFALTNS (Sequence number 70)
TYQSTYYSTTYATGQVGNQQVVFAQTNS (Sequence number 71)
TYQSTYYSTTYATGQTGNLQVTFAQTNS (SEQ ID NO: 72)
TYQSTYYSTTYATGQTGNQLVTFAQTNS (SEQ ID NO: 73)
TYQSTYYSTTYATGQTGNQQVVFAQTNS (SEQ ID NO: 74)
TYQSTYYSTTYATGQTGNLQVTYAQTNS (SEQ ID NO: 75)
TYQSTYYSTTYATGQTGNQQTTYAQTNS (SEQ ID NO: 76)

KKPKSVTDIY (SEQ ID NO: 77)

TM2 mutant
LLNQAQSDQLFVATQPFWTHY (Sequence number 78)
LLNQAQSDQQFVATQPFWTHY (Sequence number 79)
QQNLAQSDQQFVATQPFWTHY (Sequence number 80)
LQNLAQSDQQYTATQPFWTHY (SEQ ID NO: 81)
QLNLAQSDQQYTATQPFWTHY (SEQ ID NO: 82)
LLNQAQSDQQFTATQPYWTHY (SEQ ID NO: 83)
QQNLAQSDQQFTATQPYWTHY (SEQ ID NO: 84)
QQNQAQSDQQYTATQPYWTHY (SEQ ID NO: 85)

LINEKGLHNAMCK (SEQ ID NO: 86)

TM3 mutant:
YTTAYYYTGYYGSTYYTTTTST (SEQ ID NO: 87)

DRYLAIVLAANSMNNRT (SEQ ID NO: 88)

TM4 mutant:
VQHGTTTSQGTWAAATQVAAPQFMF (SEQ ID NO: 89)
VQHGVTTSQGTWAAATQTAAPQFMF (Sequence number 90)
VQHGTTTSQGVWAAATQTAAPQFMY (SEQ ID NO: 91)
VQHGTTTSQGTWAAAIQTAAPQFMY (SEQ ID NO: 92)
VQHGTTTSQGTWAAATQTAAPQFMF (SEQ ID NO: 93)
VQHGTTISQGTWAAATQTAAPQYMF (SEQ ID NO: 94)
VQHGTTTSQGTWAAATQTAAPQFMY (SEQ ID NO: 95)
TQHGTTTSQGTWAAATQTAAPQYMY (SEQ ID NO: 96)

TKQKENECLGDYPEVLQEIWPVLRNVET (SEQ ID NO: 97)

TM5 mutant:
NFLGFQQPQQIMSYCYFRIT (SEQ ID NO: 98)
NFQGFLQPQQTMSYCYFRIT (SEQ ID NO: 99)
NFQGFLQPQQTMSYCYFRTT (SEQ ID NO: 100)
NFQGFQQPQQTMSYCYYRIT (SEQ ID NO: 101)
NFQGFLQPQQTMSYCYYRTT (SEQ ID NO: 102)
NFQGYLQPQQTMSYCYFRTT (SEQ ID NO: 103)
NYQGFQQPQQTMSYCYFRTT (SEQ ID NO: 104)
NYQGYQQPQQTMSYCYYRTT (SEQ ID NO: 105)

QTLFSCKNHKKAKAIK (SEQ ID NO: 106)

TM6 mutant:
LIQQTTTTFYQFWTPYNTMTFQETL (SEQ ID NO: 107)
LIQQTTTTFYQYWTPYNVMTFQETQ (SEQ ID NO: 108)
LIQQTTTTYYQFWTPYNTMTFQETQ (SEQ ID NO: 109)
QIQQTTTTFYQYWTPYNTMTFQETQ (SEQ ID NO: 110)
LTQQTTTTYYQFWTPYNTMTFQETQ (Sequence number 111)
QIQQTTTTFFQYWTPYNTMTYQETQ (SEQ ID NO: 112)
QIQQTTTTFYQYWTPYNTMTYQETQ (SEQ ID NO: 113)
QTQQTTTTYYQYWTPYNTMTYQETQ (SEQ ID NO: 114)

KLYDFFPSCDMRKDLRL (SEQ ID NO: 115)

TM7 variant:
ALSVTETVAFSHCCQNPQIYAFAG (SEQ ID NO: 116)
AQSVTETTAFSHCCQNPLIYAFAG (SEQ ID NO: 117)
ALSVTETVAFSHCCQNPQTYAYAG (SEQ ID NO: 118)
AQSVTETTAFSHCCQNPQIYAYAG (SEQ ID NO: 119)
ALSVTETTAFSHCCQNPQTYAYAG (SEQ ID NO: 120)
ALSTTETTAYSHCCQNPQIYAFAG (SEQ ID NO: 121)
ALSVTETTAYSHCCQNPQTYAYAG (SEQ ID NO: 122)
AQSTTETTAYSHCCQNPQTYAYAG (SEQ ID NO: 123)

EKFRRYLYHLYGKCLAVLCGRSVHVDFSSSESQRSRHGSVLSSNFTYHTSDGDALLLL (SEQ ID NO: 124)

Similar to Example 1 above, the sequences before, between, and after each list of transmembrane domain variants are the N', middle, and C' intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as is known in the art. This coding sequence is then recombinantly expressed to produce SEQ ID NOs: 68, 77, 86, 88, each containing one transmembrane domain variant within each variant list between the respective intracellular and extracellular domains. , 97, 106, and 115.

The library so generated was then assayed for binding to CX3CR1 cognate ligand (CXCL1) in aqueous medium as described in Example 1. Ligand binding was detected and samples were then sequenced. Seven mutants were sequenced. The results are shown in FIG.

Example 3: CCR3 Mutants The method of Example 1 was repeated for chemokine receptor type 3 isoform 3.

All or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain were replaced with Q, T and Y (respectively) to align with the wild type (top line). Get the array (bottom line).

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain comprising the underlined amino acids of SEQ ID NO: 126. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of the extracellular and intracellular loop sequences (sequences not underlined) of SEQ ID NO: 126. Additionally or alternatively, proteins comprising TM1 herein include SEQ ID NO: 126 or one, two, three of the natural V, L, I and F amino acids set forth in SEQ ID NO: 125, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more.

The native protein sequence of CCR3 is again subjected to the method (note the differences in the N-terminal sequence). The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain variants for each transmembrane domain. The results are shown in the table below.

