JP4729764B2 - Rapid and highly efficient selection method of highly functional protein, highly functional protein obtained thereby, and production method and utilization method thereof - Google Patents

Description

  The present invention relates to a method for selecting a highly functional protein, a highly functional protein obtained thereby, and a method for producing and utilizing the same.

  The interaction of the protein that interacts with the target molecule is deeply involved in the function of the protein, and attempts have been made to obtain a protein having a superior function. For example, the high specificity and affinity for antigens of a kind of antibody molecule of a protein has high utility value in a wide range of fields, and research on the construction of a system that can create antibodies freely and effectively is actively conducted. Yes. Inherently, antibodies repeat domain shuffling and somatic mutation in B cells (affinity maturation), and only antibodies with higher specificity and affinity for an antigen can act in the immune system. it can.

  In the study of antibody affinity, monoclonal antibodies are generally used from the viewpoint of their homogeneity. Currently, the main method for preparing monoclonal antibodies is the cell fusion method (hybridoma technology) (Non-patent Document 1). . However, this cell fusion method is very time-consuming, requires expensive reagents, and takes time (about one year). Furthermore, since it is necessary to immunize animals, there are various problems that it is difficult to select antibodies against self-antigens and poisons. Recently, evolutionary molecular engineering techniques such as phage display (Non-patent document 2) and ribosome display (Non-patent document 3) (technology that directly associates nucleic acid [genetic information] and protein [functional information]) as a method to solve these problems Are used for the selection of monoclonal antibodies. That is, a target antibody can be obtained relatively easily by constructing an antibody library having great diversity and repeatedly selecting and concentrating the target antibody (corresponding molecule). In particular, a method for a single chain antibody (Non-patent Document 4) in which the C-terminal of one chain and the N-terminal of the other chain are bound with a highly flexible peptide linker is known.

However, these techniques still have some drawbacks. In phage display, the upper limit of a realistic DNA library is about 10 ⁹ (in the case of antibodies), the library size is small, and biological systems (such as infection and amplification of E. coli) are used. Unexpected bias (mutation, etc.) is applied. Furthermore, antigens that are toxic to E. coli cannot be used. Since ribosome display is a complete in vitro experimental system using a cell-free translation system, these biases are not applied, and the upper limit of the library can be 10 ¹⁰ -10 ¹¹ . However, the formation efficiency of mRNA-protein-ribosome ternary complex is as low as 0.2% (Non-patent Document 3), which is one hundredth of that of in vitro virus (IVV) method (Non-patent Document 5)). . In addition, since the complex is very unstable, there is a problem that a stronger selective pressure cannot be applied during the biopanning operation. Here, the selection pressure in panning is the most important factor in antibody concentration, and the fact that strong selection pressure cannot be applied results in the need to repeat more selection cycles. In addition, a wide variety of antibodies with different stability and affinity can be obtained for one type of antigen, so many samples must be cloned, expressed, and analyzed to obtain higher performance. . This is not only very hard work, but also takes time and money.

  So far, large-scale protein expression has been studied in wheat germ systems (Non-patent Document 6) and E. coli systems (Non-patent Document 7) as cell-free translation systems for synthesizing proteins in vitro. Along with that, 3'UTR is generally used to improve the stability and translation efficiency of mRNA as a stable translation template that can express a large amount of protein (Non-patent Document 8). A method such as substitution or modification of the chemical structure is used (Non-patent Document 9).

  In 1997, we succeeded in building “in vitro virus (IVV)”, a screening tool for evolutionary molecular engineering, for the first time in the world (Non-patent Document 10). In the IVV method, a library of RNA-protein linked molecules (IVV) in which protein and mRNA are covalently bonded via puromycin by performing cell-free translation reaction using mRNA with puromycin bound to the 3 'end as a template. Can be built. A protein that binds to a target molecule is screened in vitro from the library of corresponding molecules, and then the gene can be amplified and decoded by reverse transcription PCR. After that, we started basic research to apply the IVV method to proteome analysis, constructed IVV libraries from cDNA libraries derived from humans and mice, and used them as target molecules such as proteins, nucleic acids, and drugs. We have established a technique for screening unknown and interacting proteins in vitro rapidly and with high sensitivity. Recently, we succeeded in developing a template DNA that can express IVV and C-terminal labeled protein with high efficiency in a cell-free translation system of wheat germ extract (Non-patent Document 5). The inventors of the present invention have previously proposed a method for efficiently constructing an IVV as a mapping molecule by linking mRNA (genotype) and protein (phenotype) via puromycin in a cell-free translation system. (Patent Document 1; Patent Document 2; Patent Document 3; Patent Document 4).

  Furthermore, in the process of constructing the IVV method, we found that when a low concentration of puromycin derivative is introduced into a cell-free translation system, it binds to the C-terminus of the synthesized protein. By applying this principle and using, for example, a compound in which a fluorescent dye is linked to puromycin, it becomes possible to label the C-terminus of the protein with a fluorescent dye (Non-patent Document 11; Non-patent Document 12). . The present inventors have proposed a method for efficiently labeling the C-terminus of a protein in a cell-free translation system (Patent Document 5; Patent Document 6; Patent Document 7).

  A large amount of protein is required to analyze the detailed function and three-dimensional structure of a protein and to produce an antibody. For this reason, it is difficult to purify the endogenous target protein and obtain the required amount. Therefore, a recombinant protein in which the cDNA of the target protein is expressed in a suitable host is used. Various hosts such as Escherichia coli, yeast, insect cells and cultured cells derived from animals can be considered as hosts for expressing single chain antibodies. Among them, E. coli is convenient because a large amount of protein can be obtained in a short period of time at a low cost. In addition, when a system that infects insect cells with a recombinant baculovirus is used, protein expression, which was difficult in the E. coli system, becomes possible.

  Antibodies also play an important role in various analysis tools that have been developed for post-genome functional analysis research. One example is protein detection and quantification. For that purpose, it is indispensable to use highly specific antibodies with high specificity. Examples of protein detection methods using antibodies include Western blotting, immunostaining, fluorescent antibody staining, antibody chip method in in vitro methods, and immunoprecipitation in in vivo methods. In order to detect the protein using such a technique, the antibody is preliminarily fused with a fluorescent protein such as GFP, or the antibody is fused with an enzyme protein such as horseradish peroxidase or alkaline phosphatase, The enzyme activity is used as an index.

  Methods for detecting protein-protein interactions using antibodies include surface plasmon resonance, fluorescence resonance energy transfer, fluorescence depolarization, evanescent field imaging, fluorescence correlation spectroscopy, fluorescence imaging, solid-phase enzyme immunoassay and so on. In particular, Fluorescence Correlation Spectroscopy (FCS) requires a small amount of sample (approximately femtoliter), a short measurement time (approximately 10 seconds), and is easy to automate for HTS (actual EVOTEC is developing a device aimed at ultra HTS that can screen more than 100,000 samples a day), and is an excellent detection system (Non-patent Document 13). In addition, in Fluorescence Cross-Correlation Spectroscopy (FCCS) using two types of fluorescent dyes, interactions between molecules of the same size, which were difficult with FCS using one type of fluorescent dye, are also possible. Detection is possible, and application of protein interaction to HTS is expected. Generally, in a protein interaction detection system, it is necessary to modify a protein with a probe for immobilization or a probe such as a fluorescent dye. The present inventors have previously proposed a method of modifying the C-terminus of a protein in a cell-free translation system using a nucleic acid derivative such as puromycin (Patent Document 5, Patent Document 6, and Patent Document 3). This method has the advantage that the function of the protein is less likely to be impaired than the conventional chemical modification method and fluorescent protein fusion method.

  The IVV method is a method of constructing an mRNA-protein linked molecule in which mRNA and protein are chemically bound via puromycin on the ribosome when mRNA is expressed in a cell-free translation system or the like (Non-patent Document 10). Non-patent document 12; Non-patent document 5). Furthermore, the mRNA-protein linked molecule can be obtained by amplifying the mRNA by an in vitro test method and amplifying the mRNA portion of the selected mapping molecule by reverse transcription PCR.

Since various commercially available cell-free translation systems are supplemented with DTT (dithiothreitol), which is a reducing agent, they are not suitable for the expression of proteins having an SS bond typified by antibodies. In particular, since the wheat germ cell-free translation system requires DTT for its expression, there have been no screening and expression examples of antibodies having high binding activity from cDNA libraries of single-chain antibodies. Therefore, establishment of a wheat germ cell-free translation system that can screen for a desired antibody having high binding activity from a cDNA library even in the presence of DTT is desired.
Kohler, G. and Milstein, C. (1975) Nature 256, 495 Smith, GP (1985) Science 228, 1315-1317 Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA 94, 4937-4942 Huston, JS, Margolies, MN, Haber, E. (1996) Adv. Protein Chem., 49, 329 Miyamoto-Sato, E., et al., (2003) Nucleic Acids Res., 31, e78 Madin K, et al. (2000) Proc. Natl. Acad. Sci. USA., 97, 559-564 Shimizu, Y. et al. (2001) Nat. Biotechnol., 19, 751-755 Sachs. AB, et al. (1997) Cell 89, 831-838 Ueda. T., et al. (1991) Nucleic Acids Symp Ser. 25, 151-152 Nemoto, N., et al., (1997) FEBS Lett., 414, 405-408 Japanese Patent Laid-Open No. 10-816636 International Publication No. WO98 / 16636 Pamphlet JP 2002-176987 A International Publication No. WO02 / 48347 Pamphlet Nemoto, N., et al. (1999) FEBS Lett. 462, 43-46 Miyamoto-Sato, E., et al. (2000) Nucleic Acids Res. 28, 1176-1182 Japanese Patent Laid-Open No. 11-322781 Japanese Unexamined Patent Publication No. 2000-139468 International Publication No. WO02 / 46395 Pamphlet Masahiro Kaneshiro (1999) Protein Nucleic Acid Enzyme 44 ： 1431-1438

  An object of the present invention is to provide a method for quickly and efficiently selecting a highly functional protein or a nucleic acid encoding the same. Another object of the present invention is to provide a highly functional protein or a nucleic acid encoding the same, and a method for producing and using the same.

  When the present inventors prepared a library of nucleic acids encoding proteins using the IVV method, it was conventionally considered that the desired product could not be obtained due to inactivation of the protein in protein selection. It has been found that a high pressure can be applied by heat treatment at a high temperature, and as a result, a high-functional protein can be selected quickly and efficiently. It was also found that a highly functional protein can be selected by translating a protein having an S—S bond in a cell-free translation system that requires a reducing agent. Based on the above findings, the present invention has been completed.

Accordingly, the present invention provides the following.
(1) A method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same, comprising the following steps (a) to (d):
(a) A step of preparing a library of DNAs encoding proteins.
(b) Transcribing the library DNA prepared in (a), linking a spacer containing puromycin to the 3 ′ end of the transcribed RNA, and then mapping the genotype to the phenotype in a cell-free translation system Constructing a library of mapping molecules by preparing the molecules.
(c) A step of heat-treating the library of corresponding molecules.
(d) A step in which the mapping molecule is bound to the target molecule, sufficiently washed and then eluted, and the nucleic acid portion of the mapping molecule is amplified by reverse transcription-PCR or PCR.
(2) The selection method according to (1), wherein the target molecule is an antigen and the protein is a single chain antibody.
(3) The selection method according to (1), wherein the spacer contains polyethylene glycol.
(4) The selection method according to (1), further comprising the step of repeating the operations of (a) to (d) using the DNA amplified in (d) as the library of (a).
(5) The selection method of (1), wherein the conditions for the heat treatment are selected from the range of 50 to 100 ° C. and 1 to 30 minutes.
(6) The selection method according to (1), further comprising a step of converting the RNA portion of the corresponding molecule into an RNA-DNA hybrid by reverse transcription before the step of (d).
(7) The selection method according to (6), wherein a substance derived from a cell-free translation system that inhibits a reverse transcription reaction is removed before reverse transcription.
(8) The selection method according to (1), wherein the cell-free translation system is a cell-free translation system containing a thiol compound.
(9) The selection method according to (1), wherein the cell-free translation system is a cell-free translation system of a wheat germ extract, a rabbit reticulocyte extract, or an E. coli S-30 extract.
(10) A method for producing a protein that interacts with a target molecule, the step of selecting a nucleic acid encoding a protein that interacts with the target molecule by the selection method of any one of (1) to (9), and selection The manufacturing method including the process of translating the made nucleic acid and manufacturing protein.
(11) The production method of (10), wherein the target molecule is an antigen and the protein is a single chain antibody.
(12) The production method of (11), wherein the antigen is angiotensin II.
(13) The production method of (11), wherein the antigen is Lewis X.
(14) The method according to (11), wherein the step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound.
(15) The production method of (14), wherein the cell-free translation system is a wheat germ extract, a rabbit reticulocyte extract, or an E. coli S-30 extract.
(16) The method according to (11), wherein the step of producing a single chain antibody comprises transforming a living cell with the selected nucleic acid and expressing the single chain antibody in the living cell.
(17) The step of producing a single-chain antibody comprises producing a single-chain antibody encoded by a selected nucleic acid and a fusion protein of an enzyme or green fluorescent protein (GFP) ( 11) The production method.
(18) A method for labeling the C-terminal of a protein by translating a nucleic acid selected by the selection method of any one of (1) to (9) in a cell-free translation system in the presence of a C-terminal labeling agent .
(19) A single-chain antibody having binding activity for angiotensin II and having an amino acid sequence shown in (A) or (B) below.
(A) SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or 105 The amino acid sequence shown in.
(B) An amino acid sequence having an amino acid sequence having a homology of 90% or more with the amino acid sequence of (A).
(20) A nucleic acid encoding the single chain antibody of (19).
(21) A single chain antibody having a binding activity to Lewis X and having an amino acid sequence shown in (A) or (B) below.
(A) The amino acid sequence shown in SEQ ID NO: 107, 109, 111, 113, 115 or 117.
(B) An amino acid sequence having an amino acid sequence having a homology of 90% or more with the amino acid sequence of (A).
(22) A nucleic acid encoding the single chain antibody of (21).
(23) A method for immunologically detecting a protein using the antibody obtained by the production method of any one of (11) to (17).
(24) The detection method according to (23), which is Western blotting, immunostaining, fluorescent antibody staining, antibody chip method, or immunoprecipitation method.
(25) A molecular interaction comprising contacting a protein obtained by the production method of any one of (11) to (17) with a target molecule and detecting an interaction between the protein and the target molecule. How to detect.
(26) Detection of (25) which is fluorescence correlation spectroscopy, fluorescence imaging analyze, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method, or solid phase enzyme immunoassay method Method.
(27) Substituting the variable region encoded by the nucleic acid with the variable region of human IgG based on the sequence of the nucleic acid selected by the selection method of (2) from a human or other animal-derived DNA library. Or a human-type antibody constructed by
(28) A therapeutic agent comprising the antibody of (27) as an active ingredient.

Construction of single chain antibody cDNA library used in the present invention. IVV library selection cycle by IVV method. Chemical structural formula of angiotensin II-biotin. Lewis X-sp-Biotin chemical structural formula. The translation solution obtained in [15] was confirmed by matching molecules by 5% polyacrylamide electrophoresis in the presence of 8M urea. Above is an electrophoresis gel (electrophoresis photo). The lower bar graph shows the FITC fluorescence of the upper electrophoresis gel measured and quantified with MOLECULAR IMAGER FX (Bio-rad). MH0: library obtained in [11]; MI1: library obtained in [11] was reacted in [18] at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes to select angiotensin II as an antigen [25 MI2: MI1 was reacted in [18] at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes, angiotensin II was selected as an antigen and recovered in [25]; MI3: MI2 [18] ] At 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes, angiotensin II was selected as an antigen and recovered in [25]. The translation solution obtained in [15] was confirmed by matching molecules by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph). MH0: The library obtained in [11]; MK1: The library obtained in [11] was reacted in [18] at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes to select Lewis X as an antigen [25 ] MK2: A library obtained by reacting MK1 in [18] at 50 ° C. for 30 minutes and then 99 ° C. for 5 minutes, selecting Lewis X as an antigen and collecting in [25]. The translation solution obtained in [15] was confirmed by matching molecules by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph). MM1: The library obtained in [11] was reacted in [18] at 50 ° C. for 30 minutes, angiotensin II was selected as an antigen and recovered in [25]; MM2: MM1 in [18] at 50 ° C. Library that was reacted for 30 minutes and angiotensin II was selected as an antigen and collected in [25]; library obtained in MP1: [11] was reacted at 99 ° C for 5 minutes in [15], then [20] [21] The library collected in [25] after performing an experiment after [22] by reacting at 50 ° C for 30 minutes and then 99 ° C for 5 minutes in [18] after selection using angiotensin II as an antigen in [18]. The eluate obtained in [21] was subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis. Above is an electrophoresis gel (electrophoresis photo). The lower bar graph shows the amount of ethidium bromide in the upper electrophoresis gel measured and quantified with MOLECULAR IMAGER FX (Bio-rad). The flow obtained in [20] was diluted 1000 times, the wash obtained in [20] and the eluate (Elute) obtained in [21] were diluted 1 and 10 times in [22]. PCR and 1% agarose gel electrophoresis (electrophoresis photo). The flow obtained in [20] was diluted 1000-fold, the wash obtained in [20] and the eluate obtained in [21] were diluted 1-fold and 10-fold and PCR was performed in [22]. % Agarose gel electrophoresis. Above is an electrophoresis gel (electrophoresis photo). The lower bar graph shows the amount of ethidium bromide in the upper electrophoresis gel measured and quantified with MOLECULAR IMAGER FX (Bio-rad). F stands for Flow, W stands for Wash, E stands for 1-fold eluate, and E0.1 stands for 10-fold dilution of eluate. A phylogenetic tree created by TreeViewPPC after sequence alignment by clustalx based on the amino acid sequence of in-frame ones analyzed by sequence analysis in [31]. A surrounded by a line is converged by angiotensin II selection, and B surrounded by a line is converged by Lewis X selection. The clone number is indicated after the library abbreviation. The enlarged view of A and B enclosed with the line of FIG. A is converged by the selection of Angiotensin II, B is converged by the selection of Lewis X. Western blot result (electrophoresis photograph). The two on the right are Western blots of MI3-55 [37]. The left three are Western blot controls. A bar graph showing the result of analyzing the sequence obtained by selection using angiotensin II as an antigen and analyzing with Biacore in [39] after translating in [34]. A bar graph showing the results of analyzing the sequence obtained by selection using Lewis X as an antigen and analyzing with Biacore in [39] after translating in [34]. MI3-55 was translated in [34], treated at 4 ° C, 60 ° C or 99 ° C for 5 minutes and analyzed with Biacore of [39] in bar graph. MI3-55 was translated by [34], treated at 4 ° C, 60 ° C or 99 ° C for 5 minutes and analyzed by [40] ELISA in a bar graph.

