CZ109393A3 - Bonded proteins - Google Patents

Abstract

A non-naturally-occurring fusion protein comprising an extension peptide portion covalently linked at its C-terminus to the N-terminus of a biologically active portion is disclosed. The extension peptide portion can be removed by DPP IV cleavage. A use of fusion proteins with DPP IV cleavable extension peptide portions in medecinal preparations is disclosed. A method of purifying desired proteins from a mixture containing a fusion protein is disclosed.

Description

Linked proteins

Technical field

BACKGROUND OF THE INVENTION 1. Field of the Invention

Molecular biology techniques, particularly recombinant DNA technology, allow the production of relatively large amounts of the desired biologically active polypeptides. In addition, the genetic information encoded in the polypeptides can be engineered to produce relatively large amounts of modified polypeptides. Polypeptide modifications are often used to improve their activity or to facilitate their production or preparation. Therefore, many efforts have been made to determine which modifications desirably increase, enhance or otherwise alter the biological activity of the polypeptides of interest. In addition, much work is being done to modify the desired polypeptides to facilitate their production and purification.

Polypeptides produced by natural processes are often biosynthesized from large precursors, which are processed by a series of proteolytic cleavages to give end products. Therefore, there are a number of proteases that recognize and cleave specific amino acids or amino acid chains. These proteases are involved in the conversion of the precursor protein to the final polypeptide product.

One of these proteases is dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5). DPP IV was first introduced in Kopsu-Hav

V.K., Glenner, G. G., Histo. Chemistry 3: 197-201 (1966). It has been shown to be present in many mammalian tissues. Today, DPP IV is commercially available from Enzyme Systems Products (Dublin, California). DPP IV recognizes specific amino acid chains at the N terminus of the protein. Namely, DPP IV separates the dipeptide from the N terminus if the second amino acid from the N terminus is proline (Pro), hydroxyproline (Hyp), alanine (Ala), serine (Ser) and threonine (Thr) and at the N terminus any amino acid, the amino acid residue from the N terminus must not be proline or hydroxyproline. DPP IV is more effective when the second amino acid from the N terminus is proline or alanine, and is usually most effective when proline is at that site. The activity of DPP IV in the progressive cleavage of PRO parts of the precursor of naturally occurring peptides has been extensively documented.

Modern technology allows the production of biologically active proteins at a high level. Important polypeptides can be synthesized using peptide synthesizers or in host cells using recombinant DNA technology. Biologically active proteins are often used as medicaments. There are numerous examples in which active proteins are used as medicaments, prophylactic agents, or to enhance or suppress certain properties. Because DPP IV and other proteases degrade proteins, these drugs are susceptible to degradation. Therefore, it is a problem to use biologically active polypeptides as drugs to reduce their content in tissues, so they need to be administered more frequently. The rapid development of recombinant DNA methodology, which allowed the production of polypeptides, proteins and their analogs in large quantities in a very short time, created the need for highly efficient and reliable isolation of these proteins from complex mixtures in which all proteins are produced in host cells along with proteins of the growth environment. . Purification of a variety of polypeptides produced by host cells can be very expensive and can cause denaturation of the protein product itself. An overview of protein purification techniques is contained in the prior art review of U.S. Patent No. 4,782,137, issued Nov. 1, 1988 to Hopp et al., Which is incorporated herein by reference.

To circumvent the limitation of the technique and to find better methods, recombinant LHA technology can be used to obtain the desired polypeptides in the form of artificial proteins containing a linker peptide that can be used as a ligand or other target for detergent compositions. For example, US Patent No. 4,782,137 describes the synthesis of an artificial peptide comprising an antigenic linker peptide. The artificial protein may be applied to a column containing immobilized antibodies and thus isolated. US Patent No. 4,569,794 discloses the purification of artificial proteins containing N-terminal extensions having affinity for mobilized metals. Artificial proteins bind to immobilized metals on the column. The problem with these methods is that the linker peptide is often undesirable and can be difficult to remove.

The present invention relates to artificial fusion proteins comprising a protein core and an N-terminal extension of a cleavable DPP

IV. According to the present invention, an artificial protein is obtained whose extension attached to the parent protein does not naturally occur as an extension of the N terminus of the parent protein, therefore it is an artificial linked protein. The present invention relates to protein drugs that are DPP IV cleavable artificial proteins whose protein core is a biologically active protein. This invention relates to DPP IV cleavable artificial proteins that are useful for purification methods because N-terminal extension is a characteristic or property facilitating the purification of artificial proteins.