MTTSLDTVETFGTTSYYDDVGLLCEKADTRALMA (SEQ ID NO: 127)

TM1 mutant:
QFVPPQYSQTFTTGQQGNVTVTMTQIKY (SEQ ID NO: 128)
QFVPPQYSQTFTTGQQGNTTVTMTQIKY (SEQ ID NO: 129)
QFVPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 130)
QFTPPQYSQTYTTGQQGNVTTTMTQIKY (SEQ ID NO: 131)
QFTPPQYSQTYTTGQQGNTVTTMTQIKY (SEQ ID NO: 132)
QFTPPQYSQTYTTGQQGNTTVTMTQIKY (SEQ ID NO: 133)
QFTPPQYSQTYTTGQQGNTTTTMTQIKY (SEQ ID NO: 134)
QYTPPQYSQTYTTGQQGNTTTTMTQTKY (SEQ ID NO: 135)

RRLRIMTNIY (SEQ ID NO: 136)

TM2 variant:
LLNQATSDQQFQVTQPFWIHY (Sequence number 137)
LQNQAISDQLFQTTQPFWTHY (SEQ ID NO: 138)
QQNLAISDQQFQTTQPFWTHY (SEQ ID NO: 139)
QLNQAISDQQFQTTQPYWTHY (SEQ ID NO: 140)
QQNLAISDQQYQVTQPYWTHY (SEQ ID NO: 141)
LQNQATSDQLFQTTQPYWTHY (SEQ ID NO: 142)
QQNQAISDQQYQVTQPYWTHY (Sequence number 143)
QQNQATSDQQYQTTQPYWTHY (SEQ ID NO: 144)

VRGHNWVFGHGMCK (SEQ ID NO: 145)

TM3 mutant:
LQSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 146)
QLSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 147)
QLSGFYHTGQYSETFYTTQQTT (SEQ ID NO: 148)
QLSGFYHTGQYSETYFTTQQTT (SEQ ID NO: 149)
QLSGYYHTGQYSETFFTTQQTT (SEQ ID NO: 150)
QQSGFYHTGQYSETFFTTQQTT (SEQ ID NO: 151)
QQSGFYHTGQYSETFYTTQQTT (SEQ ID NO: 152)
QQSGYYHTGQYSETYYTTQQTT (SEQ ID NO: 153)

DRYLAIVHAVFALRART (SEQ ID NO: 154)

TM4 mutant:
TTFGTTTSTVTWGQAVQAAQPEFIF (SEQ ID NO: 155)
TTFGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO: 156)
TTYGTTTSTTTWGQAVQAAQPEFIF (SEQ ID NO: 157)
TTYGTTTSTTTWGQAVQAAQPEFTF (SEQ ID NO: 158)
TTYGTTTSTTTWGQATQAAQPEFIF (SEQ ID NO: 159)
TTFGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO: 160)
TTYGTTTSTTTWGQATQAAQPEFIY (SEQ ID NO: 161)
TTYGTTTSTTTWGQATQAAQPEYTY (SEQ ID NO: 162)

YETEELFEETLCSALYPEDTVYSWRHFHTLRM (SEQ ID NO: 163)

TM5 mutant:
TIFCQVQPQQTMATCYTGTT (SEQ ID NO: 164)
TIFCQTQPQQVMATCYTGTT (SEQ ID NO: 165)
TIFCQTQPQQTMATCYTGIT (SEQ ID NO: 166)
TIFCQTQPQQTMATCYTGTI (SEQ ID NO: 167)
TTFCQVQPQQVMATCYTGTT (SEQ ID NO: 168)
TIYCQVQPQQVMATCYTGTT (SEQ ID NO: 169)
TIFCQTQPQQTMATCYTGTT (SEQ ID NO: 170)
TTYCQTQPQQTMATCYTGTT (SEQ ID NO: 171)

KTLLRCPSKKKYKAIR (SEQ ID NO: 172)

TM6 mutant:
QTYTTMATYYTYWTPYNTATQQSSY (SEQ ID NO: 173)

QSILFGNDCERSKHLDL (SEQ ID NO: 174)

TM7 variant:
VMQVTEVTAYSHCCMNPTYAFTG (Sequence number 175)
VMQVTEVTAYSHCCMNPTTYAYVG (array number 176)
VMLTTEVTAYSHCCMNPTTYAFTG (SEQ ID NO: 177)
VMQVTETTAYSHCCMNPTYAYTG (SEQ ID NO: 178)
TMQVTETIAYSHCCMNPTYAFTG (SEQ ID NO: 179)
TMQVTETTAYSHCCMNPTTYAFVG (Sequence number 180)
VMQTTETIAYSHCCMNPTTYAYTG (SEQ ID NO: 181)
TMQTTETTAYSHCCMNPTTYAYTG (SEQ ID NO: 182)

ERFRKYLRHFFHRHLLMHLGRYIPFLPSEKLERTSSVSPSTAEPELSIVF (SEQ ID NO: 183)

Similar to Example 1 above, the sequences before, between, and after each list of transmembrane domain variants are the N', middle, and C' intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as is known in the art. This coding sequence is then recombined and expressed to produce SEQ ID NOs: 127, 136, 145, 154, each containing one transmembrane domain variant within each variant list between the respective intracellular and extracellular domains. , 163, 172, 174, and 183.

The library so generated was then assayed for binding to the CCR3 cognate ligand, CCL3, in aqueous medium as described in Example 1. Ligand binding was detected and samples were then sequenced. Eleven mutants were sequenced. The results are shown in FIG.

Example 4: CCR5 Variants The method of Example 1 was repeated for chemokine receptor type 5 isoform 3.

All or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain were replaced with Q, T and Y (respectively) to align with the wild type (top line). Get the array (bottom line).

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes a transmembrane domain comprising the underlined amino acids of SEQ ID NO: 185. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of the extracellular and intracellular loop sequences (sequences not underlined) of SEQ ID NO: 185. Additionally or alternatively, proteins comprising TM1 herein include SEQ ID NO: 185 or one, two, three of the naturally occurring V, L, I and F amino acids set forth in SEQ ID NO: 184, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more.

The native protein sequence of CCR5 is again subjected to this method (note the differences in the N-terminal sequence). The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain variants for each transmembrane domain. The results are shown in the table below.

MDYQVSSPIYDINYYTSEPCQKINVKQIAA (SEQ ID NO: 186)

TM1 mutant:
RLQPPQYSQTFTFGFTGNMQVTQTQINC (SEQ ID NO: 187)
RLQPPQYSQTFTFGYTGNMQVTQTQINC (Sequence number 188)
RQQPPQYSQTFTFGFTGNMQTTQTQINC (SEQ ID NO: 189)
RQQPPQYSQTFTYGFTGNMQTTQTQINC (SEQ ID NO: 190)
RQQPPQYSQTYTFGFTGNMQTTQTQINC (SEQ ID NO: 191)
RQQPPQYSQTFTFGYTGNMQTTQTQINC (SEQ ID NO: 192)
RQQPPQYSQTYTFGYTGNMQTTQTQINC (SEQ ID NO: 193)
RQQPPQYSQTYTYGYTGNMQTTQTQTNC (SEQ ID NO: 194)

KRLKSMTDIY (SEQ ID NO: 195)

TM2 variant:
LQNQAISDQFFQQTVPFWAHY (SEQ ID NO: 196)
LQNQAISDQFFQQTTPFWAHY (SEQ ID NO: 197)
LQNQAISDQFFQQTTPYWAHY (SEQ ID NO: 198)
LQNQAISDQFYQQTTPYWAHY (SEQ ID NO: 199)
LQNQAISDQYFQQTTPYWAHY (Sequence number 200)
LQNQATSDQFFQQTTPYWAHY (SEQ ID NO: 201)
LQNQAISDQYYQQTTPYWAHY (SEQ ID NO: 202)
QQNQATSDQYYQQTTPYWAHY (SEQ ID NO: 203)

AAAQWDFGNTMCQ (SEQ ID NO: 204)