  The method for selecting a protein that interacts with a target molecule or a nucleic acid that encodes the target molecule according to the present invention includes a method in which the nucleic acid part of the mapping molecule in the library encodes a protein that interacts with the target molecule. In addition to heat-treating the rally, it can be carried out according to a nucleic acid selection method using a normal mapping molecule (hereinafter also referred to as “IVV method”).

  “Target molecule” means a molecule that interacts with a protein to be selected, and specifically includes proteins, nucleic acids, sugar chains, low-molecular compounds, and the like. The protein is not particularly limited as long as it has an ability to interact with the protein to be selected, and may be the full length of the protein or a partial peptide including a binding active site. The protein may be a protein having a known amino acid sequence and its function or an unknown protein. These can also be used as target molecules, such as synthesized peptide chains, proteins purified from living organisms, or proteins that have been translated using a suitable translation system from a cDNA library or the like and purified. The synthesized peptide chain may be a glycoprotein having a sugar chain bound thereto. Of these, a purified protein having a known amino acid sequence, or a protein translated and purified from a cDNA library or the like using an appropriate method can be preferably used.

  The nucleic acid is not particularly limited as long as it has the ability to interact with the protein to be selected, and DNA or RNA can also be used. Further, it may be a nucleic acid with a known base sequence or function or an unknown nucleic acid. Preferably, a nucleic acid having the ability to bind to a protein and a base sequence known in the art, or a nucleic acid that has been cleaved and isolated from a genomic library or the like using a restriction enzyme or the like can be used. The sugar chain is not particularly limited as long as it has the ability to interact with the protein to be selected, and its sugar sequence or function may be a known sugar chain or an unknown sugar chain. Preferably, a sugar chain that has already been separated and analyzed and whose sugar sequence or function is known is used. The low molecular compound is not particularly limited as long as it has an ability to interact with the protein to be selected. Those having an unknown function or those having an already known ability to bind to a protein can be used.

  The “interaction” that these target molecules perform with the protein to be selected of the present invention or the “interaction” between target molecules usually means a covalent bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond between the two molecules, In addition, the term indicates an action due to a force acting between molecules resulting from at least one of bonds due to electrostatic force, but this term should be interpreted in the broadest sense and should not be interpreted in a limited way in any sense. The covalent bond includes a coordination bond and a dipole bond. In addition, electrostatic coupling includes electric repulsion in addition to electrostatic coupling. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction.

  Specific examples of interactions include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and partner molecule, binding and dissociation between enzyme and substrate, Binding and dissociation between nucleic acids and proteins that bind to them, binding and dissociation between proteins in the information transmission system, binding and dissociation between glycoproteins and proteins, binding and dissociation between sugar chains and proteins, Or the coupling | bonding and dissociation between a low molecular weight compound and protein are mentioned.

  The IVV method is much more stable than ribosome display or phage display because mRNA is covalently bound to the protein via puromycin or a derivative thereof, which is a protein synthesis inhibitor. Therefore, it is possible to perform panning at a stronger selection pressure than the two technologies of ribosome display and phage display, which allows selection of proteins that interact not only with specificity but also with more stable target molecules. Thus, since a high concentration effect is obtained, a desired protein can be obtained with fewer selection cycles. Furthermore, since it is concentrated to almost single, it is not necessary to analyze many samples. As a result, the operation is simple and the time can be greatly reduced and the cost can be reduced.

In addition, establishment of highly functional antibodies (specificity, affinity, stability, etc.) in the library depends on the size (diversity) of the library. Here, the upper limit of the library that can be selected by the IVV method can be 10 ¹³ or more if conditions are satisfied, and a library larger than any other method can be targeted. In addition, as it is a complete in vitro experimental system, as with ribosome display, no unnecessary bias is applied. Moreover, since the obtained protein can be expressed as a functional protein in a cell-free translation system containing a reducing agent such as DTT, a large number of samples can be prepared, and accordingly, high throughput can be easily achieved.

  The protein thus selected can be used in various fields depending on its function. For example, when the protein is a single-chain antibody, the high-affinity single-chain antibody selected in this way can be used for detecting a protein or detecting an interaction outside or inside the cell. In addition, the high-affinity single-chain antibody selected from the mouse antibody cDNA library is replaced with a variable region or CDR region (complementarity determining region) of the human IgG antibody, so that the chimeric IgG antibody or the humanized mouse IgG You can make antibodies. These antibodies produce little or little human anti-mouse antibody production elicited when administered to humans. Furthermore, a high affinity single chain antibody selected from a human antibody cDNA library can be replaced with the variable region of a human IgG antibody to produce a fully human monoclonal IgG antibody. This can be used as an antibody drug that does not cause anaphylactic symptoms.

The single chain antibody used as the protein to be selected of the present invention may be a normal single chain antibody (also referred to as a single chain Fv fragment (scFv)) in which a V _H chain and a V _L chain are bound via a linker peptide. . The DNA library encoding a single-chain antibody used in the present invention is not particularly limited as long as it is a library containing a large number of DNAs encoding single-chain antibodies, but theoretically any antigen. It is preferable to use a DNA containing a DNA encoding a wide variety of antibodies of 10 ⁹ or more. Such antibody DNA libraries include natural antibody DNA derived from higher vertebrates, preferably mice, humans, chickens, goats, camel spleens and B cells, which are commercially available and used in ordinary in vitro antibody selection systems. Any library can be used. For example, a mouse natural antibody cDNA library (Krebber, A. et al. (1997)) J. Immun. Methods. 201, 35-55: Engberg, J. (1996) Molecular Biotech. 6, 287-310) MRNA is extracted from the spleen of a mouse, gene fragments encoding the variable regions (V _H , V _L ) of the antibody H chain and L chain are converted to cDNA by RT-PCR, and they are amplified by PCR. Furthermore, a mouse single-chain antibody cDNA library is completed by synthesizing a DNA fragment from the obtained fragment so that the C terminus of V _H and the N terminus of _VL are connected by a (Gly ₄ Ser) ₄ linker. Here, any linker sequence may be used as long as it has a relatively high degree of freedom, but it is generally preferable to use the (Gly ₄ Ser) ₄ sequence (SEQ ID NO: 120). The order of connection may be V _L − (Gly ₄ Ser) ₄ −V _H.

In addition, any mutant antibody DNA library can be advantageously used. For example, (1) n-CoDeR: a library (n-CoDeR) in which only CDRs (3 H chains and 3 L chains) are extracted from an antibody cDNA by PCR and incorporated into an antibody frame that is easy to express in E. coli and phage. : Diversity 2 × 10 ⁹ ) (Jirholt, P. et al. (1998) GENE 215, 471-476: Soderlind, E. et al. (2000) Nature Biotechnol 18. 852-856). (2) A library in which random sequences (NNK, NNB) are introduced into the hypervariable region (CDR) of the antibody to increase its diversity (Hayashi, N. et al. (1994) Biotechniqes 17, 310-345) . (3) HuCAL: Human antibody is a combination of 7 types of H chains (subclass: VH1A, VH1B, VH2-6) and 7 types of L chains (subclass: Vκ1-Vκ4, Vλ1-Vλ3) About 95% or more are covered by things (49 types in total). A library in which random sequences are introduced into CDR3 (both H chain and L chain) of these 14 types of antibodies. (Knappik, A. et al. (2000) J. Mol. Biol. 296, 57-86: Hanes, J et al. (2000) Nature Biotech. 18, 1287-1292). (4) A library in which random mutations are introduced into a single antibody gene by combining point mutation and DNA shuffling. (Jermutus, L. (2001) Proc. Natl. Acad. Sci. USA 98, 75-80). These libraries are designed to increase panning speed, antibody stability, expression, functionality, etc., and adaptation to the present invention is more effective.

Hereinafter, each step of the selection method of the present invention will be described.
Step (a) is a step of preparing a library of DNAs encoding proteins that interact with the target molecule. A library of DNA encoding this protein can be prepared according to a usual method.

  In step (b), the DNA of the library prepared in (a) was transcribed, and a spacer containing puromycin was ligated to the 3 ′ end of the transcribed RNA (hereinafter referred to as “translation”). This is a step of constructing a library of mapping molecules by preparing a mapping molecule between a genotype and a phenotype in a cell-free translation system. This step can be performed in the same manner as in the usual IVV method. Although details will be described later, the spacer preferably contains polyethylene glycol.

  Step (c) is a step of heat-treating the library of corresponding molecules. The heat treatment here means exposure to heating conditions that are not burdened by the treatment in the normal IVV method. For example, when the mapping molecule is directly bound to the target molecule after translation, it is exposed to heating conditions that do not occur during translation, and the RNA portion of the mapping molecule is converted to an RNA-DNA hybrid by reverse transcription. When bound to a target molecule, it is exposed to heating conditions that are not between translation and reverse transcription. This temperature is usually selected from the range of 50 to 100 ° C. and 1 to 30 minutes depending on the process from translation to binding to the target molecule. In terms of strong selective pressure, the heat treatment is preferably performed at a temperature of 80 ° C. or higher, more preferably 90 ° C. or higher, whether there is only translation or translation and reverse transcription.

  Prior to the step (d), the RNA portion of the mapping molecule is preferably converted into an RNA-DNA hybrid by reverse transcription. This can prevent nonspecific binding of RNA to the carrier and target molecule. When reverse transcription is performed, it is preferable to remove a substance derived from a cell-free translation system that inhibits the reverse transcription reaction before reverse transcription. Examples of the removal method include fractionation using a gel filtration column, a spin column, a nickel column, and the like.

The order of the heat treatment, reverse transfer and fractionation is not particularly limited, but usually the order of heat treatment, fractionation and reverse transfer, or the order of fractionation, reverse transfer and heat treatment is preferred.
Step (d) is a step in which the mapping molecule is bound to the target molecule, sufficiently washed and then eluted, and the nucleic acid portion of the mapping molecule is amplified by reverse transcription-PCR or PCR. This step can be performed in the same manner as in the usual IVV method. Elution can usually be performed by elution with a solution containing a free target molecule.

  In the selection method of the present invention, it is preferable to repeat the operations (a) to (d) using the DNA amplified in (d) as the library (a). When it is repeated, the DNA amplified in (d) is processed for use as the library in (a). For example, a 5 ′ UTR sequence (amplification primer sequence-SP6 promoter sequence-Ω29 enhancer sequence) is added to the N-terminus for each cycle. However, since the sequences of the N terminus of the antibody are different, it is necessary to introduce a common sequence at the N terminus. In the examples described later, a T7 tag (MASMTGGQQMG (SEQ ID NO: 118)) is used. However, any tag may be used as long as it is generally used. A FLAG tag (DYKDDDDK (SEQ ID NO: 119)) was introduced on the C-terminal side. Generally, when constructing a library, introduction of some unfavorable mutations (STOP codon, insertion, deletion) by PCR amplification, chemical synthesis, etc. is inevitable. Therefore, it is possible to select only in-frame tags by the C-terminal tag. This tag may also be anything that is commonly used.

An example of an IVV library used in the selection method of the present invention will be described with reference to FIG. The protein is a single chain antibody. Extract the variable part of each H chain and L chain of mouse spleen-derived antibody (IgG) mRNA by RT-PCR, and connect the C terminal of the H chain and the N terminal of the L chain with a (Gly ₄ Ser) ₄ linker. A chain antibody cDNA library is used. These are converted to mRNA by in vitro transcription, and a PEG spacer having puromycin is bound to the 3 ′ end of the mRNA. When these are translated by a cell-free translation system, an IVV library in which mRNA and an antibody corresponding to the sequence are covalently bonded via puromycin is constructed. This library has a diversity of more than 10 ¹³ and is the largest library of known antibody selection methods.

  An outline of the steps of the selection method of the present invention when a single-chain antibody DNA library is used is shown in FIG. By repeating the selection cycle, that is, selection, amplification, library reconstruction, and translation, the desired antibody is concentrated. Here, by applying a certain selection pressure at the time of selection, the properties and concentration efficiency of the obtained antibody are determined. That is, a selection pressure at the protein level for removing unstable antibodies and a selection pressure for removing non-specifically bound ones. In the present invention, it has been found that by adopting the IVV method, the target antibody can be selected even when a high selection pressure is applied such that the target antibody cannot be obtained by the conventional method.

  Hereinafter, examples of the translation template, C-terminal modified protein of the present invention, C-terminal labeling method and mapping method using the translation template will be described, but the following examples give specific recognition of the present invention. The scope of the present invention is not limited in any way by the following examples.

  The coding part of the protein to be selected consists of a 5 ′ end region, an ORF region, and a 3 ′ end region, and may or may not have a Cap structure at the 5 ′ end. The 3 ′ end region of the coding part is a poly Ax8 sequence as an A sequence, an XhoI sequence as an X sequence, a sequence having (C or G) NN (C or G) with 4 or more bases, and an A sequence. There is an XA sequence as a combination of X sequences. A configuration comprising an A sequence, an X sequence, or a Flag-tag sequence as an affinity tag sequence upstream of the XA sequence is conceivable. Here, the affinity tag sequence may be any sequence that uses any means capable of detecting or purifying proteins, such as those using antigen-antibody reactions such as HA-tag and IgG protein A (z domain), and His-tag. It doesn't matter. Here, as the range that affects the translation efficiency, the combination of XA sequences is important, the first four bases are important in the X sequence, and the sequence of (C or G) NN (C or G) is What it has is preferable. The 5 'terminal region consists of a transcription promoter and a translation enhancer. T7 / T3 or SP6 can be used as the transcription promoter, and there is no particular limitation. However, in wheat cell-free translation systems, omega sequences are used as translation enhancer sequences. Or a sequence containing a part of the omega sequence is preferably used, and SP6 is preferably used as the promoter. A part of the omega sequence (O29) of the translation enhancer includes a part of the TMV omega sequence (Gallie D.R., Walbot V. (1992) Nucleic Acids Res., 20, 4631-4638).

  The polyethylene glycol (PEG) part (spacer) consists of a CCA region, a PEG region, and a donor region. The minimum required configuration is a donor region. From the viewpoint of translation efficiency, those having not only the donor region but also the PEG moiety are preferred, and further, puromycin having no ability to bind to amino acids is preferred. The molecular weight range of polyethylene glycol in the PEG region is 400 to 30,000, preferably 1,000 to 10,000, more preferably 2,000 to 6,000. In addition, the CCA region can be configured with or without puromycin.

  When used in the construction of the mapping molecule, a structure containing puromycin is used in a cell-free translation system so that a spacer can be bound to the C-terminus of the protein translated therein. Including puromycin includes including derivatives thereof. The following are mentioned as an example of a derivative | guide_body. 3'-N-aminoacylpuromycin aminonucleoside (3'-N-Aminoacylpuromycin aminonucleoside, PANS-amino acid), for example, PANS-Gly with glycine amino acid, PANS-Val with valine, PANS-Ala with alanine, and all other amino acids PANS-all amino acids corresponding to can be used. In addition, 3'-Aminoacyladenosine aminonucleoside (AANS-amino acid, 3'-Aminoacyladenosine aminonucleoside, connected by an amide bond formed as a result of dehydration condensation between the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond For example, AANS-Gly having an amino acid part of glycine, AANS-Val of valine, AANS-Ala of alanine, and other AANS-all amino acids corresponding to all amino acids can be used. Nucleosides or nucleosides and amino acid ester-bonded ones can also be used. In addition, nucleoside or a substance having a chemical structure skeleton similar to nucleoside and an amino acid or a substance having a chemical structure skeleton similar to amino acid can be used as long as they can be chemically bonded. It is preferable to have a base sequence consisting of DNA and / or RNA of one residue or more on the 5 ′ side of the CCA region (CCA). The type of base is preferably C> (U or T)> G> A in this order. The sequence is dC-Puromycin, rC-Puromycin, etc., more preferably dCdC-Puromycin, rCrC-Puromycin, rCdC-Puromycin, dCrC-Puromycin, etc., which is a CCA sequence (Philipps GR (1969) Nature 223, 374-377) is suitable.

  In a translation template used only for translation, such as in the case of C-terminal labeling, a configuration that does not include puromycin or a configuration that includes puromycin that does not have the ability to bind to amino acids is preferable. Any substance lacking the ability of the amino acid group of the puromycin derivative to bind to an amino acid, and a CCA region lacking puromycin are conceivable, but more translation is possible by including puromycin that cannot bind to proteins on the ribosome. Increases efficiency. The reason is not clear, but puromycin that cannot bind to the protein may stimulate turnover by stimulating the ribosome. For puromycin that cannot be bound, the above puromycin is bound to an amino acid by an appropriate method.