The present invention provides artificial proteins having an N-terminal extension of the cleavable DPP IV, so that exposure to the artificial protein, DPP IV results in the conversion of the artificial protein to the desired protein. When used as a protein drug, the artificial protein is converted to biologically active protein in vivo by the effect of DPP IV present in the target organism. When used in the purification process, artificial proteins can be purified using specifically designed N termini as ligands and then treated with DPP IV to remove the N termini extension and release the desired protein.

The present invention allows the production of the desired proteins as artificial proteins, which are later converted to the desired proteins by exposure to DPP IV. Protein drugs are converted to drugs over time by the patient's own DPP IV, which ensures the continued presence of the active agent in the patient's body and reduces the frequency of administration.

Pure desired proteins can be isolated according to the invention by producing and purifying the artificial proteins and then treating the artificial proteins in vitro with DPP IV to produce the desired protein.

SUMMARY OF THE INVENTION

The present invention relates to an artificial fusion protein comprising an expanding protein moiety covalently linked from its C terminus to the N terminus of a basic protein moiety, the expanding protein moiety having the formula

AXY (X '-Y) _r , where A and either is not, or means methionine, n is 0-20

X is selected from the group consisting of all naturally occurring amino acid residues

X 'is selected from the group consisting of all naturally occurring amino acid residues except proline and hydroxyproline;

Y is selected from the group consisting of proline, hydroxyproline, alanine, serine, and threonine.

The invention also relates to the use of these artificial proteins in medicament formulations and to a method of purifying desired proteins from a composition comprising such artificial proteins and impurities, comprising the steps of selectively contacting said artificial proteins with a material that immobilizes said artificial proteins, removing said impurities, separating said artificial protein from said material, contacting said artificial protein with DPP IV and isolating said desired protein.

U.S. Patent No. 4,569,794, issued February 11, 1986 to Smith et al., Relates to the purification of proteins and substances useful in these methods. The invention provides a method for isolating linked proteins having biologically active polypeptides at the C terminus and a linker at the N terminus that is a metal ion chelating member. The coupled peptide has an affinity for immobilized metal ions. Impurities can be removed by passing the protein-containing mixture through a column containing immobilized metal ions. The coupled protein associates with metal ions and only impurities are washed out. Under altered conditions, the coupled peptide is released from the immobilized metal ions to give a purified coupled protein.

U.S. Patent No. 4,782,137, issued November 1, 1988 to Hopp et al., Describes the synthesis of a fusion protein having a highly antigenic N terminus and a desired polypeptide at the C terminus. According to Hopp et al., The pooled proteins are purified from the crude supernatant by passing the crude supernatant through a column containing immobilized antibodies that distinguish the antigenic portion of the pooled protein. Immobilized antibodies retain the protein on the column while unwanted supernatant components elute. The pooled protein is then washed and concentrated.

U.S. Patent No. 4,734,399, issued March 29, 1988 to Felix et al., Relates to growth hormone releasing factor analogs. A number of analogs having Tyr-Ala and His-Ala end groups are described. However, these molecules are not linked proteins, but rather only core proteins. The terminal dipeptides of Felix aj are part of the core of the bGRF analog molecule.

European Patent Application, Publication Number 0 220 958, published May 6, 1987, relates to the selective chemical removal of residues at the N terminus. The invention relates to a method and compounds useful for removing N-terminal residues from desired polypeptides. The desired polypeptide exists in the form of a fusion protein having the desired polypeptide member at the N terminus to a linker of the formula X-Pro. Upon exposure of the coupled protein to specific buccal conditions, diketopiperazine from the X-Pro member of the coupled protein is formed and released. This produces the desired polypeptide from the fusion protein. The pooled proteins of EPO 220 958 ('958) are not included in the present invention because according to the present invention, when the N-terminal extension is only a dipeptide, that is, when A is absent, n is zero and X is a natural

Y is either alanine, serine or threonine. Thus, whenever the dipeptide is elongated, it is X-Ala, X-Ser or X-Thr. The '958 application describes chemical rather than enzymatic hydrolysis of the dipeptide X-Pro. The dipeptides X-Ala, X-Ser and X-Thr are not susceptible to the type of chemical cleavage described in the '958 application that separates the X-Pro extension from the protein.

The Australian patent application, AU-A-12709/88, discloses coupled peptides useful for affinity metal chromatography (IMAC). The disclosed affinity peptides contained at least two adjacent histidine residues. Purification of the IMAC by the disclosed compositions requires special synthetic chemistry for the preparation of nitrilotriacetic acid (NTA) resins.