TM3 mutant:
QQTGQYFTGYYSGTYYTTQQTT (SEQ ID NO: 205)
QQTGQYYTGYYSGTYYTTQQTT (SEQ ID NO: 206)

DRYLAVVHAVFALKART (SEQ ID NO: 207)

TM4 mutant:
TTYGTTTSTTTWTTATYASQPGTTY (SEQ ID NO: 208)

TRSQKEGLHYTCSSHFPYSQYQFWKNFQTLKI (SEQ ID NO: 209)

TM5 mutant:
VIQGQVQPQQVMVTCYSGIQ (Sequence number 210)
VIQGQVQPQQVMTTCYSGIQ (SEQ ID NO: 211)
VIQGQVQPQQTMTTCYSGIQ (SEQ ID NO: 212)
VTQGQVQPQQTMVTCYSGTQ (array number 213)
TIQGQVQPQQVMTTCYSGTQ (Sequence number 214)
TIQGQVQPQQTMVTCYSGTQ (Sequence number 215)
TTQGQVQPQQVMTTCYSGTQ (Sequence number 216)
TTQGQTQPQQTMTTCYSGTQ (SEQ ID NO: 217)

KTLLRCRNEKKRHRAVR (SEQ ID NO: 218)

TM6 mutant:
QTFTTMTTYYQFWAPYNIVQQLNTF (SEQ ID NO: 219)
QTFTTMTTYYQFWAPYNTVQQLNTF (SEQ ID NO: 220)
QTFTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO: 221)
QTFTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO: 222)
QTYTTMTTYYQYWAPYNTVQQLNTF (SEQ ID NO: 223)
QTFTTMTTYYQYWAPYNTTQQLNTF (SEQ ID NO: 224)
QTYTTMTTYYQYWAPYNTVQQQNTF (SEQ ID NO: 225)
QTYTTMTTYYQYWAPYNTTQQQNTY (SEQ ID NO: 225)

QEFFGLNNCSSSNRLDQ (SEQ ID NO: 226)

TM7 variant:
AMQVTETQGMTHCCINPIIYAFVG (SEQ ID NO: 227)
AMQVTETLGMTHCCTNPIIYAFTG (SEQ ID NO: 228)
AMQVTETQGMTHCCINPTIYAYVG (SEQ ID NO: 229)
AMQTTETQGMTHCCINPITYAFTG (SEQ ID NO: 230)
AMQTTETQGMTHCCINPTIYAFTG (SEQ ID NO: 231)
AMQVTETQGMTHCCTNPTIYAYVG (SEQ ID NO: 232)
AMQTTETQGMTHCCINPTTYAYVG (SEQ ID NO: 233)
AMQTTETQGMTHCCTNPTTYAYTG (SEQ ID NO: 234)

EKFRNYLLVFFQKHIAKRFCKCCSIFQQEAPERASSVYTRSTGEQEISVGL (SEQ ID NO: 235)

Similar to Example 1 above, the sequences before, between, and after each list of transmembrane domain variants are the N', middle, and C' intracellular or extracellular regions, respectively.

The above sequences were then used to generate coding sequences suitable for expression in an expression system, in this case yeast, as is known in the art. This coding sequence is then recombinantly expressed to produce SEQ ID NOs: 186, 195, 204, 207, each containing one transmembrane domain variant within each variant list between the respective intracellular and extracellular domains. , 209, 218, 226, and 235.

The library so generated was then assayed in aqueous medium for binding to the CCR5 cognate ligand, CCL5, as described in Example 1. Ligand binding was detected and samples were then sequenced. One mutant was sequenced. The results are shown in FIG.

Example 5: CXCR3 Mutants The method of Example 1 was repeated for CXC chemokine receptor type 3 isoform 2. All or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain are replaced with Q, T and Y (respectively), aligned to the wild type (SEQ ID NO: 324, upper line). The following sequence (SEQ ID NO: 325, lower line) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include SEQ ID NO: 325 or one, two, three of the natural V, L, I and F amino acids set forth in SEQ ID NO: 324, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more.

The native protein sequence of CXCR3 was subjected to the method as described above. The program output divided the native sequence into extracellular and intracellular regions and selected eight transmembrane domain variants for each transmembrane domain. The results are shown in the table below.

MVLEVSDHQVLNDAEVAALLENFSSSYDYGENESDSCCTSPPCPQDFSLNFDR (SEQ ID NO: 235)

TM1 mutant:
AFLPALYSQQFQQGQQGNGAVAATQLS (SEQ ID NO: 236)
AFQPALYSQQFQQGQQGNGAVAAVQQS (SEQ ID NO: 237)
AFQPAQYSQQFLQGQQGNGAVAATQQS (SEQ ID NO: 238)
AYQPALYSLQYQQGQQGNGATAAVQQS (SEQ ID NO: 239)
AYQPALYSQLFQQGQQGNGATAATQQS (array number 240)
AFQPALYSLQYQQGQQGNGATAATQQS (SEQ ID NO: 241)
AYQPAQYSLQYQQGQQGNGATAAVQQS (Sequence number 242)
AYQPAQYSQQYQQGQQGNGATAATQQS (Sequence number 243)

RRTALSSTD (SEQ ID NO: 244)

TM2 variant:
TFLQHLAVADTQQVQTLPQWA (SEQ ID NO: 245)
TFLQHQAVADTQLVQTQPQWA (SEQ ID NO: 246)
TFQQHLAVADTQQVQTQPQWA (SEQ ID NO: 247)
TYLQHQAVADTQQVQTQPQWA (SEQ ID NO: 248)
TYQLHQAVADTQQVQTQPQWA (Sequence number 249)
TYQQHLAVADTQQVQTQPQWA (Sequence number 250)
TYQQHQAVADTQQVQTQPQWA (Sequence number 251)
TYQQHQATADTQQTQTQPQWA (Sequence number 252)

VDAAVQWVFGSGLCK (SEQ ID NO: 253)

TM3 mutant:
TAGAQYNTNFYAGAQQQACISF (SEQ ID NO: 254)
TAGAQYNTNFYAGAQLQACTSF (SEQ ID NO: 255)
TAGAQYNTNFYAGAQQLACTSF (SEQ ID NO: 256)
TAGAQFNTNYYAGAQQQACISF (SEQ ID NO: 257)
TAGAQYNTNYYAGAQQQACISF (SEQ ID NO: 258)
TAGAQYNTNYYAGAQLQACTSF (SEQ ID NO: 259)
TAGAQYNTNYYAGAQQLACTSF (SEQ ID NO: 260)
TAGAQYNTNYYAGAQQQACTSY (SEQ ID NO: 261)

DRYLNIVHATQLYRRGPPARVT (SEQ ID NO: 262)

TM4 mutant:
LTCQAVWGQCQQFAQPDFIF (SEQ ID NO: 263)
QTCQAVWGQCQQFAQPDFIF (SEQ ID NO: 264)
QTCQATWGQCQQFAQPDFIF (SEQ ID NO: 265)
QTCQATWGQCQQYAQPDFIF (SEQ ID NO: 266)
QTCQATWGQCQQFAQPDFTF (SEQ ID NO: 267)
QTCQATWGQCQQFAQPDYIF (SEQ ID NO: 268)
QTCQATWGQCQQYAQPDYIF (SEQ ID NO: 269)
QTCQATWGQCQQYAQPDYTY (SEQ ID NO: 270)