The PEG portion can be configured to have a modifying substance. As a result, the translation template can be used as a tag for collection, reuse by purification, or immobilization. It is possible to introduce a fluorescent substance, biotin, or various separation tags such as His-tag as a modifying substance into at least one residue of DNA and / or RNA base. In addition, the 5 ′ end region of the coding part is SP6 + O29, and the 3 ′ end region is, for example, Flag + XhoI + A _n (n = 8), so that each length is about 5 ′ end region. The length is 60 bp, about 40 bp at the 3 ′ end region, and can be designed as an adapter region for PCR primers. This created a new effect. In other words, it is possible to easily create a code part with 5 'and 3' end regions by PCR from any vector, plasmid, or cDNA library, and ligate the PEG part to this code part instead of 3 'UTR. By doing so, a translation template with high translation efficiency can be obtained.

  The method for ligating the PEG part and the code part is not particularly limited, and any method may be used such as a method using a general DNA ligase or a ligation by a photoreaction. In the ligation using RNA ligase, the A sequence in the 3 ′ end region is important as a range that affects the ligation efficiency in the coding region, and a mixture of dA and / or rA of at least 2 residues or a single polyA It is a continuous chain, preferably a poly A continuous chain of 3 residues or more, more preferably 6 to 8 residues or more. The DNA and / or RNA sequence at the 5 ′ end of the donor region of the PEG part affects the ligation efficiency. In order to ligate the coding part and the PEG part with RNA ligase, it is necessary to contain at least one residue. For an acceptor having a poly A sequence, at least one residue of dC (deoxycytidyl) is required. Acid) or 2-residue dCdC (dideoxycytidylic acid) is preferred. The type of base is preferably C> (U or T)> G> A in this order. Furthermore, it is preferable to add polyethylene glycol having the same molecular weight as that of the PEG region during the ligation reaction.

  Next, C-terminal modification (labeling) of the protein to be selected will be described. A protein to be selected, which is synthesized by translation using a translation template of a protein to be selected in the presence of the modifier and is C-terminally modified with the modifier, and includes a translation template and a modifier. The feature here is in particular the configuration of the code part of the translation template. Details are described below.

The PEG part of the translation template is characterized in that puromycin cannot be linked to an amino acid. As a configuration suitable for C-terminal labeling, it is important that the 3 ′ end region is an XA sequence, and the first 4 bases are important in the X sequence (C or G ) Those having the sequence of NN (C or G) are preferred. Again, the 5 ′ end region of the coding part is SP6 + O29 and the 3 ′ end region is, for example, Flag + XhoI + A _n (n = 8), so that each length is the 5 ′ end region. The length is about 60 bp, and the length at the 3 ′ end region is about 40 bp, and can be designed as an adapter region for a PCR primer. This makes it possible to easily create a code part with a 5 'terminal region and a 3' terminal region by PCR. By ligating this code part with a PEG part instead of 3 'UTR, it is suitable for C-terminal labeling. A translation template with high translation efficiency can be obtained.

  The modifier has a peptide transfer reaction in a protein translation system, that is, an acceptor part having a group (including a residue) that can bind to a protein by a peptide transfer reaction on a ribosome, and a modification part via a nucleotide linker. It has a combined configuration. Protein synthesis is performed in the presence of this modifying agent, the resulting C-terminal modified protein is purified, and the detection of protein and interaction can be detected by using a detection system for intermolecular interaction. The modifying part contains a modifying substance as in the PEG part. Specific examples of the non-radioactive modifying substance as the modifying substance include fluorescent and non-fluorescent modifying substances. Examples of fluorescent substances include fluorescent dyes such as fluorescein series, rhodamine series, Cy3, Cy5, eosin series, NBD series, and fluorescent proteins such as green fluorescent protein (GFP). The non-fluorescent substance may be any compound that can serve as a mark, such as a coenzyme such as biotin, a protein, a peptide, a saccharide, lipids, a dye, or polyethylene glycol. In the modifying agent, the modifying part preferably has a fluorescent group, a group that binds to a protein, or both. The acceptor part is a protein translation system and has a group capable of binding to a protein by a peptide transfer reaction, and preferably has a residue of puromycin or a derivative thereof. Puromycin has a structure similar to aminoacyl-tRNA and is known as an antibiotic that inhibits protein synthesis, but is known to bind to the C-terminus of proteins at low concentrations (Miyamoto-Sato, E. et al. al. (2000) Nucleic Acids Res. 28: 1176-1182). The puromycin derivative that can be used may be any substance that has a structure similar to puromycin and can bind to the C-terminus of the protein. Specific examples include 3′-N-aminoacylpuromycin aminonucleoside, 3′-N-aminoacyl adenosine aminonucleoside and the like. Specifically, the nucleotide linker that connects between the modifying portion and the acceptor portion is a nucleic acid or nucleic acid derivative in which one or a plurality of ribonucleotides or deoxyribonucleotides are linked, and a particularly preferred example is a ribonucleotide containing a cytosine base. Examples thereof include compounds in which one or more nucleotides (-rC-) or deoxyribonucleotides (-dC-) are linked. In addition, any substance may be used as long as it can increase the yield of the modified protein by being inserted between the modified part and the acceptor part. In the modifying agent of the present invention, the nucleotide linker is 2′-deoxycytidylic acid, 2′-deoxycytidyl- (3 ′, 5 ′)-2′-deoxytidylic acid, ribocytidylic acid, or ribocytidyl- (3 ′, 5 ′)-ribocytidylic acid is preferred.

  The modifying agent can be produced by coupling the modifying part and the acceptor part through a desired nucleotide linker by a chemical bonding method known per se. Specifically, for example, the acceptor moiety protected with an appropriate protecting group is bound to a solid phase carrier, and a nucleotide phosphoramidite, deoxynucleotide phosphoramidite, and functional modifier as a nucleotide linker using a nucleic acid synthesizer As described above, the phosphoramidite bound with a fluorescent substance, biotin, or the like is sequentially bound, followed by deprotection. Depending on the type of each part or the type of bonding, they can be combined by a liquid phase synthesis method, or both can be used in combination. When a metal ion such as nickel is used as the functional modifier, a chelating reagent such as nitrilotriacetic acid or iminodiacetic acid that can coordinate with the metal ion can be bound, and then the metal ion can be coordinated. .

The PEG portion of the translation template is the same as described above except that puromycin can be linked to an amino acid. In addition, the code part is the same as described above. In particular, as a configuration suitable for association, it is important that the 3 ′ end region is an A sequence, and the efficiency of total protein association is remarkably improved. It was confirmed that the amount of free protein was drastically reduced. Again, the 5 ′ end region of the coding part is SP6 + O29 and the 3 ′ end region is, for example, Flag + XhoI + A _n (n = 8), so that each length is the 5 ′ end region. The length is about 60 bp, and the length at the 3 ′ end region is about 40 bp, and can be designed as an adapter region for a PCR primer. This makes it possible to easily create a protein coding part with 5 'and 3' end regions by PCR from any vector, plasmid, or cDNA library. A high translation template is obtained. Proteins modified with C-terminal by PEG can be detected with RNase A when not using the coding part for protein detection or interaction detection, for example, FCCS measurement, fluorescent reader, protein chip, etc. It is preferable to intentionally cut. By cleaving, it is possible to eliminate the difficulty in detecting protein-protein interactions due to interference with the coding part.

  A cell-free translation system is used to form IVV from a protein cDNA library. Specifically, a wheat germ extract, rabbit reticulocyte extract, or E. coli S-30 extract containing or not containing a thiol compound such as dithiothreitol (DTT) or β-mercaptoethanol is used. In particular, in cell-free translation systems containing DTT, it has been difficult to express an active antibody as a protein. In the present invention, an active antibody is selected by using IVV even in the presence of DTT. It turns out that it is possible. In these cell-free protein synthesis systems, the above-mentioned protein translation template is added and incubated at 25 to 37 ° C. for 1 to several hours to form IVV. In the case of C-terminal labeling, a C-terminal modified protein is synthesized by simultaneously adding 1 to 100 μM modifier. The synthesized IVV is directly used for the next heat treatment step or reverse transfer step.

  When the mapping molecule (also referred to as “IVV”) is directly heated after translation, it is usually treated at 50-100 ° C. under conditions selected from the range of 1-30 minutes. In the case where the mRNA part of IVV is converted into an RNA-DNA hybrid by reverse transcriptase in advance, it is preferable to remove it because the cell-free translation system contains a substance that inhibits the reverse transcription reaction. For the removal operation, gel filtration, preferably Sephadex G200 (manufactured by Amersham Bioscience), or spin column, preferably Ultrafree MC, 100,000 NMWL (manufactured by Millipore), or PURESYSTEM (post-genomic laboratory of E. coli) In the case of a company), nickel resin is used.

  By making the mRNA part of IVV into an RNA-DNA hybrid by reverse transcription (RT), nonspecific adsorption of the mRNA part to the carrier and antigen can be prevented. In the reverse transcription reaction, mRNA is denatured by heating at 65 ° C., cooled to 4 ° C., annealed, ReverTra Ace (manufactured by TOYOBO) is added, and reacted at 50 ° C. for 30 minutes. The reverse transcriptase may be any enzyme and any condition as long as it performs a reverse transcription reaction. It is not limited to said conditions. The IVV heat treatment conditions in which the mRNA part is RNA-DNA hybrid are also usually selected from the range of 1-30 minutes at 50-100 ° C, but higher than the maximum temperature of reverse transcription.

  The DNA polymerase used in RT-PCR and PCR is not particularly limited as long as it is used in PCR reactions, but more highly efficient and PCR fidelity is not too high. preferable. The KOD Dash (TOYOBO product) used in the present invention can be efficiently amplified even from an extremely small amount of template DNA, and can be evolved to a higher function since a suitable mutation is introduced.

  Target molecules are peptides (which may be chemically synthesized or isolated from nature, or partial digests of proteins), proteins, nucleic acids (DNA or RNA), saccharides, various low molecular compounds, metals All compounds and substances such as metal compounds are applicable.

  The target molecule used in the present invention is bound to a solid phase such as a resin or a bead depending on the embodiment. As a method for binding to the solid phase, the target molecule is bound via a modifying substance and bound by other portions. To be made.

  The modifier used for binding via a modifier is usually a molecule that specifically binds to a specific polypeptide (hereinafter sometimes referred to as a “ligand”) and is attached to the solid surface. Binds a specific polypeptide that binds to the ligand (hereinafter sometimes referred to as "adapter protein"). Adapter proteins include binding proteins, receptor proteins that constitute receptors, antibodies, and the like. Examples of adapter protein / ligand combinations include biotin-binding proteins / biotin such as avidin and streptavidin, maltose-binding protein / maltose, G protein / guanine nucleotide, polyhistidine peptide / nickel or metal ions such as cobalt, glutathione-S -Transferase / glutathione, DNA binding protein / DNA, antibody / antigen molecule (epitope), calmodulin / calmodulin binding peptide, ATP binding protein / ATP, or various receptor proteins such as estradiol receptor protein / estradiol / ligands thereof It is done.

  Among these, adapter protein / ligand combinations include biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, polyhistidine peptide / metal ions such as nickel or cobalt, glutathione-S-transferase / glutathione, The combination of streptavidin / biotin is most preferable. These binding proteins are known per se, and the DNA encoding the protein has already been cloned.

  The adapter protein can be bound to the solid phase surface by a method known per se. Specifically, for example, tannic acid, formalin, glutaraldehyde, pyruvaldehyde, bis-diazotized benzidone, toluene- A method utilizing a 2,4-diisocyanate, an amino group, a carboxyl group that can be converted into an active ester, or a hydroxyl group or amino group that can be converted into a phosphoramidide can be used.

  When binding to the solid phase by a moiety other than the modifying substance, known methods commonly used for binding proteins, nucleic acids, sugar chains, and low molecular weight compounds to the solid phase, specifically, for example, tannic acid, formalin, Use a method that uses glutaraldehyde, pyruvic aldehyde, bis-diazotized benzidone, toluene-2,4-diisocyanate, amino group, carboxyl group that can be converted to active ester, hydroxyl group or amino group that can be converted to phosphoramidide, etc. be able to.

  The solid support is preferably agarose beads or agarose beads in which a magnetic material is embedded. The distance between the solid phase surface and the antigen molecule is preferably 30 angstroms or more from a three-dimensional viewpoint.

In general, there are two methods for obtaining a highly functional protein. Hereinafter, a high affinity antibody will be described as an example. One is a method in which only the antibody at that concentration is selected by setting the antigen concentration used in the selection experiment low. This method is naturally inefficient because the amount of antibody to be recovered is small, and accordingly, the antigen concentration that can be set is limited to a recoverable concentration. The second is off-rate selection (Hawkins, R. (1992) J. Mol. Biol. 226, 889-896; Boder, E. (1997) Nat. Biotechnol. 15, 553-557; Jermutus, L. (2001 ) Proc. Natl. Acad. Sci. USA 98, 75-80), which uses the dissociation rate of antigen and antibody. The dissociation constant (Kd) is expressed as the ratio of the association rate constant (on-rate) to the dissociation rate constant (off-rate), but the biopolymer association constant is within a certain range (10 ⁴ -10 ⁶ M ⁻¹ S ⁻¹ ), the real Kd is determined by the dissociation rate constant. Therefore, an antibody with a slower dissociation rate has a higher affinity. Therefore, the longer the elution time with an antigen, the more the antibody with higher affinity can be concentrated. However, this method has a problem in that it takes a long time for a one-cycle selection experiment. As a method for solving this problem, there is a method in which the antigen or antibody is eluted by digesting it with a protease in a sequence-specific manner instead of eluting with an antigen. This protease is not limited by any protease, including those with extremely high substrate specificity such as TMV protease and factor Xa, and those with low sequence specificity such as trypsin and proteinase K. The above methods can also be applied in the present invention, and are extremely effective methods by combining them.

  A method for producing a protein using a nucleic acid selected by the selection method of the present invention, that is, a step of selecting a nucleic acid encoding a protein that interacts with a target molecule by the selection method of the present invention, and the selected nucleic acid Also provided is a production method comprising the step of producing a protein by translation.

The step of selecting a nucleic acid is as described for the selection method of the present invention.
Production of a protein using a nucleic acid can be performed according to a usual method. For example, the nucleic acid may be translated in a cell-free translation system, or a living cell such as E. coli may be transformed with the nucleic acid by using a plasmid into which the nucleic acid has been introduced, and the protein expressed in the living cell. Good. As the cell-free translation system, a cell-free translation system containing a thiol compound such as dithiothreitol or β-mercaptoethanol, preferably wheat germ extract, rabbit reticulocyte extract, or E. coli S-30 extract Can be mentioned.

  When a protein is expressed, it may be expressed as a fusion protein of a protein encoded by a selected nucleic acid and an enzyme or green fluorescent protein (GFP).

  Examples of proteins obtained by the production method of the present invention include single chain antibodies. As examples of single chain antibodies, when the antigen is angiotensin II, MI1-16 (61), MI1-4 (63), MI2-10 (65), MI2-34 (67) shown in Examples below , MI2-41 (69), MI2-48 (71), MI3-15 (73), MI3-26 (75), MI3-28 (77), MI3-34 (79), MI3-41 (81), MI3-42 (83), MI3-47 (85), MI3-5 (87), MI3-55 (89), MI3-62 (91), MI3-64 (93), MI3-66 (95), MI3 -74 (97), MK2-3 (99), MM2-11 (101), MP1-36 (103), MP1-41 (105) (the numbers in parentheses indicate the amino acid sequence number) . In addition, when the antigen is Lewis X, MI3-8 (107), MK1-15 (109), MK1-17 (111), MK1-24 (113), MK2-8 (115) described in Examples below are used. ), MK2-19 (117) (the number in parentheses indicates the amino acid sequence SEQ ID NO). Amino acid sequences highly homologous to the amino acid sequences of these antibodies (for example, 90% or more (maximum matching of homology calculation method: GENETYX-MAC Version 7.3 (SOFTWARE DEVELOPMENT CO., LTD))) or amino acids of these antibodies Those having amino acids having substitutions, deletions or insertions of one or several (usually not more than 30) residues in the sequence are expected to have many similar binding activities. Accordingly, the present invention also provides the following antibodies and nucleic acids encoding them.

A single-chain antibody having binding activity for angiotensin II and having an amino acid sequence shown in (A) or (B) below.
(A) SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or 105 The amino acid sequence shown in.
(B) An amino acid sequence having an amino acid sequence having a homology of 90% or more with the amino acid sequence of (A).

A single-chain antibody having binding activity to Lewis X, the single-chain antibody having an amino acid sequence shown in (A) or (B) below.
(A) The amino acid sequence shown in SEQ ID NO: 107, 109, 111, 113, 115 or 117.
(B) An amino acid sequence having an amino acid sequence having a homology of 90% or more with the amino acid sequence of (A).

  Also provided is a method of labeling the C-terminus of a protein by translating the nucleic acid selected by the selection method of the present invention in a cell-free translation system in the presence of a C-terminus labeling agent. Labeling can be performed as described in JP-A-11-322781, JP-A-2000-139468, and WO 02/46395. Cell-free translation systems include wheat germ extract, rabbit reticulocyte extract, and E. coli S-30 extract.