Tallon M.A. et al. Biochem. 26: 7767-7774 (1987) relates to the synthesis of extended analogues of the tridecapeptide alpha factor Saccharomyces Cerevisiae. Synthesized analogs are widespread alpha factors that represent natural pro-alpha factor chains encoded in the MF-alpha-1 structural gene.

G. Kreil et al.: Eur. J. Biochem 111, 49-58 (1980) disclose the sequential cleavage of a portion at the N terminus of the melittin precursor (Promittitt) by dipeptides with peptidase IV. Promelittin is a major component of bee venom. In the amino acid chain at the N terminus of the precursor, every second residue is either proline or alanine. When promelittin is exposed to DPP IV isolated from porcine kidney, the N region of the precursor end is cleaved sequentially to produce the mature protein. Promelittin, unlike the fusion proteins of the invention, is a naturally occurring protein.

Julius D. et al. Cell, 32: 839-852 (March 1983) refers to the role of membrane bound DPP IV to produce yeast alpha factor from a larger precursor polypeptide. The yeast alpha factor, unlike the fusion proteins of the invention, is a naturally occurring protein.

C. Mollay et al. J. Biochem., 160, 31-35 (1986) describe the isolation of DPP IV from Xenopus laevis skin secretions. DPP IV activity is discussed.

Mentlein R. FEB, 234: No. 2, 251-256 (July 1988) summarizes the role of proline residues in the maturation and degradation of peptide hormones and neuropeptides. It is reported that in mammals proline-specific proteases, such as DPP IV, are not involved in the biosynthesis of regulatory peptides, but may play an important role in their degradation. Thus, it is noted that while in vertebrates and lower vertebrates, precursor proteins rely on DPP IV to convert precursors to mature forms, processing of regulatory proteins in mammals generally uses DPP IV as a degradation protease.

Frohman, L.A. aj .: J.Clin. Invest. 78, 906-913 (1986) report that human growth hormone releasing factor (hGRF) and its analogs rapidly degenerate in vivo in man and in vitro by the effect of plasma DPP IV.

Frohman et al. J. Clin. Invest. 83, 1533-1540 (1989) report that human growth hormone releasing factor (hGRF) and its analogs rapidly degenerate in vivo in human and in vitro plasma DPP IV.

Kubiak T..M. aj. : Drug Metabolism and Disposition, 17, No. 4, 393-397 (1989) report on the metabolic degradation of in vitro bovine growth hormone releasing factor (bGRF) analogues in bovine and porcine plasma and its correlation with plasma DPP IV activity. The bGRF analogs tested had an Ala residue at position 2- from the N terminus. Metabolic degradation (bGRF) in plasma is reported to be induced by the presence of plasma DPP IV.

Hong W. et al. Biochemistry, 28: 8474-8479 (1989) discloses the term for enzymatically active DPP IV in Chinese hamster ovary cells after transfection.

Kreil G. TIBS 15: 23-26 (January 1990) summarizes the gradual cleavage of dipeptides by DPP in converting precursors to end products. The precursors described by Kreil are naturally occurring proteins. The coupled proteins of the invention are artificial proteins.

Boman et al. J.Biol. Chem. 264: 5852-5860 (1989) showed that dipeptidyl peptidase isolated from cecropia pupae (with similar specificity to DPP IV) was able to remove the natural chains at the N-terminus of Ala-Pro-Glu-Pro from the N-terminus of synthetic copies of natural precursors of cecropia A and B. Preprocecropin described by Boman is a naturally occurring protein.

Dalboge H. et al. Bio / Technology 5: 161-164 (February 1987) describe the conversion of in vitro human growth hormone (hGH) precursors produced by E. coli to authentic hGH. The terminal extension of the precursors is removed by dipeptidyl peptidase I.

Dalboge H. et al FEBS, 246 (1,2): 89-93 (March 1989) describe the cloning and expression of IL-β precursors and their conversion to IL-β by removing the spread of precursors at the N terminus of dipeptide 1 by peptidase I.

Dalboge H. et al. FEBS, 266 (1,2): 1-3 (June 1990) disclose in vivo treatment of N terminal methionine in E. coli. It is reported that the removal of the N terminal methionine from expanded human growth hormone was dependent on the amino acid adjacent to methionine.

Hopp T.P. aj. Bio / Technology 6: 1204-1210 (October 1988) describe the attachment of an eight amino acid peptide to the N terminus of a recombinant lymphokine to create an antigenic N terminus that could be used for immunoaffinity purification. This publication corresponds. U.S. Patent No. 4,782,137 described above.