LSAHHDERLNATHCQYNFPQVGR (SEQ ID NO: 271)

TM5 mutant:
TAQRTQQQTAGYQQPQQTMAY (Sequence number 272)

CYAHILAVLLVSRGQRRLRAMR (SEQ ID NO: 273)

TM6 mutant:
QVTTTVAFAQCWTPYHQVVQV (SEQ ID NO: 274)
QVTTTVAFAQCWTPYHQTVQV (SEQ ID NO: 275)
QVTTTTTAFAQCWTPYHQTVQV (SEQ ID NO: 276)
QVTTTTTAYAQCWTPYHQTVQV (SEQ ID NO: 277)
QVTTTTTAFAQCWTPYHQTTQV (SEQ ID NO: 278)
QTTTTVAFAQCWTPYHQTTQV (SEQ ID NO: 279)
QVTTTTTAYAQCWTPYHQTTQV (SEQ ID NO: 280)
QTTTTTTAYAQCWTPYHQTTQT (SEQ ID NO: 281)

DILMDLGALARNCGRESRVDV (SEQ ID NO: 282)

TM7 variant:
AKSVTSGQGYMHCCLNPLQYAFV (SEQ ID NO: 283)
AKSVTSGQGYMHCCLNPQLYAFT (SEQ ID NO: 284)
AKSVTSGQGYMHCCLNPLQYAFT (SEQ ID NO: 285)
AKSTTSGQGYMHCCLNPQQYAFV (SEQ ID NO: 286)
AKSTTSGQGYMHCCQNPLQYAFV (SEQ ID NO: 287)
AKSTTSGQGYMHCCQNPQLYAFV (SEQ ID NO: 288)
AKSTTSGQGYMHCCQNPLQYAFT (SEQ ID NO: 289)
AKSTTSGQGYMHCCQNPQQYAYT (SEQ ID NO: 290)

GVKFRERMWMLLLRLGCPNQRGLQRQPSSSRRDSSWSETSEASYSGL (SEQ ID NO: 291)

The above sequences can be used to generate coding sequences suitable for expression in expression systems, in this case yeast, as known in the art. This coding sequence is then recombined and expressed to produce multiple intracellular and extracellular loops, each containing one transmembrane domain variant in each variant list between its respective intracellular and extracellular domains. A library containing the following proteins was created.

The library so produced can then be assayed for binding to the cognate ligand in aqueous medium as described in Example 1.

Example 6: (CCR-1) CC chemokine receptor type 1
Example 1 was repeated for the title protein.

Aligned to wild type (upper line, SEQ ID NO: 292) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 293) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three of the naturally occurring V, L, I and F amino acids as described in the depicted protein or in the wild type sequence. one or more additional transmembrane regions (underlined sequences) in homologous sequences retaining one or optionally four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 7: (CCR-2) CC Chemokine Receptor Type 2 Isoform A
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 294) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 295) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three of the naturally occurring V, L, I and F amino acids as described in the depicted protein or in the wild type sequence. one or more additional transmembrane regions (underlined sequences) in homologous sequences retaining one or optionally four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 8: (CCR-4) CC chemokine receptor type 4
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 296) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 297) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three of the naturally occurring V, L, I and F amino acids as described in the depicted protein or in the wild type sequence. one or more additional transmembrane regions (underlined sequences) in homologous sequences retaining one or optionally four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 9: (CCR-6) CC chemokine receptor type 6
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 298) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 299) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 10: (CCR-7) CC Chemokine Receptor Type 7 Precursor Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 300) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 301) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 11: (CCR-8) CC chemokine receptor type 8
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 302) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 303) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 12: (CCR-9) CC Chemokine Receptor Type 9 Isoform B
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 304) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 305) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 13: (CCR-10) CC chemokine receptor type 10
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 306) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 307) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more. The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 14: (CXCR1) Chemokine receptor type 1
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 308) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 309) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 15: (CXR1) CXR chemokine receptor type 1
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 310) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 311) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three of the naturally occurring V, L, I and F amino acids as described in the depicted protein or in the wild type sequence. one or more additional transmembrane regions (underlined sequences) in homologous sequences retaining one or optionally four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 16: (CXCR2) Chemokine receptor type 2
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 312) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 313) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 17: (CCR-10) CC chemokine receptor type 10
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 314) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 315) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 18: (CXCR6) Chemokine receptor type 6
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 316) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 317) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 19: (CXCR7) Chemokine receptor type 7
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 318) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 319) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 20: (CLR-1a) Chemokine-like receptor 1 isoform a
Example 1 was repeated for the title protein. Aligned to wild type (upper line, SEQ ID NO: 320) with all or substantially all of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively) The following sequence (lower line, SEQ ID NO: 321) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences, optionally retaining four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 21: DARIA Duffy antigen/chemokine receptor isotype a
Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 322) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 323) is obtained.

Each predicted transmembrane region is underlined and exemplifies a fully modified domain of the invention. Thus, for example, the invention includes transmembrane domains that each include the underlined domains. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three, or One or more additional transmembrane regions (sequences underlined) in homologous sequences optionally retaining four or more. The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 22: CD81 Antigen CD81 may play an important role in the control of lymphoma cell proliferation, interacting with the 16 kDa Leu-13 protein to form a complex possibly involved in signal transduction. CD81 may function as a viral receptor for HCV.

Example 1 was repeated for the title protein. The following sequence aligned to the wild type (upper line, SEQ ID NO: 324) with each of the hydrophobic amino acids L, I, V and F in its transmembrane domain replaced by Q, T and Y (respectively). (lower line, SEQ ID NO: 325) is obtained.

Predicted transmembrane regions exemplify the modified domains of the invention and include the following (SEQ ID NO: 326, 327, 328, 329, 330, 331, 332, 333, respectively).

Thus, for example, the invention includes transmembrane domains, each including a modified or "mt" domain. Preferably, the TM1-comprising proteins herein include one or more (eg, all) of extracellular and intracellular loop sequences (sequences not underlined). Additionally or alternatively, proteins comprising TM1 herein include one, two, three of the naturally occurring V, L, I and F amino acids as described in the depicted protein or in the wild type sequence. one or more additional transmembrane regions (underlined sequences) in homologous sequences retaining one or optionally four or more.

The wild-type sequence can be subjected to the methods described above to select additional transmembrane domain variants as described in Example 1. Coding sequences can be designed and recombined to express proteins. Expressed proteins can be assayed for ligand binding as described herein.

Example 23: E. coli expression of QTY and CXCR4-QTY mutants

1. Large-scale production of CXCR4-QTY in E. coli BL21(DE3) The water-soluble GPCR CXCR4 was produced in E. coli with an estimated yield of approximately 20 mg of purified protein per liter of routinely used LB medium. . Estimated production cost is approximately $0.25 per milligram. Advantageously, this approach can be used to easily obtain gram quantities of water-soluble GPCRs, which then facilitates their structural determination.