  Specific examples of expressing a large amount of protein using living cells include bacteria such as Escherichia coli, Bacillus subtilis, thermophile and yeast, insect cells, cultured cells such as mammals, nematodes, Drosophila, zebrafish, mice Any cell can be used. In these cells, both the C-terminal labeled or matched modified proteins can be directly introduced, or the translation template is introduced and C-terminal labeled at the same time, 1-100 μM. The modifying protein is introduced into cells by electroporation, microinjection or the like, and the modified protein is synthesized by incubating at the optimal growth temperature of the cells for several hours. In the case of association, the above-mentioned translation template is introduced, and the association molecule is synthesized by incubating at the optimal growth temperature of the cells for several hours. Both modified proteins synthesized can be recovered by disrupting the cells and subjected to subsequent purification or detection processes. Moreover, it is also possible to use for a detection process in a cell as it is. As the translation template, an appropriate one is selected according to the translation system to be used.

  In order to express a large amount of protein in E. coli, it is necessary to introduce the target cDNA into an expression vector. At this time, the most important thing contained in the expression vector is a variety of promoters for transcription of cDNA into RNA. Since the protein to be expressed may be toxic to E. coli as a host, most vectors are designed to control expression in some form. This control is important in order not to prevent the growth of E. coli during the period from the transformation of E. coli with a vector incorporating the gene encoding the expressed protein until the induction of expression.

  A ribosome binding site and a base sequence called Shine-Dalgarno sequence are included immediately downstream of the promoter and immediately upstream of the cloning site. In general, those associated with the downstream region of the promoter used are often used as they are. This part is transcribed into mRNA and then binds to ribosomal RNA together with the initiation codon AUG. It is said that translation efficiency is best when the distance between the Shine-Dalgarno sequence and the start codon is 5 to 10 bases. At present, since it is almost always expressed as a fusion protein, the promoter optimized for the commercially available vector and the start codon of the fusion protein existing immediately downstream thereof are used as they are.

  From the viewpoint of efficient translation, the higher order structure of mRNA near the translation start point is important. For this reason, it may be necessary to convert the nucleotide sequence encoding the vicinity of the N-terminus of the target gene into an adenine- or thymine-rich one as much as possible without amino acid substitution. Furthermore, if high-efficiency expression is expected, it is also important to replace the codon that is optimal for E. coli, considering the frequency of amino acid codon usage in E. coli.

  In addition, although the expression system in E. coli is usually carried out in the cytoplasm, the cytoplasm is in a reducing environment, and therefore a protein having an S—S bond such as an antibody becomes an inclusion body. Thus, in the case of antibodies, E. coli expression is generally performed in the periplasm under an oxidative environment. In this case, it is achieved by introducing a signal sequence (for example, pel B leader or ompT) at the N-terminus of the antibody. However, the expression level in the periplasm is extremely low compared to the cytoplasm. Here, since the antibody obtained in the present invention is selected in a reducing environment where DTT is present, it should be expressed as a water-soluble functional antibody even when expressed in the cytoplasm of E. coli. Can do.

  The single chain antibody obtained by the production method of the present invention can be used for immunological detection of proteins. Examples of immunological detection methods include Western blotting, immunostaining, fluorescent antibody staining, antibody chip method, and immunoprecipitation.

  In addition, the protein interaction can be detected by bringing the single-chain antibody obtained by the production method of the present invention into contact with a protein and detecting the interaction between the single-chain antibody and the protein. Methods for detecting protein interactions include fluorescence correlation spectroscopy, fluorescence imaging analyze, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, and solid-phase enzyme immunoassay. It is done.

  In order to detect peptides and proteins using single-chain antibodies, single-chain antibodies and peptides and proteins can be modified with some modifying agent, such as fluorescent dyes, or fusion proteins with enzymes and fluorescent proteins (GFP). Or need to be detected. The method for modifying single-chain antibodies, peptides, and proteins with fluorescent dyes has been described above. The fusion protein is constructed by fusing GFP or an enzyme, preferably alkaline phosphatase or horseradish peroxidase, to the C-terminus or N-terminus of a single chain antibody. Such a single-chain antibody fusion protein is obtained by expression and purification in a cell-free translation system or E. coli, baculovirus, or animal cell system. In the case of GFP, fluorescence is detected; in the case of alkaline phosphatase, visible light is detected; in the case of horseradish peroxidase, chemiluminescence is detected by a luminometer. For protein detection, a secondary antibody with a single-chain antibody tag (Flag-tag, T7-tag, HA-tag, etc.) and a fusion protein of GFP or alkaline phosphatase or horseradish peroxidase are used. Also good. Below, the detection method of the peptide and protein using a single chain antibody is described.

(1) Immunostaining with antibodies One of the means for estimating the function of a molecule to be studied is to examine its localization. Various methods have been developed to investigate the expression of the target molecule, and all of them require a lot of time and labor to prepare experimental materials and establish experimental systems. Compared with these methods, tissue staining using an antibody is relatively easy to operate, and only an antibody that recognizes the target molecule is prepared. When you start analyzing a molecule, antibodies that recognize that molecule are often available in some way, so even the first person can start an experiment right away. Another advantage of this method is that the presence of the protein can be confirmed directly rather than in the form of gene expression. However, not all antibodies can be used for tissue staining. Good results cannot be expected with antibodies that cannot recognize the structure that the molecule takes in vivo. Whether or not an antibody suitable for tissue staining can be prepared is a turning point.

  The basic principle is the same as Western blotting, but in the case of tissue staining, the protein is chemically fixed in the tissue with formaldehyde or the like to detect its localization. A fixed target protein is detected using a primary antibody and a secondary antibody. When the signal is weak and difficult to detect, a method for specifically amplifying only the target signal has been developed. .

(2) Observation of intracellular localization of endogenous proteins by fluorescent antibody staining Localization of endogenous proteins in cells using an antibody that specifically recognizes the molecule when analyzing proteins with unknown functions Observing is often very useful information. Unlike when expressed as a GFP fusion protein, it is not possible to observe the movement of the molecule as it is in a living cell, but localization of the target protein in various states such as response to stimulation and drug treatment and cell movement. Can be observed. Even if there is no antibody against the target protein, the target molecule can be localized in the same manner using the antibody against the tag by using a method such as transient expression in the cell with the tag attached. Can be observed. However, the localization of the tagged protein observed in this case is not always coincident with the actual localization of the protein, and is often the result of overexpression. Although the operation of the experiment is relatively easy, depending on the location of the antibody and target protein, it may be necessary to examine the conditions of the fixation method, and above all, it is possible to prepare a specific antibody for the protein of interest. It is an important point.

  After fixing the cells by an appropriate fixation method, permealize the membrane with a surfactant or the like, and treat with a specific antibody (primary antibody) against the target molecule. Thus, a target protein-antibody complex is formed in the cell. Many non-specifically bound antibodies remain in the cells, which are removed by washing. Next, the target protein is detected using an antibody (secondary antibody) that recognizes the primary antibody and is labeled with a fluorescent substance. -A primary antibody-secondary antibody complex is formed. As with the primary antibody, the non-specifically bound secondary antibody is washed away, encapsulated, and observed with a microscope.

(3) Western blotting Western blotting refers to a work of electrically transferring a protein after electrophoresis to a membrane or a series of experiments including such work.

  A commonly used method in protein electrophoresis is SDS-PAGE. When the sample is subjected to heat treatment by adding SDS as a reducing agent and 2-mercaptoethanol having a negative charge, the higher order structure of the protein is almost completely destroyed. In addition, since SDS binds to the protein at a protein molecular weight ratio (1: 1.4), a uniform charge state is obtained. When this is run in a polyacrylamide gel, it can be separated according to the molecular weight of the protein.

  After SDS-PAGE, the protein developed in the gel is electrophoretically transferred to the membrane and reacted with an antibody specific for the target protein in the sample on the membrane to detect the target protein immunologically. .

  The Western blot can be transferred onto the membrane with little diffusion of the protein bands developed in the gel. It is also characterized by high specificity of antigen-antibody reaction and high sensitivity. Therefore, if an antibody against the target protein is available, it can be said that it is an easy and good method that can detect a specific protein with high sensitivity.

  Here, the method of Western blot using SDS-PAGE and enzyme activity is described, but it is not particularly limited to this protocol. The flow of Western blot operation is shown below. (1) After denaturing a protein sample with a sample buffer containing SDS or the like, the protein is separated according to molecular weight by SDS-PAGE. (2) After electrophoresis, the gel is overlapped with the membrane and set in a transfer device, and the protein band developed in the gel is electrically transferred (blotted) onto the membrane. (3) After blocking to prevent non-specific adsorption to the protein, a primary antibody reaction is carried out with an antibody that specifically recognizes the target protein. (4) The secondary antibody is reacted with a secondary antibody (with chromogenic enzyme) that specifically recognizes the primary antibody molecule. (5) The target protein is detected by color reaction using the enzyme activity of the chromogenic enzyme attached to the secondary antibody, for example, peroxidase activity or alkaline phosphatase activity.

(4) Immunoprecipitation method Immunoprecipitation method (immunoprecipitation, IP) is a specific antigen-antibody reaction and the C-terminal part of a single chain antibody is modified with biotin, or various tags such as Flag -tag, T7-tag, HA-tag, His-tag, etc. (This is easily separated from the solution by centrifugation because it binds to streptavidin or tag antibody covalently bound to agarose beads) In this method, only the antigenic protein corresponding to the antibody used is separated and purified. At present, however, not only the antigen protein alone is separated and purified, but rather the strength of “washing” and the surfactant of the lysate used are moderately weakened to form a complex together with the antigen protein that directly binds to the antibody. It is most often used to detect the protein being made and to establish protein-protein interactions in vivo. Therefore, the significance of detection is greater than that of pull-down assays that can only demonstrate in vitro binding. However, in performing immunoprecipitation, an antibody having high specificity and affinity is required. In this respect, the high-affinity antibody selected in the present invention is suitable for the purpose. Generally used antibodies require higher titers (10 ⁷ to 10 ⁹ mol ⁻¹ ) than Western blots, and a certain degree of specificity is required.

  Further, as described above, the aspect of the protein complex that can be separated varies depending on the buffer used for washing, the number of washings, the composition of the solubilizing buffer, and the like. The protocol is shown below, but is not particularly limited. (1) Lysate recovery: As a preparatory stage for immunoprecipitation, it is first necessary to obtain a protein solution from cells or tissues. The result of the subsequent immunoprecipitation varies depending on the concentration and composition of the solution. Lysate contains a variety of proteins. (2) Antigen-antibody reaction: An antigen-antibody reaction is caused by adding a single-chain antibody that recognizes the target protein to the lysate. (3) Binding between beads and antibody: Since the antibody in the solution cannot be separated as it is, agarose beads to which streptavidin or a tag antibody is covalently bonded are added so as to make it possible. Streptavidin or tag antibody specifically recognizes and binds to C-terminal modified biotin or tag of single-chain antibody, so that this operation results in single-chain antibody bound to beads and can be separated. become. (4) Separation and purification: The beads are dropped by centrifugation and separated by removing the supernatant. Further, wash buffer is added and mixed to remove some non-specifically bound protein. (5) Analysis of the purified protein complex: The protein complex contained in the protein complex is eluted from the beads and analyzed by SDS-PAGE. As a detection method, Western blotting (when it has an antibody in a known case), CBB staining, or the like is used. If an unknown band can be detected by CBB staining, it can be fixed by MS.

  In the method for analyzing the interaction between molecules using a protein that interacts with the target molecule obtained by the present invention (hereinafter also referred to as “functional protein”), the modified functional protein obtained above is usually The target molecule is brought into contact with the appropriate combination depending on the type of modifier and the type of reaction system, and the change in the signal generated based on the interaction between the two molecules in the signal generated by the modified functional protein or target molecule is measured. To analyze the interaction. In addition, the analysis of the interaction can be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analyze method, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, surface plasmon resonance method, or solid phase enzyme immunoassay method Is done. In addition, the two sets of modified functional proteins obtained above and the target molecule are brought into contact with each other in an appropriate combination depending on the type of the modifying substance, the type of reaction system, etc. The interaction is analyzed by measuring the change in the signal generated based on the action. The analysis of the interaction is performed by, for example, fluorescence correlation spectroscopy, fluorescence imaging analyze, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or solid phase enzyme immunoassay. Is called. Details of these methods are described below. The target molecule and the interaction are as described above.

  The functional protein to be used can be used after being modified with a modifying substance depending on the embodiment. The modifying substance is usually selected from non-radioactive modifying substances such as fluorescent substances. Fluorescent substances include various fluorescent dyes that have free functional groups (for example, carboxyl group, hydroxyl group, amino group, etc.) and can be linked to the above single-chain antibodies such as proteins and nucleic acids, such as fluorescein series, rhodamine series, Cy3 , Cy5, eosin series, NBD series, etc. In addition, as long as it is a modifiable compound such as a dye, the type and size of the compound are not limited.

As these modifiers, those suitable for measuring or analyzing a change in signal generated based on the interaction between the target molecule and the modified functional protein are appropriately used.
Binding of the modifying substance to the functional protein can be performed using an appropriate method known per se. Specifically, for example, the above-described method for modifying the C-terminus can be used. In addition, the modified functional protein or the target molecule used in the present invention may be bound to a solid phase depending on the embodiment. As a method of binding to the solid phase, a method of binding via a modifying substance, What is combined by parts other than is mentioned.

  The modifier used for binding via a modifier is usually a molecule that specifically binds to a specific polypeptide (hereinafter sometimes referred to as a “ligand”) and is attached to the solid surface. Binds a specific polypeptide that binds to the ligand (hereinafter sometimes referred to as "adapter protein"). Adapter proteins include binding proteins, receptor proteins that constitute receptors, antibodies, and the like. Examples of adapter protein / ligand combinations include biotin-binding proteins / biotin such as avidin and streptavidin, maltose-binding protein / maltose, G protein / guanine nucleotide, polyhistidine peptide / nickel or metal ions such as cobalt, glutathione-S -Transferase / glutathione, DNA binding protein / DNA, antibody / antigen molecule (epitope), calmodulin / calmodulin binding peptide, ATP binding protein / ATP, or various receptor proteins such as estradiol receptor protein / estradiol / ligands thereof It is done.

  Among these, adapter protein / ligand combinations include biotin-binding proteins such as avidin and streptavidin, maltose-binding protein / maltose, polyhistidine peptide / metal ions such as nickel or cobalt, glutathione-S-transferase / glutathione, The combination of streptavidin / biotin is most preferable. These binding proteins are known per se, and the DNA encoding the protein has already been cloned.

  The adapter protein can be bound to the solid phase surface by a method known per se. Specifically, for example, tannic acid, formalin, glutaraldehyde, pyruvaldehyde, bis-diazotized benzidone, toluene- A method using a 2,4-diisocyanate, an amino group, a carboxyl group that can be converted into an active ester, a hydroxyl group or an amino group that can be converted into a phosphoramidide, or the like can be used.

  When binding to the solid phase by a moiety other than the modifying substance, known methods commonly used for binding proteins, nucleic acids, sugar chains, and low molecular weight compounds to the solid phase, specifically, for example, tannic acid, formalin, Use a method that uses glutaraldehyde, pyruvic aldehyde, bis-diazotized benzidone, toluene-2,4-diisocyanate, amino group, carboxyl group that can be converted to active ester, hydroxyl group or amino group that can be converted to phosphoramidide, etc. be able to.

  “Measurement” is a means for collecting changes in the signal used for analysis and should not be construed as limiting in any way. Examples of the measurement method used include molecular correlation such as fluorescence correlation spectroscopy, fluorescence resonance energy transfer method, evanescent field molecular imaging method, fluorescence depolarization method, fluorescence imaging analyze method, surface plasmon resonance method, solid phase enzyme immunoassay method, etc. Any system capable of detecting intermolecular interactions is available.

  Fluorescence Correlation Spectroscopy (FCS): Eigen, M., et al., Proc. Natl. Acad. Sci. USA, 91, 5740-5747 (1994)) In the present invention, the original modified molecule 1 is determined by the interaction between the modified functional protein (C-terminal modified functional protein) and the antigen molecule. By measuring the change in the translational Brownian motion of the molecule, the interacting molecule can be measured. Specifically, sample particles are excited by excitation light to emit fluorescence in a part of the sample liquid volume, and the emitted light is measured to obtain a photon ratio. This value varies with the number of particles present in the spatial volume observed at a particular time. The various parameters described above can be calculated from this signal variation using an autocorrelation function. An apparatus for performing this FCS is also commercially available from Carl Zeiss, etc., and in this method, analysis can be performed using these apparatuses. When measuring or analyzing functional protein-target molecule and target molecule interactions using this method, it is necessary to provide either the C-terminal modified functional protein or the target molecule as a solution (liquid phase method). ). The target molecule need not be modified. In addition, a molecule having a molecular weight much smaller than that of the C-terminal modified functional protein to be examined for interaction does not affect the Brownian motion of the C-terminal modified functional protein, and thus is not suitable in this method.

  However, fluorescence cross-correlation spectroscopy (FCCS) using two types of fluorescent dyes can also detect interactions between proteins with similar molecular weights that were difficult with FCS using one type of fluorescent dye. As another method using two types of fluorescent dyes, the fluorescence resonance energy transfer (FRET) method is known, but in order for FRET to occur, the two fluorescent dyes must be close to each other within 40 to 50 mm, and protein Depending on the size and position of the fluorescent dye, FRET may not be observed even if they interact. In the FCCS method, the detection of cross-correlation does not depend on the distance between fluorescent dyes, so there is no such problem. On the other hand, the FCCS method has advantages such as a small amount of sample, a short detection time, and easy automation for HTS as compared with the fluorescence detection method that is another detection system. Furthermore, since the FCCS method provides extremely basic information such as the size and number of fluorescently labeled molecules, it may be used for general purposes such as the surface plasmon resonance method. The difference between the two is that the surface plasmon resonance method detects the interaction while the protein is immobilized, whereas the FCCS method allows the interaction in a solution closer to the natural state to be observed. . In the FCCS method, it is necessary to label the protein with a fluorescent dye instead of immobilizing the protein, but the present invention has made it possible to overcome this problem.