Smith M.C. J.Biol. Chem. Vol. 263,15: 7211-7215 (1988) describe experimental results supporting the hypothesis that selective metal chelating peptides at the NH2 end of a small peptide can be used to purify this protein by ion affinity chromatography. This reference discloses specific data relating to one example of the above-described US Patent No. 4,569,794. Namely, the use of a His-Trp metal chelating peptide coupled to either the luteinizing hormone releasing hormone or proinsulin allows the chimeric peptide to be purified by IMAC, whereas controlled molecules lacking the His-Trp member cannot be obtained in a similar manner.

Hochuli E. et al. Chromato. 411: 177-184 (1987) described an absorbent with nitrilotriacetic acid useful for metal ion affinity chromatography. It is reported that the absorbent described, when saturated with Ni 2+, is useful for binding peptides and proteins containing contiguous histidine residues.

Ljuhgquist C. et al. J. Biochem. 186: 563-569 (1989) describe the use of Ala-His-Gly-HisArg-Pro metal chelating peptide in two, four and eightfold multiplicities together with a column containing immobilized Zn- ions. According to Ljungquist, the use of this metal chelating peptide is zinc-coated. unexpectedly good purification of the pooled protein.

As used herein, the terms artificial fusion protein, artificial fusion polypeptide, fusion proteins, and fusion polypeptides refer interchangeably to non-naturally occurring proteins and polypeptides that comprise the core portion of the protein and the extension portion.

As used herein, the term core protein and core portion of a protein refer to a portion of a linked polypeptide that is located at the C terminus of the molecule and which, without the extending portion, would be the desired polypeptide and / or biologically active protein, including naturally occurring biologically active proteins, polypeptides, and analogs and mutants thereof.

As used herein, the term N-terminal extension refers to the first of up to about 45 amino acids beginning at the N terminus that are not part of the core protein.

As used herein, the term protein drug refers to coupled proteins whose biologically active protein is useful as a drug.

As used herein, the terms biologically active protein and biologically active polypeptides refer interchangeably to proteins and polypeptides having biological activity.

As used herein, the terms desirable protein and desirable polypeptide refer interchangeably to proteins and polypeptides to be obtained in pure form.

As used herein, the term extension portion refers to a portion of a fusion protein that is an N-terminal extension and which is not part of a biologically desirable portion.

As used herein, the term extending portion

N-terminal cleavable DPP IV refers to an extension portion of a fusion protein having an amino acid chain that can be removed by sequential digestion with DPP IV.

In the Chain Description, some amino acid residues are designated Xaa in Seq ID. The following description applies to them:

IN Seq ID No. 3 Xaa ² * represents amidated arginyl residue at the C end. IN Seq ID No. 4 Xaa29 represents amidated arginyl residue at the C end. IN Seq ID No. 5 Xaa ²⁹ represents amidated arginyl residue at the C end. IN residue Seq ID No. at the C end. 14 ⁹ represents amidated arginyl IN Seq ID No. 18 Xaa 31 represents amidated arginyl residue at the C end. IN Seq ID No. 19 Dec Xaa ^{3 3} represents amidated arginyl residue to C conc. IN Seq ID No. 20 May Xaa ^{3 9} represents amidated arginyl residue at the C end. IN residue Seq ID No. at the C end. 21 Xaa ^{4 5} represents amidated arginy1 IN Seq ID No. 24 Xaa ^{2 7} represents amidated arginy1 residue at the C end. IN Seq ID No. 25 Xaa ^{3 3} represents amidated arginyl residue at the C end. IN Seq ID No. 26 Xaa ^{3 3} represents amidated arginyl residue at the C end. IN residue Seq ID No. to C conc. 27 Mar: Xaa ^{3 5} represents amidated arginyl

⁷ represents an amidated arginyl

IN Seq ID No. 28 Xaa residue to C conc. IN Seq ID No. 29 Xaa residue at the C end. IN Seq ID No. 30 Xaa residue at the C end. IN residue Seq ID No. at the C end. 31 Xaa IN Seq ID No. 32 Xaa residue at the C end. IN Seq ID No. 33 Xaa residue at the C end. IN Seq ID No. 34 Xaa residue at the C end. IN residue Seq ID No. at the C end. 35 Xaa IN Seq ID No. 36 Xaa residue at the C end. IN Seq ID No. 37 Xaa residue at the C end. IN Seq ID No. 38 Xaa residue at the C end. IN residue Seq ID No. at the C end. 39 Xaa IN Seq ID No. 40 Xaa residue at the C end. IN Seq ID No. 41 Xaa residue at the C end. IN Seq ID No. 42 Xaa