2. Determination of the location where soluble CXCR4-QTY is produced in E. coli cells Soluble CXCR4-QTY was cloned into a pET vector. We first conducted a small scale E. coli culture study to assess the location of the produced CXCR4-QTY protein (150 ml culture). After culturing cells induced with IPTG for 4 hours at 24°C, we harvested and sonicated these cells and divided them into two fractions by centrifugation at 14,637 x g (12,000 rpm). Divided into minutes. We then detected the location of CXCR4-QTY protein using Western blot analysis of a specific anti-rho tag monoclonal antibody. We observed that CXCR4-QTY protein was in the supernatant fraction and no protein was in the pellet fraction, thus suggesting that the protein was completely water soluble.

3. Estimated yield of CXCR4-QTY produced in the soluble fraction of E. coli cells Next, the present inventors separately cultured 150 ml and obtained CXCR4-QTY purified with about 6 mg of 1D4 monoclonal antibody. Because we underestimated the yield (we did not expect the surprisingly high yield), we Not sufficient affinity rho-1D4 tag monoclonal antibody beads were used. Therefore, a significant amount of CXCR4-QTY protein did not bind to the beads because not enough beads were added during purification and the protein was in the flow lane and was further washed away. Despite the significant loss, we are still able to obtain approximately 6 mg for a 150 ml culture, as can be seen from lanes 8-10 (eluted fractions).

4. Determination of Thermal Stability of Purified Water-Soluble CXCR4-QTY Protein In most cases, structure determines function in a protein. Therefore, it is important to know whether the purified CXCR4-QTY protein produced in E. coli is still correctly folded into a typical α-helical structure with approximately 50% α-helices. The present inventors performed secondary structure measurements using circular dichroism (CD). The present inventors observed CD spectra of CXCR4-QTY protein purified at various temperatures. The present inventors measured the thermal stability of purified CXCR4-QTY protein. We observed that the purified CXCR4-QTY protein was relatively stable up to 55° C., with only partial gradual denaturation of the protein and CD signal reduction of approximately 15%. From 55°C to 65°C, the denaturation increased towards 65°C, a denaturation transition occurred from 65°C to 75°C, and at 75°C the protein was almost completely denatured.

We plotted temperature versus ellipticity at 222 nm to obtain the melting temperature (Tm) of purified water-soluble CXCR4-QTY protein. From this plot, we estimated that the Tm of purified CXCR4-QTY protein was approximately 67°C. This Tm suggests that purified water-soluble CXCR4-QTY protein is very stable compared to many other soluble proteins. This thermal stability property makes it easier to obtain diffractive crystals, since it is known that the better the thermal stability, the better the crystal lattice packing and thus the greater the chances of obtaining a structure.

5. Additional G-Protein Coupled Receptors The present inventors selected ten G-protein-coupled receptors (GPCRs) and used Zhang et al.'s “Water Soluble Membrane Proteins and Methods for the Preparation and Use Thereof.” The water-soluble form was designed using the QTY method described in US Patent Publication No. 2012/0252719A ("Zhang") entitled ``Methods of Preparation and Use''. Alternatively, proteins described herein can be selected.

6. Molecular Cloning of Genes The present inventors successfully confirmed the native and QTY genes of GPCRs in the cell-free protein expression plasmid vector pIVex2.3d and the E. coli pET28a and pET-duet-1 plasmid vectors.

7. Production of water-soluble GPCRs We produced several native and QTY proteins. When producing native GPCRs in a cell-free system, the detergent Brij35 is required; without detergent, the protein precipitates as soon as it is produced. On the other hand, we tested QTY variants in the presence and absence of detergent. Without detergent, the cell-free system produced soluble protein.

We cloned QTY mutants into the E. coli in vivo expression system pET28a and pET-duet-1 plasmid vectors for E. coli cellular protein production in the E. coli BL21(DE3) strain. We purified several water-soluble GPCR proteins, including CXCR4 and CCR5, which we used for secondary structure analysis. We performed ligand binding studies on CXCR4 using its natural ligand CCL12 (SDF1a). The present inventors produced and purified a water-soluble GPCR CCR5e mutant in E. coli. The CCR5e variant had 58 amino acid changes (approximately 18% change). Soluble GPCR CCR5e mutants were purified to homogeneity using a specific monoclonal antibody rhodopsin tag. The blue strain showed a single band on the SDS gel indicating its purity. As deduced from the protein's size markers, it appears to be a pure homodimer (the native membrane-bound CXCR4 crystal structure was dimeric). Western blot confirmed monomers and homodimers of common CCR5e variants in GPCRs.

8. Secondary structure study of QTY CCR5e The present inventors obtained a water-soluble QTY mutant of GPCR CCR5e. We then performed secondary structure analysis using an Aviv model 410 circular dichroism instrument and confirmed that the GPCR QTY CCR5-e variant had a typical α-helical structure. We also determined the Tm of the CCR5e variants, ie, the thermal stability of water-soluble CCR5e variants, by conducting experiments at various temperatures. From these experiments, we determined that the Tm of the CCR5e mutant was approximately 46°C. This Tm is good for crystal screening experiments.

9. Ligand binding studies of CXCR4 using CCL12 (SDF1a) to ensure that the designed water-soluble QTY GPCRs still retain their biological function, i.e. recognize and bind their natural ligands. We first used ELISA measurements to study water-soluble CXCR4 with its natural ligand CCL12 (also referred to as SDF1a). Assay concentrations range from 50 nM to 10 μM. The measured Kd is approximately 80 nM. The Kd of native membrane-bound CXCR4 with SDF1a is approximately 100 nM. Therefore, the Kd of water-soluble CXCR4 is within an acceptable range. Further experiments using more sensitive SPR or other measurements may be performed to generate a more accurate Kd.

Although the invention has been illustrated and described with particular reference to preferred embodiments thereof, it will be appreciated that various changes may be made in form and detail thereof without departing from the scope of the invention as encompassed by the appended claims. will be understood by those skilled in the art.