  In this method, in the system of two sets of protein-target molecules modified with two kinds of fluorescent dyes, any contact method can be used as long as both target molecules are in contact with each other sufficiently. There may be two C-terminals labeled with fluorescent dyes that do not overlap with each other at a suitable concentration in a buffer solution or the like that is usually used biochemically in a measurement well of a commercially available FCCS device. This is performed by a method in which a solution in which the modified functional protein is dissolved is added, and a solution in which two kinds of unlabeled target molecules are dissolved in an appropriate concentration in the same buffer.

  In this method, as a method for performing a large number of analyzes at the same time, for example, a plurality of different C-terminal modified functional proteins are introduced into each measurement well of the above-described measurement apparatus for FCCS, and this is bound to each functional protein. Either a target molecule solution to be obtained or a specific C-terminal modified functional protein is introduced, and a plurality of different target molecule solutions are introduced into each well.

  In the fluorescence imaging analysis method (antibody chip method), a modified molecule is brought into contact with a solid phased molecule, and the fluorescence emitted from the modified molecule remaining on the solid phased molecule due to the interaction of both molecules, It is a method of measuring or analyzing using a commercially available fluorescence imaging analyzer.

  When measuring or analyzing functional protein-target molecule interaction or target-target molecule interaction using this method, either the C-terminal modified functional protein or the target molecule is immobilized on the solid phase by the above-described method. It is necessary to be. When the target molecule is used in the form of a solid phase, both modified and unmodified molecules can be used. Moreover, when using it without making it solid-phased, it needs to be modified with the above-mentioned modifying substance. The C-terminal modified functional protein is immobilized via a modified part, or is immobilized by a part other than the modified part, such as a fusion protein such as GST, Flag-tag, His-tag, etc. Can also be used.

  As a substrate for immobilizing C-terminal-modified functional protein or target molecule, a nitrocellulose membrane or nylon membrane usually used for immobilizing proteins, nucleic acids, etc., or a slide glass or plastic microplate Etc. can also be used.

  In this method, the method for bringing the modified target molecule or functional protein into contact with the immobilized molecule may be any method as long as it is a method that allows both molecules to interact with each other. A method of preparing a solution in which a modified target molecule or a C-terminal modified functional protein is dissolved in an appropriate concentration in a buffer that is usually used biochemically and bringing it into contact with the solid surface is preferred.

  After bringing both molecules into contact, the target molecule or C-terminal modified functionality that has remained on the solid phase is preferably washed with the same buffer or the like in the excess of the modified target molecule or C-terminal modified functional protein. Using a commercially available imaging analyzer, a fluorescent signal emitted from a protein modifier or a mixture of fluorescence emitted from a modified molecule immobilized on a solid phase and fluorescence emitted from a modified molecule remaining on the solid phase By measuring or analyzing, a molecule that interacts with the immobilized molecule can be identified.

  In this method, as a method for performing a large number of analyzes simultaneously, for example, a method of immobilizing a plurality of C-terminal modified functional proteins or modified or unmodified target molecules on the solid phase surface, or one type Or a method of contacting a plurality of C-terminal modified functional proteins or modified target molecules not immobilized on the C-terminal modified functional protein or modified or unmodified target molecule. When contacting a plurality of types of C-terminal modified functional proteins or modified target molecules, the molecules remaining on the solid phase are obtained by dissociation due to differences in buffer concentration, etc., and analyzed by a known method. Can be identified.

  The fluorescence resonance energy transfer (FRET) method is well known as another intermolecular interaction detection method using two types of fluorescent dyes. FRET means that when there is an overlap between the fluorescence spectrum of one of the two types of fluorescent dye (energy donor) and the absorption spectrum of the other (energy acceptor), if the distance between the two fluorescent dyes is sufficiently small, A phenomenon in which the probability that the excitation energy excites the acceptor before the light emission from the body occurs. Therefore, if the two proteins whose interaction is to be detected are labeled with a fluorescent dye serving as a donor and acceptor, respectively, and the donor is excited, the distance between the fluorescent dyes does not interact with the two proteins. FRET does not occur and the donor's fluorescence spectrum is observed, but when the two proteins interact and the distance between the fluorescent dyes decreases, the acceptor's fluorescence spectrum is observed by FRET. The presence or absence of protein-protein interaction can be determined from the difference in spectral wavelength. As a fluorescent dye, a combination in which a donor is fluorescein and an acceptor is rhodamine is often used. Recently, an attempt has been made to detect interactions by observing FRET in cells using a combination of mutants having different wavelengths of fluorescent green protein (GFP). The disadvantage of this method is that the two fluorescent dyes need to be close to each other within 40 to 50 mm for FRET to occur, so depending on the size of the protein and the position where the fluorescent dye is attached, FRET There is a point that there is a risk that will not be observed.

  The evanescent field molecular imaging method is a method described in Funatsu, T., et al., Nature, 374, 555-559 (1995), etc. This is a method in which a second molecule is brought into contact, a light source such as a laser beam is irradiated at an angle at which an evanescent field is generated, and the generated evanescent light is measured or analyzed by a detector. These operations can be performed using an evanescent field fluorescence microscope apparatus known per se.

  When measuring or analyzing a protein-protein interaction using this method, either the C-terminal modified functional protein or the target molecule needs to be immobilized by the method described above. The target molecule does not need to be modified when immobilized, but needs to be modified with the above-described modifying substance when used without being immobilized.

  As the substrate for immobilizing the C-terminal modified functional protein or the target molecule, a substrate made of a material such as glass is used, and quartz glass is preferably used. In addition, it is preferable to ultrasonically clean the surface in order to prevent scattering of laser light and the like.

  In this method, the C-terminal modified functional protein or the modified target molecule that has not been immobilized can be brought into contact with the immobilized molecule as long as both molecules are in contact with each other to a sufficient extent to interact with each other. A solution prepared by dissolving a non-immobilized C-terminal modified functional protein or modified target molecule in an appropriate concentration in a biochemically used buffer is preferably prepared. A method of dropping on the surface is preferred.

  After bringing both molecules into contact with each other, by measuring the fluorescence excited by the evanescent field illumination using a detector such as a CCD camera, a molecule that interacts with the solid-phased molecule can be identified.

  In this method, as a method for performing a large number of analyzes simultaneously, for example, a method in which a plurality of C-terminal modified functional proteins or modified target molecules are addressed and immobilized on the substrate is used.

  Fluorescence polarization (Perran, J., et al., J. Phys. Rad., 1, 390-401 (1926)) allows fluorescent molecules excited by fluorescence polarization to remain stationary during the excited state. If the excited molecules undergo rotational Brownian motion during the excited state, the emitted fluorescence will be in a different plane from the excitation light. It is a method of using. The movement of the molecule is affected by its size, and when the fluorescent molecule is a polymer, there is little movement of the molecule during the excited state, whereas the emitted light remains polarized. Thus, in the case of a low-molecular fluorescent molecule, since the movement speed is high, the polarization of the emitted light is canceled. Therefore, the intensity of fluorescence emitted from a fluorescent molecule excited by plane-polarized light is measured on the original plane and a plane perpendicular to it, and the mobility of this molecule and its existence state are determined from the ratio of the fluorescence intensity on both planes. Information can be obtained. According to this method, the behavior of a target molecule that interacts with a fluorescently modified molecule can be traced without being affected by impurities. This is because a change in the degree of polarization is measured only when the fluorescence-modified molecule interacts with the target molecule.

For example, BECON (manufactured by Panyera) is commercially available as an apparatus for performing this method, and this method can also be performed by using these apparatuses.
When measuring or analyzing protein-protein interactions using this method, it is necessary to provide either a C-terminal modified functional protein or a target molecule as a solution. The target molecule need not be modified. In addition, a molecule having a molecular weight much smaller than that of the C-terminal modified functional protein to be examined for interaction does not affect the Brownian motion of the C-terminal modified functional protein, and thus is not suitable in this method.

  In this method, as a method of bringing two sets of C-terminal modified functional protein-target molecules into contact, any method can be used as long as both molecules are brought into contact with each other to a sufficient extent, and preferably A solution in which the C-terminal-modified functional protein is dissolved at an appropriate concentration is put into a buffer solution or the like that is usually used biochemically in a measurement well of a commercially available fluorescence depolarizer, and further a target at an appropriate concentration in the same buffer solution. This is performed by a method of adding a solution in which molecules are dissolved.

  As for the interaction between two targets measured in this method, it is effective to quantify the degree of interaction in order to detect the optimum combination. As an index indicating the degree of interaction, for example, the value of the minimum target concentration that gives the maximum degree of fluorescence polarization for a constant concentration of C-terminal modified functional protein can be used.

  Surface plasmon resonance is a method in which surface plasmon is excited by molecules interacting at the metal / liquid interface and this is measured by the change in intensity of reflected light (Cullen, DC, et al., Biosensors, 3 (4 ), 211-225 (1987-88)). When measuring or analyzing a protein-target molecule interaction using this method, the C-terminal modified protein needs to be solid-phased by the method described above, but the target molecule does not need to be modified.

  As the substrate for immobilizing the C-terminal modified functional protein, a substrate in which a metal thin film such as gold, silver, or platinum is formed on a transparent substrate such as glass is used. As the transparent substrate, any material can be used as long as it is usually used for a surface plasmon resonance device, and it is generally made of glass or the like as a material transparent to laser light, A thickness of about 0.1 to 5 mm is used. The thickness of the metal thin film is suitably about 100 to 2000 mm. A commercially available solid substrate for such a surface plasmon resonance apparatus can also be used. The immobilization of the C-terminal modified functional protein on the substrate can be performed by the method described above.

  In this method, the method for bringing the target molecule into contact with the C-terminal modified functional protein may be any method as long as the two molecules are brought into contact with each other to an extent sufficient to interact with each other. A method can be used in which a C-terminal-modified functional protein immobilized on a solid solution is brought into contact with a solution dissolved at an appropriate concentration in a buffer usually used biochemically.

  These processes may be performed by a commercially available surface plasmon resonance apparatus such as BIAcore 2000 (Pharmacia Biosensor). After contacting both molecules, the immobilized antigen molecule and the single-chain antibody are immobilized by measuring the temporal change in the relative intensity of each reflected light using a known surface plasmon resonance apparatus. Can be analyzed.

  In this method, as a method of performing a large number of analyzes simultaneously, for example, on a substrate used in the surface plasmon resonance apparatus, a plurality of target molecules are addressed and solidified, and one type of functional protein is contacted, Alternatively, a method of bringing a plurality of types of target molecules into contact with one type of immobilized C-terminal modified functional protein is used.

  In Enzyme Linked Immunosorbent Assay (ELISA): Crowther, JR, Methods in Molecular Biology, 42 (1995), a solution containing an antibody is brought into contact with an antigen immobilized on a solid phase. Fluorescence emitted from a modified molecule (such as IgG) that specifically binds an antibody that has remained on a solid-phased antigen due to the interaction between both molecules (antigen-antibody reaction), or a dye that uses the modified molecule as a substrate Is a method of measuring or analyzing a signal emitted from the sensor using a commercially available detector (ELISA reader).

  When measuring or analyzing a protein-antigen molecule interaction using this method, it is necessary that the C-terminal modified functional protein serving as the antigen is immobilized on the solid phase by the method described above. Further, the target molecule to be an antibody needs to be modified with the above-described modifying substance.

As a substrate for immobilizing the C-terminal modified functional protein serving as an antigen, a plastic microplate or the like usually used for ELISA can also be used.
In this method, the modified target molecule to be an antibody can be brought into contact with the solid phase molecule as long as it is a method that allows both molecules to interact with each other to a sufficient extent. A method in which a solution in which molecules are dissolved in a buffer solution usually used biochemically at an appropriate concentration is prepared and injected into a microplate is preferable.

  After bringing both molecules into contact with each other, a step of washing the modified molecules that are not bound to the excessively present immobilized molecules with the same buffer or the like is preferably performed, so that the fluorescence emitted from the modified molecules remaining on the solid phase is emitted. A molecule that interacts with the immobilized antigen molecule can be identified by measurement or analysis using a commercially available ELISA reader or the like.

In this method, as a method for performing a large number of analyzes simultaneously, for example, a method of immobilizing a plurality of different modified target molecules in each hole of the microplate is used.
As a method for identifying interacting molecules, a target molecule that is measured by each of the above-mentioned methods and that has been recognized to interact with the target molecule can be identified by an appropriate method known per se when the primary structure of the molecule is unknown. The structure can be analyzed. Specifically, when the target molecule in which the interaction is recognized is a protein, the primary structure can be identified by analyzing the amino acid sequence with an amino acid analyzer or the like. When the target molecule is a nucleic acid, the base sequence can be determined by an auto DNA sequencer or the like according to the base sequence determination method.

  As an apparatus for immobilizing a C-terminal modified functional protein or target molecule, in order to perform the immobilization method to the solid phase via the modified portion of the C-terminal modified functional protein or target molecule described above The apparatus can also be constructed by combining known appropriate means. Each means in this apparatus is known per se, and operations such as holding the substrate, adding the C-terminal modified protein solution, and washing in these means may be performed by methods known per se. By combining these operations, a fully automatic or semi-automatic apparatus for immobilizing a C-terminal modified protein can be constructed.

  As an apparatus for measuring protein-protein interaction, an apparatus can be constructed by combining known appropriate means in order to perform the protein-target molecule interaction measurement described above. Each means in this apparatus is known per se, and operations such as substrate holding, antigen molecule addition, washing, and signal detection in these means may be performed by methods known per se. By combining these operations, a fully automatic or semi-automatic apparatus for measuring protein-protein interaction can be constructed.

  A high-affinity single-chain antibody selected from the mouse antibody cDNA library by the method of the present invention replaces the variable region or CDR region (complementarity determining region) of the human IgG antibody, thereby producing a chimeric IgG antibody or a humanized antibody. Mouse IgG antibody can be made. These antibodies produce little or little human anti-mouse antibody production elicited when administered to humans. Furthermore, a high affinity single chain antibody selected from the human antibody cDNA library by the method of the present invention can be replaced with a variable region of a human IgG antibody to produce a complete human monoclonal IgG antibody. This can be used as an antibody drug that does not cause anaphylactic symptoms.

  Therefore, it is constructed by substituting the variable region encoded by the nucleic acid with the variable region of human IgG based on the sequence of the nucleic acid selected from the DNA library derived from human or other animal by the selection method of the present invention. Human or human-type antibodies are provided. Also provided is a therapeutic agent comprising this antibody as an active ingredient. Antibodies that bind to angiotensin II may be used as neutralizing antibodies for the treatment of hypertension. That is, since the physiological action of angiotensin II is a blood pressure raising action, the antibody is considered to bind to angiotensin II and suppress the blood pressure raising action. In addition, antibodies that bind to Lewis X may be used to treat cancer. When cells become cancerous, Lewis X is expressed on the cell surface, and antibodies can be used as targets. For example, anticancer drugs that kill cancer cells and toxins such as ricin are bound and administered systemically or locally, and the antibodies attack cancer cells intensively like missiles, making cancer-specific and effective missile treatment It can be performed. Further, as a kind of preparation used for missile treatment, an immunoliposome preparation in which a drug is placed in a lipid bilayer vesicle and an antibody is bound to the surface thereof may be used.

  Hereinafter, examples of the present invention will be specifically described. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. It is not something.

Selection of nucleic acid encoding single chain antibody (I) Creation of library [1] A newly created library was used for the production of a single chain antibody cDNA library.

  First, an H chain DNA solution was prepared. Mouse spleen Poly A + RNA (5 μg / μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-Free water 11 μl, 5 × RT buffer (TOYOBO) 22 μl, 10 mM dNTPs ( TOYOBO) 11 μl, MulgG1 / 2 forward primer (SEQ ID NO: 48) (1 pmol / μl) 27.5 μl, MulgG3 forward primer (SEQ ID NO: 47) (1 pmol / μl) 27.5 μl were mixed and reacted immediately at 65 ° C. for 9 minutes. After cooling to 4 ° C and allowing to stand at 4 ° C for 2 minutes, add 5.5 μl of ReverTra Ace (TOYOBO) and 5.5 μl of RNase inhibitor (TOYOBO), and react at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes. Was synthesized.

  [2] 5 μl of the cDNA-H solution synthesized in [1], 2.5 μl of each HB primer shown in the following HB primer list (1 pmol / μl), 2.5 μl of 10 × PCR buffer (TOYOBO), MulgG1 / 2 forward primer (SEQ ID NO: 48) (1 pmol / μl) 1.25 μl, MulgG3 forward primer (SEQ ID NO: 47) (1 pmol / μl) 1.25 μl, KOD DASH polymerase (TOYOBO) 0.25 μl are mixed, and RNase-Free water is added to the total volume 25 μl each was subjected to a PCR reaction.