C-terminal residue represents amidated arginyl ⁵ represents amidated arginyl ⁷ represents amidated arginyl ⁹ represents amidated arginyl s represents amidated arginyl represents amidated arginyl ⁵ represents amidated arginyl ¹ represents amidated arginyl ¹ represents amidated arginyl ¹ represents amidated arginyl i represents amidated arginyl ³ represents an amidated arginyl ¹ represents an amidated arginyl

In Seq ID No. Xaa ³³ represents an amidated C-terminal arginyl residue.

The present invention relates to improved proteins and polypeptides. According to the invention, biologically active polypeptides are first prepared as coupled proteins comprising two portions: a first portion representing the core portion of the protein and a second portion, the N-terminal extension, which is covalently linked at its carboxyl end to the amino terminus of the first part. The N-terminal portion of the fused polypeptide has an amino acid sequence that makes it sensitive to cleavage by dipeptidyl peptidase IV (DPP IV).

The fusion protein of the present invention has the formula: extension portion-core portion of the protein wherein the extension portion represents the N-terminal extension of DPP IV cleavable, represents a covalent peptide bond, and the core portion of the protein represents the desired peptide that is released from the extension by DPP IV processing.

The extending portion of the fusion protein of the invention has an amino acid sequence of the following formula

AXY (X'-Y) _n where A and either is not, or represents methionine, n represents the number of linearly linked (X'-Y) groups.

This number is from 0 to 20 such groups, preferably 0 to 10 groups.

X is selected from the group consisting of all naturally occurring amino acids,

Y is selected from the group consisting of proline, hydroxyproline, alanine, serine, and threonine, except when n = 0, then Y is selected from the group consisting of ala15 nine, serine, and threonine.

X 'is selected from the group consisting of all naturally occurring amino acids except proline and hydroxyproline;

According to this formula, when n = 1, there are two Y residues. Further, it is possible to have up to twenty-one Y residues and twenty-X residues in one embodiment. The individual Y and X 'moieties may be any moiety from the group from which they are selected. That is, the individual residues Y need not be identical in the embodiment. Similarly, in an embodiment with more than one X 'residue, each individual X' residue present may be any amino acid except proline and hydroxyproline, no matter what residue is each other X 'residue. Each individual Y and X 'residue must conform to the rules for that particular group and all that is necessary is that the different individual residues at specific positions comply with the rules defined above.

The coupled proteins in which (A) is present as methionine (Met) represent chains useful for producing biologically active proteins using recombinant DNA techniques in E. coli. Typically, the Met chain present in these precursors is processed by the E. coli enzymatic system or by some other means that can be used by those of ordinary skill in the art. Protein synthesis in E. coli is normally initiated by the translation initiation codon AUG, encoding pro. Met. As a result, the newly synthesized polypeptides have a methionine residue as their N terminal amino acid. E. coli has an enzymatic activity capable of effectively removing Met at the N terminus when the Met residue at the N terminus 16i is adjacent to an amino acid with a relatively small branch chain such as Gly, Ala or Ser as well as Pro. Highly specific removal of Met from the N-terminus is possible with Met cyanogen bromide. However, for this procedure to be successful, the Met at the N terminus must be the only Met in the entire protein chain, otherwise it will be cleaved after each Met chain. Therefore, for linked proteins containing internal Met chains, the second amino acid from the N terminus must be Pro, Gly, Ala or Ser if Met is to be removed by the E. coli enzymatic system.

In addition to linked polypeptides. The invention relates to molecules having recombinant DNA strands encoding linked polypeptides, methods of using recombinant DNA, methods of using linked polypeptides, including methods of purifying desired polypeptides, and methods of administering drugs involving the use of protein drugs that are converted from precursors to biologically active forms by sequential proteolytic cleavage of the N-terminal extension in vivo.

Production of linked polypeptides can be accomplished by standard peptide synthesis or by using recombinant DNA techniques, both methods are well known to those of skill in the art. Peptide synthesis is the preferred method for preparing polypeptides that contain less than 50 amino acids. For larger molecules, it is preferred to use the recombinant DNA technique in host cells.