This application may include the following inventions.
(1)
A computer-implemented method for performing a procedure for selecting water-soluble variants of a G protein-coupled receptor (GPCR), the method comprising:
inputting the sequence of the GPCR for analysis;
Obtaining a variant of the GPCR in which a plurality of hydrophobic amino acids within the transmembrane (TM) domain α-helical segment (“TM region”) of the GPCR are substituted, the step comprising:
(a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) the leucine (L) is each independently substituted with glutamine (Q), asparagine (N) or serine (S);
(c) the isoleucine (I) and the valine (V) are each independently substituted with threonine (T), asparagine (N), or serine (S), and (d) the phenylalanine is each substituted with tyrosine (Y ), followed by obtaining α-helical secondary structure results for said mutant to confirm maintenance of α-helical secondary structure within said mutant;
obtaining transmembrane region results for said mutant to confirm water solubility of said mutant, thereby selecting water-soluble mutants of said GPCR.
(2)
The method according to claim (1), wherein step (3) is performed before, simultaneously with, or after step (4).
(3)
in step (2), substituting a subset of the plurality of hydrophobic amino acids in one and the same TM region of the GPCR to create a member of a variant candidate library, and 2. The method of claim 1 or 2, wherein one or more different subsets of amino acids are substituted to create further members of the library.
(4)
10. The method of claim 1, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. The method described in (3).
(5)
2. The method of claim 1, further comprising ranking the variants using a ranking function.
(6)
The method of claim 1, further comprising performing the method using a data processor.
(7)
7. The method of claim 6, further comprising a memory coupled to the data processor.
(8)
6. The method of claim 5, wherein the ranking function includes secondary structure components and water-soluble components.
(9)
9. The method of claim 8, wherein the ranking function includes weighting values for the secondary structure component and/or the water-soluble component.
(10)
further comprising selecting N members with the highest combination scores to form a first library of variant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).
(11)
11. The method of claim (10), further comprising generating one library of variant candidates for one, two, three, four, five or all six other TM regions of the GPCR.
(12)
12. The method of claim 11, further comprising replacing two or more TM regions of the GPCR with corresponding TM regions in the mutant candidate library to create a combinatorial mutant library.
(13)
13. The method of any one of claims (1) to (12), wherein substantially all (eg all) of the leucine is replaced with glutamine.
(14)
14. The method of any one of claims (1) to (13), wherein substantially all (eg all) of the isoleucine is replaced with threonine.
(15)
15. The method of any one of claims (1) to (14), wherein substantially all (eg all) of the valine is replaced with threonine.
(16)
16. The method of any one of claims (1) to (15), wherein substantially all (eg all) of the phenylalanines are substituted with tyrosine.
(17)
17. The method of any one of claims (1) to (16), wherein one or more (eg 1, 2 or 3) of the leucines are unsubstituted.
(18)
18. The method of any one of claims (1) to (17), wherein one or more (eg 1, 2 or 3) of the isoleucines are unsubstituted.
(19)
19. The method of any one of claims (1) to (18), wherein one or more (eg 1, 2 or 3) of the valines are unsubstituted.
(20)
The method of any one of claims (1) to (19), wherein one or more (eg 1, 2 or 3) of the phenylalanines are unsubstituted.
(21)
The method according to any one of claims (1) to (20), further comprising the step of producing/expressing the combination variant.
(22)
Claim 1 further comprising the step of testing the combination variants for ligand binding (e.g., in a yeast two-hybrid method), selecting those that have substantially the same ligand binding compared to a GPCR. The method according to any one of (21) to (21).
(23)
Claims 1 to 3 further comprising the step of testing said combination variants for the biological function of said GPCR, selecting those having substantially the same biological function as compared to said GPCR. The method according to any one of (22).
(24)
24. The method of any one of claims (1)-(23), wherein the combinatorial variant library contains less than about 2 million members.
(25)
The method according to any one of claims (1) to (24), wherein the sequence of the GPCR includes information regarding the TM region of the GPCR.
(26)
The method of any one of claims (1) to (25), wherein the sequence of the GPCR is obtained from a protein structure database (eg PDB, UniProt).
(27)
The method according to any one of claims (1) to (26), wherein the TM region of the GPCR is predicted based on the sequence of the GPCR.
(28)
28. The method of claim 27, wherein the TM region of the GPCR is predicted using a TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) software module.
(29)
29. The method of claim 28, wherein the TMHMM 2.0 software module utilizes a dynamic baseline for peak search.
(30)
The method of any one of claims (1) to (29), further comprising the step of providing a polynucleotide sequence of each variant of said GPCR.
(31)
The polynucleotide sequence is codon-optimized for expression in a host (e.g., a bacteria such as E. coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cells, a non-human mammalian cell or a human cell). 31. The method of claim 30.
(32)
32. The method of any one of claims 1 to 31, wherein the scripting procedure comprises a VBA script.
(33)
The scripted procedure is operable by a Linux system (e.g. Ubuntu 12.04 LTS), a Unix system, a Microsoft Windows operating system, an Android operating system or an Apple iOS operating system. ) to (32).
(34)
A water-soluble variant of a G protein-coupled receptor (GPCR) in which multiple hydrophobic amino acids in the transmembrane (TM) domain α-helical segment (“TM region”) of the GPCR are substituted, comprising:
(a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) the leucine (L) is each independently substituted with glutamine (Q), asparagine (N) or serine (S),
(c) said isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S), and (d) said phenylalanine is each replaced with tyrosine (Y). A water-soluble mutant characterized in that the α-helical secondary structure is maintained by all seven TM regions of the mutant, and there is no predicted transmembrane region.
(35)
Claim (34) comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64. water-soluble variants described in .
(36)
The water-soluble variant according to claim (35), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57.
(37)
The water-soluble variant according to claim (35) or (36), which binds to a CXCR4 ligand.
(38)
Claim (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114 and 116-123. water-soluble variants of.
(39)
The water-soluble variant according to claim (38), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124.
(40)
The water-soluble variant according to claim (38) or (40), which binds to a CX3CR1 ligand.
(41)
Claim (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-182. water-soluble variants of.
(42)
The water-soluble variant according to claim (41), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 127, 136, 145, 154, 163, 172, 174 and 183.
(43)
The water-soluble variant according to claim (41) or (42), which binds to a CCR3 ligand.
(44)
Claim (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234. water-soluble variants of.
(45)
The water-soluble variant according to claim (44), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 186, 195, 204, 207, 209, 218, 226 and 235.
(46)
The water-soluble variant according to claim (44) or (45), which binds to a CCR5 ligand.
(47)
Claim (34), comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290. water-soluble variants of.
(48)
The water-soluble variant according to claim (47), further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291.
(49)
The water-soluble variant according to claim (47) or (48), which binds to a CXCR3 ligand.
(50)
Any one of SEQ ID NO: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323 or 325 35. A water-soluble variant according to claim (34), comprising one or more transmembrane domains as described in .
(51)
51. The water-soluble variant according to claim 50, wherein the water-soluble variant is water-soluble and binds to a ligand of a homologous natural transmembrane protein.
(52)
(a) culturing the bacteria in a growth medium under conditions suitable for protein production;
(b) dividing the bacterial lysate into fractions to produce a soluble fraction and an insoluble pellet fraction;
(c) isolating the protein from the soluble fraction;
(1) The protein is a variant of the G protein-coupled receptor (GPCR) according to any one of claims 29 to 46,
(2) producing a protein in a bacterium (e.g., E. coli), wherein the yield of the protein is at least 20 mg/L (e.g., 30 mg/L, 40 mg/L, 50 mg/L or more) of the growth medium; How to produce.
(53)
48. The method of claim 47, wherein the bacterium is E. coli BL21 and the growth medium is LB medium.
(54)
49. The method of claim 47 or 48, wherein the protein is encoded by a plasmid within the bacterium.
(55)
The method according to any one of claims (47) to (49), wherein the expression of the protein is under the control of an inducible promoter.
(56)
51. The method of claim 50, wherein the inducible promoter is inducible by IPTG.
(57)
The method according to any one of claims (47) to (51), wherein the lysate is produced by ultrasonication.
(58)
The method of any one of claims (47) to (52), wherein the lysate is centrifuged at 14,500 x g or higher to generate the soluble fraction.
(59)
A non-transitory computer-readable medium having stored thereon a set of instructions for performing the method according to any of claims (1) to (33).
(60)
A data processing system operative to select a water-soluble variant of a membrane protein, comprising a data processor operative to perform amino acid substitutions, the system comprising: a ranking function comprising a secondary structure component and a water-soluble component; A system for ranking protein variants.
(61)
61. The system of claim (60), further comprising a library of membrane proteins for processing by the system.
(62)
61. The system of claim 60, further comprising a memory coupled to the data processor for storing coded instructions for executing a replacement processor.
(63)
A system according to claim (60), operative for steps (a), (b), (c) and (d) of the method according to claim 1.
(64)
61. The system of claim (60), further comprising a ranking function that is a weighted combination based on the secondary structure components.
(65)
61. The system of claim 60, communicating with an external program via a network.
(66)
61. The system of claim (60), further comprising a database for storing water-soluble variants.
(67)
61. The system of claim 60, further comprising instructions for performing dynamic baseline processing.
(68)
61. The system of claim 60, further comprising an interface for selecting parameters of the method.
(69)
System according to claim (60), further comprising the step of inputting the sequences as described in claims 35-50.
(70)
A computer-implemented method for performing steps for selecting water-soluble variants, the method comprising:
processing the data to identify membrane protein sequences for analysis;
obtaining a variant of the membrane protein in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helical segment (“TM region”) of the membrane protein are substituted;
The data processor is
determining α-helix secondary structure results of the mutant to confirm maintenance of α-helix secondary structure in the mutant;
A method characterized in that the transmembrane region results of the mutant are determined to confirm the water solubility of the mutant, and a water-soluble mutant of the membrane protein is selected.
(71)
The said substitution is
(a) selecting a hydrophobic amino acid from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) substituting leucine (L) with glutamine (Q), asparagine (N) or serine (S), each independently;
(c) isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S); and (d) said phenylalanine is each replaced with tyrosine (Y). 71. The method of claim (70), comprising substituting.
(72)
substituting one subset of said plurality of hydrophobic amino acids in one and the same TM region of a GPCR to create a member of a variant candidate library, and one or more different subsets of said plurality of hydrophobic amino acids; 72. The method of claim 71, wherein further members of the library are created by substituting .
(73)
10. The method of claim 1, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. (70).
(74)
71. The method of claim 70, further comprising ranking the variants using a ranking function.
(75)
71. The method of claim 70, further comprising a memory coupled to the data processor.
(76)
75. The method of claim 74, wherein the ranking function includes secondary structure components and water soluble components.
(77)
77. The method of claim 76, wherein the ranking function includes weighting values for the secondary structure components and/or the water-soluble components.
(78)
further comprising selecting N members with the highest combination scores to form a first library of variant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more).
(79)
79. The method of claim (78), further comprising generating a library of variant candidates for 1, 2, 3, 4, 5 or all 6 other TM regions of the GPCR.
(80)
80. The method of claim 79, further comprising replacing two or more TM regions of the GPCR with corresponding TM regions in the variant candidate library to create a combinatorial variant library.