  PCR was performed at 96 ° C. for 5 minutes, followed by 25 cycles of 96 ° C., 30 seconds, 50 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis and treated with phenol / chloroform. That is, add the same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1), mix well, centrifuge at 4,200 ° C for 13,200 rpm for 5 minutes, transfer only the aqueous layer to a new tube, and again add the same amount of phenol: chloroform: Isoamyl alcohol (25: 24: 1) was added and mixed well, centrifuged at 4 ° C. and 13,200 rpm for 5 minutes, and only the aqueous layer was transferred to a new tube. The obtained solution was subjected to ethanol precipitation. In other words, add 1 μl of 20 mg / ml glycogen solution (Nacalai Tesque), 0.1 volume of 3 M sodium acetate (pH 5.2), 3 volumes of 100% ethanol, leave it on ice for 15 minutes, and centrifuge at 13,200 rpm for 20 minutes. The supernatant was removed, and the pellet was washed with 1 ml of cooled 70% ethanol, and then centrifuged again at 13,200 rpm for 2 minutes to remove the supernatant. Then, after centrifugation for about 15 minutes, each DNA (19 species) was dissolved in 20 μl of RNase-free water.

  [3] Each DNA solution (19 types) synthesized in [2] 1 μl, each corresponding HB primer shown in the HB primer list (10 pmol / μl) 2 μl, 10 × PCR buffer (TOYOBO) 10 μl, (2 mM) dNTPs (TOYOBO) 10 μl, VH forward primer HF1: HF2: HF3: HF4 (1: 1: 1: 1) mixture (10 pmol / μl) 2 μl shown in the following HF primer list, KOD DASH polymerase (TOYOBO) 0.5 μl And RNase-Free water was added to make a total volume of 100 μl, and each was subjected to PCR reaction.

  PCR was performed at 96 ° C. for 5 minutes, followed by 20 cycles of 96 ° C., 30 seconds, 50 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a 500-900 bp band by 2% agarose gel electrophoresis, followed by phenol / chloroform treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (19 species) was dissolved in 10 μl of RNase-free water.

  [4] The 19 kinds of DNA obtained in [3] were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and each band was cut out. The excised agarose gel fragment and 100 μl of RNase-free water were added to the tube, heated at 70 ° C. for 15 minutes, and then treated with TE saturated phenol. That is, add the same amount of TE saturated phenol (containing 8-quinolinol), mix well, centrifuge at 13,200 rpm for 5 minutes at 4 ° C, transfer only the aqueous layer to a new tube, and again add the same amount of TE saturated phenol (containing 8-quinolinol). ) Was added and mixed well, followed by centrifugation at 13,200 rpm for 5 minutes at 4 ° C., and only the aqueous layer was transferred to a new tube. The obtained aqueous layer was treated with phenol / chloroform, and ethanol precipitation was performed on the resulting solution. The obtained pellet was centrifuged and dried for about 15 minutes, and then each DNA (19 species) was dissolved in 10 μl of RNase-free water. The absorption at 260 nm was measured for each DNA solution, and HB1: HB2: HB3: HB4: HB5: HB6: HB7: HB8: HB9: HB10: HB11: HB12: HB13: HB14: HB15: HB16: HB17: HB18: HB19 (8: 9: 4: 4: 12: 4: 1: 4: 12: 4: 4: 2: 2: 4: 4: 8: 6: 1: 1) and a total of 0.5 pmol of H chain DNA solution did.

  [5] Next, an L chain DNA solution was prepared. Mouse spleen Poly A + RNA (5 μg / μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-Free water 10 μl, 5 × RT buffer (TOYOBO) 20 μl, 10 mM dNTPs ( TOYOBO) 10 μl, MuCK forward primer (SEQ ID NO: 49) (1 pmol / μl) 50 μl was mixed, reacted at 65 ° C. for 9 minutes, immediately cooled to 4 ° C., allowed to stand at 4 ° C. for 2 minutes, and then ReverTra Ace (TOYOBO) 5 μl and 5 μl of RNase inhibitor (TOYOBO) were added and reacted at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes to synthesize cDNA-L.

  [6] To 5 μl of the cDNA-L solution synthesized in [5], 2.5 μl of each LB primer (1 pmol / μl) shown in the following LB primer list, 2.5 μl of 10 × PCR buffer (TOYOBO), MuCK forward primer ( SEQ ID NO: 49) (1 pmol / μl) 2.5 μl and KOD DASH polymerase (TOYOBO) 0.25 μl were added, and RNase-free water was added to make a total volume of 25 μl.

  PCR was performed at 96 ° C. for 5 minutes, followed by 25 cycles of 96 ° C., 30 seconds, 48 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis and treated with phenol / chloroform. The obtained solution was subjected to ethanol precipitation. Then, after centrifugation for about 15 minutes, each DNA (18 species) was dissolved in 20 μl of RNase-free water.

  [7] 1 μl of each DNA solution (18 species) synthesized in [6], 2 μl of each corresponding LB primer (10 pmol / μl) shown in the LB primer list, 10 μl of 10 × PCR buffer (TOYOBO), 2 mM dNTPs ( TOYOBO) 10 μl, LH forward primer shown in the following LF primer list LF1: LF2: LF3: LF4: LFλ (1: 1: 1: 1) mixture (10 pmol / μl) 2 μl, KOD DASH polymerase (TOYOBO) 0.5 μl And RNase-Free water was added to make a total volume of 100 μl, and each was subjected to PCR reaction.

  PCR was performed at 96 ° C. for 5 minutes, followed by 20 cycles of 96 ° C., 30 seconds, 48 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a 500-900 bp band by 2% agarose gel electrophoresis, followed by phenol / chloroform treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 species) was dissolved in 10 μl of RNase-free water.

  [8] The 18 DNAs obtained in [7] were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and each band was cut out. The excised agarose gel fragment and 100 μl of RNase-free water were added to the tube, heated at 70 ° C. for 15 minutes, and then treated with TE saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, and ethanol precipitation was performed on the resulting solution. The obtained pellet was centrifuged and dried for about 15 minutes, and then each DNA (18 species) was dissolved in 10 μl of RNase-free water. The absorption at 260 nm was measured for each DNA solution, and LB1: LB2: LB3: LB4: LB5: LB6: LB7: LB8: LB9: LB10: LB11: LB12: LB13: LB14: LB15: LB16: LB17: LBλ (2: 4: 8: 8: 8: 16: 12: 4: 4: 8: 16: 16: 12: 4: 4: 2: 2: 1) and mixed to give a total 0.5 pmol L-chain DNA solution.

  [9] Further PCR was performed to unify the H and L chains. 0.5 pmol of the H chain DNA solution synthesized in [4], 0.5 pmol of the L chain DNA solution synthesized in [8], 0.5 μl of 5′UTR (SEQ ID NO: 57) (1 pmol / μl), McD-Linker + (SEQ ID NO: 56) (1 pmol / μl) 0.5 μl, McD 3′UTR (His Tag) (SEQ ID NO: 55) (1 pmol / μl) 0.5 μl, 10 × PCR buffer (TOYOBO) 5 μl, 2 mM dNTPs (TOYOBO) 5 μl, KOD DASH polymerase (TOYOBO) 0.25 μl was mixed, and RNase-free water was added to make a total amount of 45.75 μl, followed by PCR reaction.

PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C for 30 seconds, followed by a 5-minute slope at 58 ° C. After 10 cycles of 58 ° C, 30 seconds, 72 ° C and 1 minute, 72 ° C, 5 ° C The reaction was performed for a minute.
[10] Subsequently, to the PCR reaction solution 45.75 μl of [9], McD-F (SEQ ID NO: 52) (1 pmol / μl) 2 μl, McD-R (His Tag) (SEQ ID NO: 50) (1 pmol / μl) 2 μl Then, 0.25 μl of KOD DASH polymerase (TOYOBO) was added and further PCR reaction was carried out.

PCR was performed at 96 ° C. for 5 minutes, followed by 15 cycles of 96 ° C., 30 seconds, 58 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes.
[11] The DNA obtained in [10] was subjected to 1% low melting point agarose gel (Sigma) electrophoresis to cut out each band. The excised agarose gel fragment and 100 μl of RNase-free water were added to the tube, heated at 70 ° C. for 15 minutes, and then treated with TE saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, and ethanol precipitation was performed on the resulting solution. The obtained pellet was centrifuged and dried for about 15 minutes, and then the DNA was dissolved in 10 μl of RNase-free water.

(II) Library transcription [12] DNA obtained in [11] or DNA obtained in [25] 1 pmol, 5 × SP6 buffer 4 μl, ATP (100 mM) 1 μl, CTP (100 mM) 1 μl, UTP ( 100 μM) 1 μl, GTP (10 mM) 2 μl, cap analog (m7G (5 ′) PPP (5 ′) G) (Invitrogen) (40 mM) 2.5 μl, enzyme mix SP6 RNA polymerase (Promega) 2 μl, and RNase-free water Was added to make a total volume of 20 μl, reacted at 37 ° C. for 2 hours and 30 minutes, 5 μl of RQ1 RNase-Free DNase (Promega) was added, and further reacted at 37 ° C. for 1 hour.

  [13] RNA obtained in [12] was purified by RNeasy Mini kit (Qiagen). That is, add RNase-Free water to the transcription reaction solution to make the total volume 100 μl, add RLT buffer solution (Qiagen) 350 μl, 2-mercaptoethanol 5 μl, 100% ethanol 250 μl, and apply to RNeasy mini spin column at 4 ° C., 12,000 Remove the solution drained after centrifugation at rpm for 16 seconds, add 500 μl of RPE buffer (Qiagen) to the same column, remove the solution drained after centrifugation at 12,000 rpm for 16 seconds at 4 ° C, and then add RPE buffer ( Add 500 μl (Qiagen) to the same column, remove the solution discharged after centrifugation at 4 ° C, 12,000 rpm for 2 minutes, replace the column with a new tube, centrifuge at 4 ° C, 12,000 rpm for 1 minute, and re-insert the same column. The tube was replaced with a new tube, and 30.5 μl of RNase-Free water was added to the same column. After standing on ice for 10 minutes, the solution was centrifuged at 4 ° C., 14000 rpm for 1 minute and recovered as an RNA solution.

Ligation with PEG spacer [14] RNA solution obtained in [13] 29.5 μl, T4 ligation 10 × buffer 5 μl, 0.1 M DTT 1 μl, 40 mM ATP 0.5 μl, 100% DMSO 5 μl, 0.1% BSA 1 μl, RNase Inhibitor (TOYOBO) 1 μl, spacer molecule containing polyethylene glycol (PEG) (JP 2002-176987; _p (dC) ₂ T (Fl) _p PEG (2000) _p (dC _p ) ₂ Puro (As described in 2002-276987) (10 nmol) 1 μl, polyethylene glycol (PEG) 2000 (Japanese fat and oil) (30 nmol) 1 μl, T4 RNA ligase (Takara) (250 U / μl) 5 μl The reaction was allowed to proceed at 15 ° C. for 12-15 hours under light-shielded conditions or for about 40 hours at 4 ° C. under light-shielded conditions, and the RNA to which the spacer molecules were bound was purified by RNeasy Mini kit (Qiagen).

(III) Translation [15] RNA 2.5 pmol with spacer molecule obtained in [14], wheat germ extract (including 5 mM DTT) (Promega) 12.5 μl, Amino Acid mixture, Complete (1 mM) (Promega) Mix 2 μl, 2 μl of RNase inhibitor (TOYOBO), 2 μl of CH ₃ COOK (1M) (Promega), add RNase-free water to make a total volume of 25 μl, and react for 1 hour at 25 ° C. under light-shielded conditions for translation. .

(IV) Reverse transcription of library [16] Pretreatment of reverse transcription reaction of translation solution was performed. Column of 1 ml Sephadex G200 (Amersham Biosciences) gel prepared with RNase-Free water and swollen and equilibrated with PP6T buffer with 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 25 μl of the translation solution obtained in [15] was applied to the one filled in (Bio-Rad), and 2 drops were collected in a tube and collected from the 5th to the 8th.

[17] or UltrafreeMC 100,000NWL (Millipore) was subjected to 25 μl of the translation solution obtained in [15] and centrifuged at 4 ° C., 13,200 rpm for 20 minutes to recover the lower layer solution.
[18] Pretreatment solution of either [16] or [17], 5 × RT buffer (TOYOBO) 80 μl, (10 mM) dNTPs (TOYOBO) 40 μl, McD-R (His Tag) (SEQ ID NO: 50) (10 pmol/μl) Mix 20μl, add RNase-Free water to make 360μl, react at 65 ° C for 9 minutes, immediately cool to 4 ° C and leave at 4 ° C for 2 minutes, then ReverTra Ace (TOYOBO) 20 μl and RNase inhibitor (TOYOBO) 20 μl were added. The reaction was carried out at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes, or at 50 ° C. for 30 minutes.

(V) Immobilization of the target antigen [19] Resin (UltraLink Immobilized Neutr Avidin Plus) (Pierce) Take 50 μl in a 1.5 ml tube, suspend with 500 μl of RNase-free water, 4 ° C, 3,000 rpm, 1 minute The supernatant was removed by centrifugation, suspended again with 500 μl of RNase-free water, and centrifuged at 4 ° C. and 3,000 rpm for 1 minute to remove the supernatant. Add 500 μl of (0.4 μM) angiotensin II-biotin or (0.4 μM) Lewis X-sp-biotin dissolved in ELISA buffer with a composition of 100 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.1% Tween 20. The mixture was stirred with a rotary mixer (Nissin) for 1 hour at 25 ° C. FIG. 3 shows the chemical structural formula of angiotensin II-biotin, and FIG. 4 shows the chemical structural formula of Lewis X-sp-biotin. 7 μl of 5 mM biotin dissolved in ELISA buffer was added and further stirred with a rotary mixer (Nissin) at 25 ° C. for 30 minutes. Suspend in 500 μl of ELISA buffer, centrifuge at 4 ° C., 3,000 rpm for 1 minute to remove supernatant, resuspend in 500 μl of ELISA buffer, centrifuge again at 4 ° C., 3,000 rpm for 1 minute to remove supernatant did.

  Mix 10 ml maleic acid buffer 4 ml (Dig wash and block buffer set) (Roche), blocking 10 x blocking solution 4.45 ml (Dig wash and block buffer set) (Roche), and 36 ml RNase-free water The suspension was suspended with 500 μl of blocking agent, centrifuged at 4 ° C., 3,000 rpm for 1 minute to remove the supernatant, 600 μl of the same blocking agent was added again, and the resin was transferred to a 2 ml tube.

(VI) Selection of an IVV antibody that recognizes the target antigen [20] Add 400 μl of the reverse-transcribed library obtained in [18] to the resin suspended in 600 μl of the blocking agent, and then at 4 ° C. for 15 minutes. The mixture was stirred with a mini disc rotor (Bio craft).

  Syringe 5mL (Terumo), Wizard Minicolumn (Promega) and syringe needle 18G x 1/2 "(Terumo) were connected in series, and 1ml of the above suspension was applied and extruded with a syringe. The solution was stored at 0 ° C. An injection needle was inserted into the PP6T buffer solution, and 5 ml was sucked up by a syringe and pushed out again.

  Further, the above operation was performed twice with PP6T buffer containing angiotensin II (final concentration 1 nM) or Lewis X (final concentration 1 nM). The final eluate of this operation was partially stored at 4 ° C. as Wash. Angiotensin II (100 mM) 20 μl, Ribonucleic acid-core from torula Yeast RNA Type II-C (5 μg / μl) 2 μl, PP6T buffer 178 μl or Lewis X 0.5 mg 93.6 μl dissolved in PP6T buffer The mixture was mixed with 0.95 μl of Ribonucleic acid-core from torula Yeast RNA Type II-C (5 μg / μl) to make about 94.6 μl, the Terumo syringe and the Terumo needle were removed, and the mixture was suspended in the Wizard Minicolumn to collect the resin. This resin suspension was rotationally stirred with a minidisc rotor (Biocraft) at 4 ° C. for 1 hour.

  [21] The suspension obtained in [20] was applied to Ultra Free and centrifuged at 10,000 rpm for 3 minutes to recover the solution in the lower layer. 200 μl or 94.6 μl of the eluate was applied to Microcon YM-50, which was added with 100 μl of RNase-Free water in advance and centrifuged at 10,000 rpm for 3 minutes, and centrifuged at 12,000 rpm for 1.5-2.5 minutes. Further, 100 μl of RNase-free water was added, and centrifugation was repeated several times at 12,000 rpm for 1.5-2.5 minutes to remove angiotensin II or Lewis X.

(VII) Recovery of cDNA library by PCR 1 μl of elution solution obtained in [22] [21], 10 μl of 10 × PCR buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), T7-tag-F (SEQ ID NO: 53) (10 pmol / μl) 2 μl, McD-R his tag (SEQ ID NO: 50) (10 pmol / μl) 2 μl, KOD DASH polymerase (TOYOBO) 1 μl are mixed, and RNase-Free water is added to make the total volume 100 μl. PCR reaction was performed.

  PCR was carried out at 96 ° C. for 5 minutes, followed by 25-30 cycles of 96 ° C., 30 seconds, 58 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a 900-1000 bp band by 1% agarose gel electrophoresis.

  [23] The amplified gene obtained in [22] was purified by Wizard Plus Minipreps DNA Purification System (Promega). That is, the PCR reaction product was transferred to a 1.5 ml tube, 100 μl of Direct purification buffer (Promega) and 1 ml of DNA purification resin (Promega) were added and mixed, and subjected to Wizard Minicolumn (Promega) using a 2.5 ml syringe (Terumo). The solution was pushed out with a syringe and discarded (80%). 2 ml of isopropanol was added and the solution was pushed out again and discarded. The Wizard Minicolumn was placed in a new 1.5 ml tube, centrifuged at 4 ° C, 10,000 rpm for 2 minutes, then reattached to a new 1.5 ml tube, added with 60 µl of RNase-Free water, and left at room temperature for 10 minutes. The mixture was centrifuged at 4 ° C., 10,000 rpm for 2 minutes, the Wizard Minicolumn was discarded, the lower layer was collected, and further centrifuged at 4 ° C., 10,000 rpm, 5 minutes. The resulting precipitate was removed and the supernatant was collected in a new 1.5 ml tube.

The absorbance at 260 nm was measured for the DNA solution to estimate the concentration.
(VIII) Addition of omega sequence by PCR [24] 0.2 pmol of DNA solution obtained in [23] to add omega sequence by PCR, 10 x PCR buffer (TOYOBO) 10 μl, 2 mM dNTPs (TOYOBO) 10 μl, McD 5′UTR (SEQ ID NO: 54) (1 pmol / μl) 1 μl, McD-F (SEQ ID NO: 52) (10 pmol / μl) 2 μl, McD-R his tag (SEQ ID NO: 50) (10 pmol / μl) 2 μl, 0.5 μl of KOD DASH polymerase (TOYOBO) was mixed, and RNase-free water was added to make a total volume of 100 μl, followed by PCR reaction.

  PCR was performed at 96 ° C. for 5 minutes, followed by 7-10 cycles of 96 ° C., 30 seconds, 58 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a band of about 1000 bp by 1% agarose gel electrophoresis, followed by phenol / chloroform treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, the DNA was dissolved in 10 μl of RNase-free water.

  [25] The DNA obtained in [24] was subjected to 1% low melting point agarose gel (Sigma) electrophoresis to cut out each band. The excised agarose gel fragment and 100 μl of RNase-free water were added to the tube, heated at 70 ° C. for 15 minutes, and then treated with TE saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, and ethanol precipitation was performed on the resulting solution. The obtained pellet was centrifuged and dried for about 15 minutes, and then the DNA was dissolved in 10 μl of RNase-free water.

  FIG. 5 shows the result of confirming the corresponding molecules by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8M urea. In the figure, the lower bar graph shows the result of measuring and quantifying the FITC fluorescence of the upper electrophoresis gel with MOLECULAR IMAGER FX (Bio-rad). From the left, Nega is one of the library MH0 clones obtained in [11], MH0-15, Posi is one of the library MI3 clones, MI3-55, and MH0 is the library obtained in [11]. MI1 is a library obtained by reacting the library obtained in [11] in [18] at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes, angiotensin II is selected as an antigen, and recovered in [25]. In [18], 50 ° C., 30 minutes, then 99 ° C., reacted for 5 minutes, angiotensin II was selected as an antigen and collected in [25], MI3 was MI2 in [18] at 50 ° C., 30 minutes, then 99 ° C., The library recovered after [25] was selected by reacting for 5 minutes and selecting angiotensin II as an antigen.

  MH0 contains a significant percentage of things that are not translated into proteins due to stop codons or mutations, so the efficiency of mapping is as low as 15%, compared to 25% for MI1, 37% for MI2, and MI3 The efficiency of matching has increased to 41%. Nega and Posi are in-frame clones, and the association efficiencies in this case are 34% and 40%, respectively, but MI2 or MI3 have values almost equal to the association efficiencies. Thus, the protein level selection was successful, indicating that the library has an increased in-frame rate.

  FIG. 6 shows the result of confirming the corresponding molecule by subjecting each translation solution obtained in the above step to 5% polyacrylamide electrophoresis in the presence of 8M urea as in FIG. From left to right, MH0 is the library obtained in [11], and MK1 is the library obtained in [11] in [18] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes to select Lewis X as an antigen. The library recovered in [25], MK2 represents the library recovered in [25] after selecting Mx1 as an antigen by reacting MK1 in [18] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes. MH0 contains a significant percentage of things that are not translated into proteins due to stop codons or mutations, so the efficiency of mapping is as low as 15%, whereas MK1 maps to 41% and MK2 maps to 36% The efficiency of has increased.

  FIG. 7 shows the result of confirming the corresponding molecule by subjecting each translation solution obtained in the above step to 5% polyacrylamide electrophoresis in the presence of 8M urea as in FIG. From the left, MM1 is a library obtained by reacting the library obtained in [11] at 50 ° C for 30 minutes in [18], selecting angiotensin II as an antigen and recovered in [25], and MM2 is MM1 at 50 ° C in [18] A library that was reacted for 30 minutes and angiotensin II was selected as an antigen and recovered in [25], and MP1 was reacted in [15] at 99 ° C for 5 minutes in [15], then [20] [21] Shows the library recovered in [25] after selection using angiotensin II as an antigen in [18], followed by reaction at [18] for 5 minutes at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes. . The efficiency of the association is increased to 25% for MM1 and 33% for MM2. In MM1, [18] is similar to MI1, which was reacted at 50 ° C for 30 minutes and then reacted at 99 ° C for 5 minutes, and MM2 also showed a matching efficiency comparable to that of MI2. Selection at the level is considered successful. On the other hand, MP1 had an extremely low associating efficiency of 8%, and it was considered that some denaturation occurred in RNA or the corresponding molecule by reacting at 99 ° C for 5 minutes in [15].

  FIG. 8 shows the results obtained by subjecting the eluate obtained in [21] to PCR in [22] and subjecting to 1% agarose gel electrophoresis. The lower bar graph shows the absorption of ethidium bromide in the upper electrophoresis gel by MOLECULAR IMAGER. It shows the value measured and measured with FX (Bio-rad). From the left, Nega is one of the library MH0 clones obtained in [11], MH0-15, Posi is one of the library MI3 clones, MI3-55, and MI1 is the library obtained in [11]. [18] was reacted at 50 ° C. for 30 minutes and then 99 ° C. for 5 minutes, and angiotensin II was selected as an antigen and recovered in [25]. MI2 was MI1 at [18] at 50 ° C. for 30 minutes and then 99 ° C. The library that was reacted for 5 minutes and angiotensin II was selected as an antigen and recovered in [25], and MI3 was reacted at [18] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes to select angiotensin II as an antigen [ 25] shows the collected library. Nega shows that the amount of DNA recovered is very small and contains almost no molecules with affinity for Angiotensin II, while Posi recovers a large amount of DNA with an affinity for Angiotensin II. It shows that it is the majority. In MI1 and MI2, the amount of DNA recovered by selection is still small, and it is considered that the ratio of molecules having an affinity for angiotensin II contained in the library is low. In contrast, in MI3, the amount of recovered DNA increased rapidly, and the proportion of molecules with an affinity for angiotensin II contained in the library increased rapidly, indicating that enrichment occurred due to repeated selection. It is considered a thing.

  FIG. 9 shows the results obtained by diluting the Flow obtained in [20] 1000 times and the Wash obtained in [20] and the eluate (Elute) obtained in [21] diluted 1 and 10 times. The results are shown in [22] and subjected to 1% agarose gel electrophoresis. Regarding Elute, x1 stands for 1-fold dilution of the eluate, and x0.1 stands for 10-fold dilution of the eluate. MK1 was obtained by reacting the library obtained in [11] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes in [18], and Lewis X was selected as an antigen and recovered in [25]. 18] shows a library collected by reacting Lewis X as an antigen after reacting at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes. As shown in Flow, a sufficiently large excess of molecules is added to the selection, but in Wash, almost no DNA band is visible, indicating that the selection can wash out molecules with low affinity to Lewis X. . In MK1, the amount of DNA recovered by selection is still small, and it is thought that the proportion of molecules having affinity for Lewis X contained in the library is low. In contrast, in MK2, the amount of recovered DNA increased rapidly, the proportion of molecules with affinity for Lewis X contained in the library increased rapidly, indicating that enrichment occurred due to repeated selection. It is considered a thing.

  FIG. 10 shows a flow obtained by diluting the flow obtained in [20] 1000 times and a wash obtained in [20] and a dilute solution obtained in [21] by 1 and 10 times [22]. 2 shows the results of PCR and 1% agarose gel electrophoresis, and the results of measuring and quantifying the ethidium bromide absorption of the electrophoresis gel with MOLECULAR IMAGER FX (Bio-rad). F stands for Flow, W stands for Wash, E stands for 1-fold eluate, and E0.1 stands for 10-fold dilution of eluate.

  As shown in Flow, a sufficiently large excess of molecules was added to the selection, but in Wash, almost no DNA band was visible, indicating that the molecules with low affinity for Angiotensin II were sufficiently washed out in the selection. .

  MI1 is a library obtained in [11] and reacted in [18] at 50 ° C. for 30 minutes and then 99 ° C. for 5 minutes, angiotensin II is selected as an antigen and recovered in [25], and MM1 is [11] The library obtained in [18] was selected at [18] for 30 minutes at 50 ° C. and angiotensin II was selected as an antigen and recovered in [25].

  In MI1, the amount of DNA recovered by selection is still small, and it is considered that the proportion of molecules having an affinity for angiotensin II contained in the library is low. In contrast, in MM1, the amount of recovered DNA is very large, and it is considered that the proportion of molecules having an affinity for angiotensin II contained in the library is large. In other words, MM1 was only reacted at 50 ° C for 30 minutes at [18], whereas MI1 was stable at 99 ° C by reacting at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes in [18]. This indicates that only molecules with a high molecular weight are selected and those that are unstable tend to be removed.

Sequence analysis (I) Cloning [26] TOPO cloning kit (Invitrogen) was used to obtain clones from the library. DNA obtained in [11] or DNA obtained in [25] 0.05-1 pmol, taq polymerase 10 × buffer (TOYOBO) 2 μl, dATP (40 mM) 2 μl, taq polymerase (Taq Pol) (TOYOBO) 0.5 μl was mixed, RNase-Free water was added to make a total volume of 20 μl, and phenol / chloroform treatment and ethanol precipitation were performed at 72 ° C. for 15 minutes. Then, after centrifugation for about 15 minutes, the DNA was dissolved in 4 μl of RNase-free water.

  [27] 4 μl of the DNA obtained in [26], 0.5 μl of Topo vector (Invitrogen) and 1 μl of Salt solution (Invitrogen) were mixed and allowed to stand at room temperature for 20 minutes. In order to transform into Escherichia coli, 5.5 μl of the above mixed solution was placed in a competent cell dissolved on ice, and left on ice for 30 minutes, followed by heat shock at 43 ° C. for 45 seconds. Place 250 μl of SOC (Invitrogen) in the cell and incubate on a shaking incubator for 1 hour and 30 minutes at 37 ° C. Two agar plates (5 g tryptone, 2.5 g yeast extract, 5 g NaCl, 7.5 g agar, Ampicillin (25 mg) (Petri dish 9 cm) was cultured overnight at 37 ° C.

(II) Determination of the base sequence A portion of the colony formed on the agar plate cultured in [28] and [27] is suspended with a toothpick, suspended in 20 μl of RNase-free water, heated at 99 ° C for 5 minutes, and then rapidly cooled. Stored as a colony suspension at 20 ° C.

  [29] Colony suspension obtained in [28] 1 μl, 10 × PCR buffer (TOYOBO) 5 μl, 2 mM dNTPs (TOYOBO) 5 μl, M13FII (SEQ ID NO: 58) (10 pmol / μl) 1 μl, M13RII ( SEQ ID NO: 59) (10 pmol / μl) 1 μl and KOD DASH polymerase (TOYOBO) 0.25 μl were mixed, and RNase-free water was added to make the total volume 50 μl.

  PCR was performed at 96 ° C. for 5 minutes, followed by 30 cycles of 96 ° C., 30 seconds, 58 ° C., 30 seconds, 72 ° C. and 1 minute, and then reacted at 72 ° C. for 5 minutes. Each amplified gene was confirmed to have a 900-1000 bp band by 1% agarose gel electrophoresis. The amplified gene was purified by Wizard Plus Minipreps DNA Purification System (Promega).

[30] DNA 16 ng obtained in [29], M13FII (SEQ ID NO: 58) (1.6 pmol / μl) 2 μl, or M13RII (SEQ ID NO: 59) (1.6 pmol / μl) 2 μl, DTCS kit Premix (Beckman coulter) 6 μl was mixed, and RNase-free water was added to make a total volume of 10 μl, followed by PCR reaction.
PCR was performed at 96 ° C. for 5 minutes, followed by 30 cycles of 96 ° C., 20 seconds, 58 ° C., 20 seconds, 60 ° C., 4 minutes, and then reacted at 72 ° C. for 5 minutes.

  [31] Transfer the PCR reaction product obtained in [30] to a 1.5 ml tube, mix well with 1 μl of 3 M NaOAc, 1 μl of 0.1 M EDTA, and 1 μl of a 20 mg / ml glycogen solution (Nacalai Tesque). 60 μl of% ethanol was added and mixed. After centrifuging at 4 ° C., 14000 rpm for 15 minutes to remove the supernatant and washing the pellet with 70% ethanol, centrifugation was again carried out at 14000 rpm for 2 minutes to remove the supernatant twice. Then, after centrifugal drying for 30-40 minutes, 40 μl of deionized formamide (Beckman coulter) was added and mixed well. Sequence analysis was performed with CEQ 2000 DNA Analysis System (Beckman coulter).

  In Table 5, from the left lane, “Name” is the type and number of selections, “In frame” is the number of in-frames, “Mutation” is the number of proteins that are not translated into proteins by stop codons or mutations, and “Total” Is the sum of In frame and Mutation, “In frame (%)” is 100 × In frame / Total, “C” is the number of elements having an array converged to A or B in FIGS. 11 and 12, and “C (%)” “100 × number of elements having an array converged to A or B in FIG. 11 and FIG. 12 / In frame”, “IVV (%)” represents the association efficiency calculated from FIG. 5, FIG. 6 and FIG. Show.

  In addition, MH0 is the library obtained in [11], MI1 is the library obtained in [11] in [18], reacted at 50 ° C. for 30 minutes and then 99 ° C. for 5 minutes to select angiotensin II as an antigen [ 25], the library collected in MI2, MI1 was reacted in [18] at 50 ° C. for 30 minutes and then 99 ° C. for 5 minutes, angiotensin II was selected as the antigen, and the library recovered in [25], MI3 18] at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes, angiotensin II was selected as an antigen and recovered in [25], MK1 was obtained in [11] at 50 ° C. in [18] The library collected by reacting Lewis X as an antigen for 30 minutes and then at 99 ° C for 5 minutes and recovered in [25], MK2 was reacted with MK1 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes. As an antigen MM1 is a library collected in [25], MM1 is a library obtained in [11], reacted at 50 ° C. for 30 minutes in [18], selected as angiotensin II as an antigen, and collected in [25], MM2 indicates a library obtained by reacting MM1 in [18] at 50 ° C. for 30 minutes, selecting angiotensin II as an antigen, and recovering in [25].

  100 × (number of those having sequences converged to A in FIG. 11 and FIG. 12) / In frame is 33% for MI1, 57% for MI2, and 87% for MI3. Selection with II as the antigen showed a rapid convergence to the sequence shown in A. 100 × (number of those having sequences converged to B in FIGS. 11 and 12) / In frame indicated by C (%) / In frame shows 50% for MK1 and 40% for MK2, and shows a high value by rotating the selection. Selection with Lewis X as an antigen showed convergence to the sequence shown in B. On the other hand, 100 × (number of those having sequences converged to A in FIGS. 11 and 12) / In frame indicated by C (%) is 0% for MM1 and 13% for MM2, and the selection is performed once. As a result, none of the sequences shown in A was found, and only one clone was found in the sequence shown in A by turning the selection twice. MM1 and MM2 were reacted at 50 ° C for 30 minutes at [18], whereas MI1, MI2, and MI3 were reacted at 50 ° C for 30 minutes at [18] and then reacted at 99 ° C for 5 minutes. Therefore, by reacting at 99 ° C for 5 minutes, the sequence shown in A suddenly converged, but in the case of MM1 and MM2 not reacting at 99 ° C for 5 minutes, the sequence shown in A can be found, but it is very efficient. I found it bad.

  MP1 was reacted with the library obtained in [11] at 99 ° C for 5 minutes in [15], then selected with angiotensin II as an antigen in [20] and [21], and then in [18] at 50 ° C for 30 minutes. This was a library collected at [25] after reacting at 99 ° C for 5 minutes and performing the experiment after [22]. As a result of analyzing the sequence analysis by [31], two in-frame, stop codon or mutation 11 were not translated into protein, 13 were in total, and 2 were those with sequences converged to A in FIGS. In the case of MP1, it was found that the in-frame ratio is extremely low and the efficiency shown in A can be found although it cannot be said that efficiency is good.

  FIG. 11 and FIG. 12 show the phylogenetic tree created by TreeViewPPC after sequence alignment by clustalx with the amino acid sequence for the in-frame ones analyzed by sequence analysis in [31]. A surrounded by a line is converged by angiotensin II selection, and B surrounded by a line is converged by Lewis X selection. The clone number is indicated after the library abbreviation. A surrounded by a line shows 90% or more homology as a result of analysis by GENETYX-MAC at the protein level compared to MI3-55, and B surrounded by a line analyzed by protein level GENETYX-MAC compared to MK1-17 As a result, 90% or more homology was shown.

Some of the sequenced clones (MI1-16, MI1-4, MI2-10, MI2-34, MI2-41, MI2-48, MI3-15, MI3-26, MI3-28, MI3-34, MI3 -41, MI3-42, MI3-47, MI3-5, MI3-55, MI3-62, MI3-64, MI3-66, MI3-74, MK2-3, MM2-11, MP1-36, MP1-41 , MI3-8, MK1-15, MK1-17, MK1-24, MK2-19, MK2-8 ), the nucleotide sequence and the amino acid sequence encoded by the nucleotide sequence are shown in the even SEQ ID NOs: 60 to 116. The amino acid sequence is shown in the odd sequence number of 61-117. The amino acid number of CDR was determined as follows according to Martin, ACR Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133.