Claims (80)

A computer-implemented method for performing a procedure for selecting water-soluble variants of a G protein-coupled receptor (GPCR), the method comprising:
inputting the sequence of the GPCR for analysis;
Obtaining a variant of the GPCR in which a plurality of hydrophobic amino acids within the transmembrane (TM) domain α-helical segment (“TM region”) of the GPCR are substituted, the step comprising:
(a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) the leucine (L) is each independently substituted with glutamine (Q), asparagine (N) or serine (S);
(c) the isoleucine (I) and the valine (V) are each independently substituted with threonine (T), asparagine (N), or serine (S), and (d) the phenylalanine is each substituted with tyrosine (Y ), followed by obtaining α-helical secondary structure results for said mutant to confirm maintenance of α-helical secondary structure within said mutant;
obtaining transmembrane region results for said mutant to confirm water solubility of said mutant, thereby selecting water-soluble mutants of said GPCR. 2. The method of claim 1, wherein step (3) is performed before, simultaneously with, or after step (4). in step (2), substituting a subset of the plurality of hydrophobic amino acids in one and the same TM region of the GPCR to create a member of a variant candidate library, and 3. The method of claim 1 or 2, wherein one or more different subsets of amino acids are substituted to create further members of the library. 10. The method of claim 1, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. The method described in 3. 2. The method of claim 1, further comprising ranking the variants using a ranking function. The method of claim 1, further comprising performing the method using a data processor. 7. The method of claim 6, further comprising a memory coupled to the data processor. 6. The method of claim 5, wherein the ranking function includes secondary structure components and water soluble components. 9. The method of claim 8, wherein the ranking function includes weighting values for the secondary structure components and/or the water-soluble components. further comprising selecting N members with the highest combination scores to form a first library of variant candidates for the TM region, where N is a predetermined integer (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). 11. The method of claim 10, further comprising generating one library of candidate variants for one, two, three, four, five or all six other TM regions of the GPCR. 12. The method of claim 11, further comprising replacing two or more TM regions of the GPCR with corresponding TM regions in the variant candidate library to create a combinatorial variant library. 13. The method of any one of claims 1-12, wherein substantially all (eg all) of the leucine is replaced with glutamine. 14. The method of any one of claims 1-13, wherein substantially all (eg all) of the isoleucine is replaced with threonine. 15. The method of any one of claims 1-14, wherein substantially all (eg all) of the valine is replaced with threonine. 16. The method of any one of claims 1-15, wherein substantially all (eg all) of the phenylalanines are substituted with tyrosine. 17. The method of any one of claims 1-16, wherein one or more (eg 1, 2 or 3) of the leucines are unsubstituted. 18. The method of any one of claims 1-17, wherein one or more (eg 1, 2 or 3) of the isoleucines are unsubstituted. 19. The method of any one of claims 1-18, wherein one or more (eg 1, 2 or 3) of the valines are unsubstituted. 20. The method of any one of claims 1-19, wherein one or more (eg 1, 2 or 3) of the phenylalanines are unsubstituted. 21. The method of any one of claims 1 to 20, further comprising the step of producing/expressing the combination variant. Claims 1-21 further comprising the step of testing said combination variants for ligand binding (e.g., in a yeast two-hybrid method), selecting those having substantially the same ligand binding compared to a GPCR. The method according to any one of the above. 23. The method of claims 1-22 further comprising testing the combination variants for the biological function of the GPCR, selecting those having substantially the same biological function as compared to the GPCR. The method described in any one of the above. 24. The method of any one of claims 1-23, wherein the combinatorial variant library contains less than about 2 million members. 25. The method according to any one of claims 1 to 24, wherein the sequence of the GPCR comprises information regarding the TM region of the GPCR. 26. The method according to any one of claims 1 to 25, wherein the sequence of the GPCR is obtained from a protein structure database (eg PDB, UniProt). 27. The method according to any one of claims 1 to 26, wherein the TM region of the GPCR is predicted based on the sequence of the GPCR. 28. The method of claim 27, wherein the TM region of the GPCR is predicted using a TMHMM2.0 (Transmembrane Prediction Using Hidden Markov Models) software module. 29. The method of claim 28, wherein the TMHMM 2.0 software module utilizes a dynamic baseline for peak search. 30. The method of any one of claims 1 to 29, further comprising the step of providing a polynucleotide sequence of each variant of said GPCR. The polynucleotide sequence is codon-optimized for expression in a host (e.g., a bacteria such as E. coli, a yeast such as Saccharomyces cerevisiae or fission yeast, an insect cell such as Sf9 cells, a non-human mammalian cell or a human cell). 31. The method of claim 30, wherein: 32. A method according to any preceding claim, wherein the scripting procedure comprises a VBA script. The scripted procedure is operable by a Linux system (e.g. Ubuntu 12.04 LTS), a Unix system, a Microsoft Windows operating system, an Android operating system or an Apple iOS operating system. 33. The method according to any one of 32. A water-soluble variant of a G protein-coupled receptor (GPCR) in which multiple hydrophobic amino acids in the transmembrane (TM) domain α-helical segment (“TM region”) of the GPCR are substituted, comprising:
(a) the hydrophobic amino acid is selected from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) the leucine (L) is each independently substituted with glutamine (Q), asparagine (N) or serine (S),