  MI1-16, MI1-4, MI2-10, MI2-34, MI2-41, MI2-48, MI3-15, MI3-26, MI3-28, MI3-34, MI3-41, MI3-42, MI3- 47, MI3-5, MI3-55, MI3-62, MI3-64, MI3-66, MI3-74, MK2-3, MM2-11, MP1-36, MP1-41 CDRs are the following amino acid numbers: is there.

  The CDRs of MI3-8, MK1-15, MK1-17, MK1-24, MK2-19, and MK2-8 have the following amino acid numbers.

Preparation of Antibody Colony suspension obtained in [32] [28] 1 μl, 10 × PCR buffer (TOYOBO) 10 μl, 2 mM dNTPs (TOYOBO) 10 μl, 25 mM MgSO _{4 4} μl, McD-F (SEQ ID NO: 52 ) (10 pmol / μl) 3 μl, McD-R (His Tag) -stop (SEQ ID NO: 51) (10 pmol / μl) 3 μl, KOD PLUS polymerase (TOYOBO) 2 μl are mixed, and RNase-Free water is added to make the total amount. Each 100 μl was subjected to PCR reaction.

  PCR was performed at 94 ° C. for 5 minutes, followed by 25-30 cycles of 94 ° C., 30 seconds, 58 ° C., 30 seconds, 68 ° C., 2 minutes, and then reacted at 68 ° C. for 5 minutes. Each amplified gene was confirmed to have a 900-1000 bp band by 1% agarose gel electrophoresis.

  [33] Transcription of clone DNA was performed. DNA obtained in [32] 1 pmol, 5 × SP6 buffer 4 μl, ATP (100 mM) 1 μl, CTP (100 mM) 1 μl, UTP (100 mM) 1 μl, GTP (10 mM) 2 μl, cap analog (m7G (5 ') PPP (5 ') G) (Invitrogen) (40 mM) 2.5 μl, enzyme mix SP6 RNA polymerase (Promega) 2 μl was mixed, RNase-Free water was added to make a total volume of 20 μl, and reacted at 37 ° C. for 2 hours 30 minutes. 5 μl of RQ1 RNase-Free DNase (Promega) was added and further reacted at 37 ° C. for 1 hour. The obtained RNA was purified by RNeasy Mini kit (Qiagen).

  [34] RNA 20 pmol obtained in [33], wheat germ extract (including 5 mM DTT) (Promega) 50 μl, Amino Acid mixture, Complete (1 mM) (Promega) 8 μl, RNase inhibitor (TOYOBO) 8 μl, CH3COOK (1M) (Promega) 8 μl was mixed, RNase-free water was added to make the total volume 100 μl, and the reaction was carried out under light-shielded conditions at 25 ° C. for 1-3 hours for translation.

  [35] 1 ml of Sephadex G75 (Amersham Biosciences) gel prepared with Milli-Q water and swollen and equilibrated with PP6T buffer with 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 200 μl of the translation solution obtained in [34] was applied to the column (Bio-Rad) packed, and 4 drops were collected in a tube and the 3rd to 6th were collected.

  [36] or Sephadex G75 (Amersham Biosciences) gel 1 prepared with Milli-Q water and swollen and equilibrated with His Tag A buffer having a composition of 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole pH 7.4 200 μl of the translation solution obtained in [34] was applied to a column (Bio-Rad) packed in ml, and 4 drops were collected in a tube and 3rd to 6th were collected. To this solution, 100 μl of Ni-NTA agarose (Qiagen) resin equilibrated with His Tag A buffer was added, and the mixture was stirred with a rotary mixer (Nissin) at 25 ° C. for 1 hour. Suspend in 500 μl of His Tag A buffer, centrifuge at 4 ° C, 3,000 rpm for 1 minute and remove the supernatant three times, collect the resin, collect the resin, and the composition is 20 mM sodium phosphate, 500 mM NaCl Then, 150 μl of His Tag B buffer (475 mM imidazole pH 7.4) was added, and the mixture was rotated and stirred with a rotary mixer (Nissin) at 25 ° C. for 30 minutes to 1 hour. The resin suspension was applied to Urtra Free and centrifuged at 10,000 rpm for 3 minutes to collect the solution in the lower layer.

  [37] Micro Spin G-25 column (Amersham Biosciences) equilibrated with PP6T buffer was centrifuged at 735 g for 1 minute at 4 ° C to remove the eluate in the lower layer, and the solution obtained in [36] 50 μl was applied to the column. Centrifugation was performed at 4 ° C. and 735 g for 2 minutes to recover the lower layer solution.

  Alternatively, 1 ml of Sephadex G75 (Amersham Biosciences) gel prepared with milliQ water and swollen and equilibrated with PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 (column) 150 μl of the solution obtained in [36] was applied to the one filled in (Bio-Rad), and 4 drops were collected in a tube and the 3rd to 6th bottles were collected.

  FIG. 13 shows the results of Western blotting of [37] of MI3-55 (two on the right). The left three are Western blot controls. From left to right, Carboxy-terminal FLAG-BAP fusion protein (Sigma) 100 ng, 50 ng, 20 ng, Biotynylated SDS-PAGE Standards low range (Bio-rad) 2 μl, MI3-55 [37] 15 μl and 5 μl collected up to the 6th were subjected to 15% polyacrylamide electrophoresis and then transferred to a PBDF membrane, anti-FLAG M2-Mouse (Sigma) was the primary antibody, Goat anti-mouse IgG (H + L) -HRP Western blot detected with ECL Western Blotting Detection Reagents (Amersham biosciences) using conjugate (Sigma) and Avidin-HRP conjugate (Bio-rad) as secondary antibodies. MI3-55 detected a band with a molecular weight of about 31,000 daltons, which was consistent with the calculated value for single-chain antibodies.

Confirmation of coupling by via core [38] The via core 3000 system was used as the via core. The sensor chip B1 (CM4) was immobilized by the amine coupling method. Flow cells 1 and 2 were activated with a solution containing 0.2 M N-ethyl-N′-dimethylaminopropylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) for 10 minutes. Next, mix 20 μl of 50 μM streptavidin (Sigma) (0.02 M potassium phosphate buffer pH 6.5) with flow cell 2 and 10 mM acetate buffer pH 5.0 980 μl for 10 minutes, and flow cell 1 with 10 mM acetate buffer. Solution pH 5.0 was run for 10 minutes. Subsequently, the flow cells 1 and 2 were blocked with 1M ethanolamine pH 8.5 for 10 minutes. In the flow cell 2, a 2024 or 2305 response unit was bonded to the sensor chip surface. Further, 50 μM NaCl and 1 μM NaCl 10 μl were flowed through the flow cells 1 and 2 five times, and then 10 μl of angiotensin II-biotin (0.1 μM) was flowed into the flow cell 2 in which the 2024 response unit was bound to the sensor chip surface. 3 μl of Lewis-X-sp-biotin (0.1 μM) was passed through the flow cell 2 in which the 2305 response unit was bound to the sensor chip surface. Finally, as a result of 10 μl of Glycine 1.5 flowing into flow cell 1 and 2, 35.0 response unit was bound to the sensor chip surface in flow cell 2 of angiotensin II-biotin, and 31.6 response unit was bound to flow cell 2 of Lewis-X-sp-biotin. . The theoretical Rmax was estimated to be 801 for angiotensin II-biotin flow cell 2 or 1081 for Lewis-X-sp-biotin flow cell 2.

  [39] Biacore was analyzed using PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20, and a flow rate of 20 μl / min. Samples obtained in [35] or [37] were injected with KINJECT. Regeneration was performed with 20 μl of Glycine 1.5, 10 μl of 50 mM NaCl, and 1 M NaCl. The response curve was calculated by subtracting the flow cell 1 from the flow cell 2 to obtain a combined response unit.

  FIG. 14 shows MI3-55, MI3-42, MI3-28, MI3-41, MI3-34, MI3 among the sequences that converge to the sequences shown in FIG. 11 and FIG. 12A by selection using angiotensin II as an antigen. -5, MI3-15, and MI3-26 are translated by [34] and then analyzed by Biacore and calculated KD as a bar graph. The single-chain antibody selected by the method of the present invention showed a very high affinity value of 0.4-1.5 nM and was found to have a very high affinity.

  FIG. 15 shows translation of MK1-1, MK1-24, MK2-19, and MK1-17 with [34] among the sequences that converge to the sequences shown in FIG. 11 and FIG. Is a bar graph showing the calculated KD after Biacore analysis. The single chain antibody selected by the method of the present invention showed a value of 20-43 nM and was found to be a high affinity antibody.

  FIG. 16 is a bar graph showing the results of analyzing [39] Biacore after MI3-55 was translated at [34], treated at 4 ° C, 60 ° C or 99 ° C for 5 minutes and treated at [35]. It is shown by. From the right lane, 1 indicates sample stock solution, 0.5 indicates 1/2 dilution of stock solution, 0.25 indicates 1/2 dilution of 0.5, and 0.125 indicates 1/2 dilution of 0.25. When the stock solution was treated at 99 ° C for 5 minutes, the response was 56% compared to that treated at 4 ° C for 5 minutes, but the response treated at 4 ° C for 5 minutes was equivalent to that diluted to 0.25. Therefore, even when MI3-55 is treated at 99 ° C for 5 minutes and treated at [35], it has a binding activity of about 25%, and the single chain antibody selected by the method of the present invention is very heat-resistant. It has been shown.

Confirmation of binding by ELISA [40] Add 200 μl of angiotensin II-biotin or Lewis-X-sp-biotin (100 nM) / ELISA buffer per well of Nunc streptavidin-coated 96-well microplate at 50 ° C for 1 hour Shake gently and fix. The plate solution was discarded and water was thoroughly drained, and 200 μl of ELISA buffer was added to wash the plate three times. After adding 200 μl of ELISA buffer and allowing to stand for 5 minutes, the plate was washed again with 200 μl of ELISA buffer three times. Blocking one (Nacalai Tesque Co., Ltd.): Blocking agent diluted with MilliQ (1: 4) was added and blocking was performed overnight at 4 ° C or for 1 to several hours at 25 ° C. Next, the operation of washing the plate with 200 μl of PBS (Nacalai Tesque) was performed 4 times, and the sample obtained in [35] or [37] was added to the 100 μl plate. At this time, when examining antagonistic inhibition, angiotensin II or Free Lewis X previously mixed with a single chain antibody sample was added. Shake gently at 25 ° C for 0.5-2 hours, and then wash the plate with 200 µl of PBS (Nacalai Tesque, Inc.) four times to obtain anti-FLAG M2-Mouse (Sigma): Blocking one as the primary antibody solution. Company): 100 μl of ELISA buffer (1: 10: 190) was added. Gently shake at 25 ° C for 0.5-2 hours, and then wash the plate with 200 µl of PBS (Nacalai Tesque) 4 times to obtain the secondary antibody solution Goat anti-mouse IgG (H + L) -HRP conjugate ( (Sigma): 100 μl of Blocker casein in TBS (PIERCE) (1: 400) was added. TMB Peroxidase EIA substrate kit (Bio rad) (A: B = 9: 1) 100 After gently shaking at 25 ° C for 0.5-2 hours, wash the plate 4 times with 200 µl PBS (Nacalai Tesque) μl was added. The reaction was stopped by gently shaking at 25 ° C. for 5-60 minutes and adding 100 μl of 1 NH ₂ SO ₄ . Absorbance was measured at 450 nm and 630 nm with a Multiskan JX (Dainippon Pharmaceutical Co., Ltd.) 96-well microplate reader, and the value at 630 nm was subtracted from the value at 450 nm.

  FIG. 17 is a bar graph showing the results of the analysis of [40] after the translation of MI3-55 with [34], followed by treatment at 4 ° C, 60 ° C or 99 ° C for 5 minutes and [35]. It is shown by. From the right lane, 1 indicates sample stock solution, 0.5 indicates 1/2 dilution of stock solution, 0.25 indicates 1/2 dilution of 0.5, 0.125 indicates 1/2 dilution of 0.25, 0.0625 indicates 1/2 dilution of 0.125 . The undiluted solution treated at 99 ° C for 5 minutes was 21% of the value treated at 4 ° C for 5 minutes, and the 0.5 dilution was treated at 99 ° C for 5 minutes compared to the one treated at 4 ° C for 5 minutes. However, it was equivalent to that obtained by diluting the sample treated at 4 ° C. for 5 minutes to 0.125. Therefore, even when MI3-55 is treated at 99 ° C. for 5 minutes and treated at [35], it has a binding activity of about 12.5% or more, and the single chain antibody selected by the method of the present invention is extremely heat-resistant. It was shown that there is.

Industrial applicability

  The present invention is a technique for applying protein functional maturation to an evolutionary molecular engineering technique (IVV method) to quickly and inexpensively select proteins in a test tube. Specifically, a desired protein can be obtained by constructing a protein cDNA library, converting to an assigned molecule (IVV) library, selecting, recovering genes, and repeating this cycle several times. Furthermore, by using a library obtained by adding mutation (point mutation, DNA shuffling) to a known protein gene, it is possible to select a protein with higher functionality, higher stability, and higher expression.

According to the present invention, protein selection is performed under a strong selective pressure environment (heat treatment at a high temperature), so that a very high concentration effect is exhibited and a protein having a strong binding force with respect to a target molecule can be quickly selected. . The obtained protein has an extremely wide range of applications such as application to diagnostics and therapeutics as well as use in basic molecular biological research. In particular, the following excellent effects can be achieved.
(1) Pre-heat treatment before binding the library to the antigen to denature unstable proteins and maintain a stable three-dimensional structure, or a protein with high stability Only groups can be selected.
(2) When the reverse transcription reaction is performed simultaneously, nonspecific binding to the carrier or target molecule in the RNA portion is suppressed, and the concentration efficiency is dramatically increased.
(3) The obtained protein can be expressed in a large amount in the cytoplasm of E. coli in a cell-free translation system or in a reducing environment in the presence of a reducing agent such as DTT, and is also stable against the reducing agent. .

Claims (23)

A method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same, comprising the following steps (a) to (d):
(a) A step of preparing a library of DNAs encoding proteins.
(b) Transcribing the DNA of the library prepared in (a), linking a spacer containing puromycin to the 3 ′ end of the transcribed RNA, and then mapping the genotype and phenotype in a cell-free translation system Building a library of molecules.
(c) A step of heat-treating the library of corresponding molecules at 80-100 ° C. for 1-30 minutes.
(d) A step of binding the corresponding molecule to the target molecule, thoroughly washing and then elution, and amplifying the nucleic acid part by reverse transcription-PCR or PCR. The selection method according to claim 1, wherein the target molecule is an antigen and the protein is a single-chain antibody. The selection method according to claim 1 or 2, wherein the spacer comprises polyethylene glycol. The selection method according to any one of claims 1 to 3, further comprising the step of repeating the operations (b) to (d) using the DNA amplified in (d) as the library of (a). The selection method according to any one of claims 1 to 4, further comprising a step of reversely transcribing the RNA portion of the mapping molecule into an RNA-DNA hybrid before the step (d). 6. The selection method according to claim 5, wherein a substance derived from a cell-free translation system that inhibits the reverse transcription reaction is removed before reverse transcription. The selection method according to any one of claims 1 to 6, wherein the cell-free translation system is a cell-free translation system containing a thiol compound. The selection method according to any one of claims 1 to 7, wherein the cell-free translation system is a cell-free translation system of a wheat germ extract, a rabbit reticulocyte extract, or an E. coli S-30 extract. A method for producing a protein that interacts with a target molecule, the step of selecting a nucleic acid encoding a protein that interacts with the target molecule by the selection method according to any one of claims 1 to 8, and the selected nucleic acid The manufacturing method including the process of translating and manufacturing a protein. The method according to claim 9, wherein the target molecule is an antigen and the protein is a single chain antibody. The method according to claim 10 , wherein the antigen is angiotensin II. Process according to claim 10, wherein the antigen is a Lewis X. The production method according to any one of claims 10 to 12, wherein the step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound. The production method according to claim 13, wherein the cell-free translation system is a wheat germ extract, a rabbit reticulocyte extract, or an E. coli S-30 extract. The method according to any one of claims 10 to 12, wherein the step of producing a single-chain antibody comprises transforming a living cell with the selected nucleic acid and expressing the single-chain antibody in the living cell. The step of producing a single chain antibody comprises producing as a fusion protein of a single chain antibody encoded by a selected nucleic acid and an enzyme or green fluorescent protein (GFP). The manufacturing method in any one of. A nucleic acid is selected by the selection method according to any one of claims 1 to 8, and the selected nucleic acid is translated in a cell-free translation system in the presence of a C-terminal labeling agent to label the C-terminus of the protein. how to. A method for producing a single-chain antibody by the production method according to claim 10 and immunologically detecting a protein using the produced single-chain antibody. The detection method according to claim 18, which is a Western blot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, or an immunoprecipitation method. A protein is produced by the production method according to claim 9, the produced protein is brought into contact with a target molecule, and the interaction between the protein and the target molecule is detected. How to detect the effect. The detection method according to claim 20, which is a fluorescence correlation spectroscopy, a fluorescence imaging analyze method, a fluorescence resonance energy transfer method, an evanescent field molecular imaging method, a fluorescence depolarization method, a surface plasmon resonance method, or a solid phase enzyme immunoassay method. . The human or selection method according to claim 2, wherein the DNA library derived from other animals and select single chain antibodies involve replacing the variable regions of the IgG of human single chain antibodies selected, human Alternatively, a method for constructing a human antibody. A method for producing a therapeutic agent, comprising constructing an antibody by the construction method according to claim 22, and formulating the constructed antibody as an active ingredient.

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