(c) said isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S), and (d) said phenylalanine is each replaced with tyrosine (Y). A water-soluble mutant characterized in that the α-helical secondary structure is maintained by all seven TM regions of the mutant, and there is no predicted transmembrane region. 35. Comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 4-11, 13-20, 22-29, 31-38, 40-47, 49-56 and 58-64. water-soluble variants of. 36. The water-soluble variant of claim 35, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 3, 12, 21, 30, 39, 48 and 57. 37. A water-soluble variant according to claim 35 or 36, which binds to CXCR4 ligand. The aqueous solution according to claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 69-76, 78-85, 87, 89-96, 98-105, 107-114 and 116-123. Sex mutant. 39. The water-soluble variant of claim 38, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 68, 77, 86, 88, 97, 106, 115 and 124. 41. A water-soluble variant according to claim 38 or 40, which binds to CX3CR1 ligand. 35. The aqueous solution according to claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 128-135, 137-144, 146-153, 155-162, 164-171, 173 and 175-182. Sex mutant. 42. The water-soluble variant of claim 41, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 127, 136, 145, 154, 163, 172, 174 and 183. 43. A water-soluble variant according to claim 41 or 42, which binds to a CCR3 ligand. The aqueous solution according to claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 187-194, 196-203, 205-206, 208, 210-217, 219-225, 227-234. Sex mutant. 45. The water-soluble variant of claim 44, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NO: 186, 195, 204, 207, 209, 218, 226 and 235. 46. A water-soluble variant according to claim 44 or 45, which binds to CCR5 ligand. 35. The aqueous solution according to claim 34, comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 236-243, 245-252, 254-261, 263-270, 272, 274-281 and 283-290. Sex mutant. 48. The water-soluble variant of claim 47, further comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 235, 244, 253, 262, 271, 273, 282 and 291. 49. A water-soluble variant according to claim 47 or 48, which binds to CXCR3 ligand. Any one of SEQ ID NO: 2, 67, 126, 185, 327, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323 or 325 35. The water-soluble variant of claim 34, comprising one or more transmembrane domains as described in . 51. The water-soluble variant of claim 50, wherein the water-soluble variant is water-soluble and binds to a homologous natural transmembrane protein ligand. (a) culturing the bacteria in a growth medium under conditions suitable for protein production;
(b) dividing the bacterial lysate into fractions to produce a soluble fraction and an insoluble pellet fraction;
(c) isolating the protein from the soluble fraction;
(1) The protein is a variant of the G protein-coupled receptor (GPCR) according to any one of claims 29 to 46,
(2) producing a protein in a bacterium (e.g., E. coli), wherein the yield of the protein is at least 20 mg/L (e.g., 30 mg/L, 40 mg/L, 50 mg/L or more) of the growth medium; How to produce. 48. The method of claim 47, wherein the bacterium is E. coli BL21 and the growth medium is LB medium. 49. The method of claim 47 or 48, wherein the protein is encoded by a plasmid within the bacterium. 50. The method of any one of claims 47-49, wherein expression of the protein is under the control of an inducible promoter. 51. The method of claim 50, wherein the inducible promoter is inducible by IPTG. 52. A method according to any one of claims 47 to 51, wherein the lysate is produced by sonication. 53. The method of any one of claims 47 to 52, wherein the lysate is centrifuged at 14,500 xg or higher to generate the soluble fraction. A non-transitory computer-readable medium having stored thereon a set of instructions for performing the method of any of claims 1-33. A data processing system operative to select a water-soluble variant of a membrane protein, comprising a data processor operative to perform amino acid substitutions, the system comprising: a ranking function comprising a secondary structure component and a water-soluble component; A system for ranking protein variants. 61. The system of claim 60, further comprising a library of membrane proteins for processing by the system. 61. The system of claim 60, further comprising a memory coupled to the data processor that stores coded instructions for executing a replacement processor. 61. The system of claim 60, operative for steps (a), (b), (c) and (d) of the method of claim 1. 61. The system of claim 60, further comprising a ranking function that is a weighted combination based on the secondary structure components. 61. The system of claim 60, communicating with an external program via a network. 61. The system of claim 60, further comprising a database for storing water soluble variants. 61. The system of claim 60, further comprising instructions for performing dynamic baseline processing. 61. The system of claim 60, further comprising an interface for selecting parameters of the method. 61. The system of claim 60, further comprising inputting a sequence as described in claims 35-50. A computer-implemented method for performing steps for selecting water-soluble variants, the method comprising:
processing the data to identify membrane protein sequences for analysis;
obtaining a variant of the membrane protein in which a plurality of hydrophobic amino acids in the transmembrane (TM) domain α-helical segment (“TM region”) of the membrane protein are substituted;
The data processor is
determining α-helix secondary structure results of the mutant to confirm maintenance of α-helix secondary structure in the mutant;
A method characterized in that the transmembrane region results of the mutant are determined to confirm the water solubility of the mutant, and a water-soluble mutant of the membrane protein is selected. The said substitution is
(a) selecting a hydrophobic amino acid from the group consisting of leucine (L), isoleucine (I), valine (V) and phenylalanine (F);
(b) substituting leucine (L) with glutamine (Q), asparagine (N) or serine (S), each independently;
(c) isoleucine (I) and said valine (V) are each independently substituted with threonine (T), asparagine (N) or serine (S); and (d) said phenylalanine is each replaced with tyrosine (Y). 71. The method of claim 70, comprising substituting. substituting one subset of said plurality of hydrophobic amino acids in one and the same TM region of a GPCR to create a member of a variant candidate library, and one or more different subsets of said plurality of hydrophobic amino acids; 72. The method of claim 71, wherein additional members of the library are created by substituting . 10. The method of claim 1, further comprising the step of ranking all members of the library based on a combined score, the combined score being a weighted combination of the α-helix secondary structure prediction result and the transmembrane region prediction result. 70. 71. The method of claim 70, further comprising ranking the variants using a ranking function. 71. The method of claim 70, further comprising a memory coupled to the data processor. 75. The method of claim 74, wherein the ranking function includes secondary structure components and water soluble components. 77. The method of claim 76, wherein the ranking function includes weighting values for the secondary structure components and/or the water-soluble components. further comprising selecting N members with the highest combination scores to form a first library of variant candidates for the TM region, where N is a predetermined integer (e.g., 3, 74. The method of claim 73, wherein the method is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). 79. The method of claim 78, further comprising generating a library of candidate variants for one, two, three, four, five or all six other TM regions of the GPCR. 80. The method of claim 79, further comprising replacing two or more TM regions of the GPCR with corresponding TM regions in the variant candidate library to create a combinatorial variant library.

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