Fused polypeptides having extension. The N termini recognized and cleaved by DPP IV are more useful and advantageous than unmodified polypeptides having only a protein core. The present invention describes two areas of particular utility. The first provides coupled polypeptides called proteinaceous drugs that include biologically active polypeptides useful as drugs covalently associated with the N-terminal extension of cleavable DPP IV. These prodrugs can be converted to biologically active forms by cleavage of DPP IV in the human or animal body into which they have been administered as a protein drug. Thus, the invention relates to fused polypeptides useful as protein drugs, to the use of fused polypeptides in medical preparations, and to a method of administering biologically active polypeptides to a patient. A second use of the linked polypeptides of the invention is protein purification, wherein the N-terminal extension is a component of the polypeptides that allows purification thereof and a component removable by DPP IV cleavage. Thus, the present invention relates to linked polypeptides useful for purification processes and to a method of purifying desired polypeptides. This serves as examples illustrating the usefulness of the invention and is not intended to limit the invention in any way.

In both uses, the core protein portion of the fusion protein is released from the extension portion by DPP IV activities. In the case of pooled proteins used for purification methods, it is undesirable that the nuclear proteins are substrates for digestion by DPP IV. That is, it is preferred that DPP IV is unable to cleave the nuclear protein after cleavage of the extension portion. It is often highly desirable that when a nuclear protein is a substrate for DPP IV, it is administered in the form of a protein drug. In such cases, the protein drug can prolong the life of the nuclear protein because the in vivo portion of DPP IV (i.e., in plasma, and kidney and liver tissue) will be used to process the end-to-end and thus delay protein degradation. That is, the N-terminal extension may act as a substrate for DPP IV and a competitive inhibitor, delaying temporarily the action of DPP IV on the nuclear protein and thereby temporarily protecting it.

As used herein, a protein drug means a fusion protein comprising an N-terminal extension portion of a cleavable DPP IV and covalently bound to a nuclear protein that is a biologically active polypeptide useful as a drug. The protein drugs of the invention may be administered as individual formulations or in combination with other compounds. A preferred embodiment is a well defined individual form of a protein drug. In all cases protein. drugs are processed by naturally occurring DPP IV, normally found in the body.

The advantage of administering a protein drug in medical preparations is that it slows down the activity or ensures the prolonged presence of the biologically active protein. Thus, protein drugs may remain active longer than unmodified molecules. Protein drugs may exist in an inactive state until such time that a sufficient portion of the extension portion is degraded and the molecule becomes active. Protein drugs can therefore act as delayed drug release systems. In addition, different N-terminal extensions degrade at different rates, depending on their length and specific residues present in their amino acid chain. Combinations of different forms of protein drugs can be formed, having multiple N-terminal extensions that can provide a sustained, stable level of active drug in the patient for a long time. Thus, protein drugs can act as delayed drug release systems.

As described above, DPP IV cleaves a dipeptide from the N terminus of polypeptides when certain residues occupy certain positions. As used herein, position one refers to the position of the amino acid residue at the N terminus. As used herein, position two refers to the position of the amino acid residue immediately adjacent to position one and which is the other from the N terminus. As used herein, position three refers to the position of the amino acid residue immediately adjacent to position two and which is the third from the N terminus. Cleavage that removes the dipeptide from the N terminus occurs between position two and position three if amino acid three is not proline or hydroxyproline and amino acid two is proline (Pro), hydroxyproline (Hyp), alanine (Ala), serine (Ser) and threonine ( Thr).

DPP IV cleaves residues at the N terminus at different rates, depending on which of the four amino acids is at position two. In most cases, DPP IV cleaves most efficiently when position two is occupied by Pro, and further most effective when position two is occupied by Ala. If position one is occupied by tyrosine, phenylalanine or histidine, DPP IV operates at approximately the same speed when position two is occupied by Pro or Ala. DPP IV is then most effective when position two is occupied by Ser. It is least effective if position two is occupied by Thr.

Using this information, various N-terminal extensions can be designed that will cleave at different speeds. Thus, preparations containing either a specific protein drug or a combination of forms of protein drugs can be administered. Protein drugs having a N-terminal extension fan will be processed at a rate that will depend on their amino acid chain. A combination of protein drugs can be formulated to contain a series of protein drugs that will be processed into active peptides over a period of time.

Amino acid chain length and modification are controlling factors in DPP IV cleavage rates. Extensions containing all or most alternating Y = Pro will be processed the fastest, while extensions containing Y - Thr will be converted the slowest. It is further known that the dipeptide units X-Pro, where X is either Glu or Asp, cleave much more slowly than their analogs, where X is a neutral or basic amino acid residue. Because extensions can contain different residues (of four) at each extension position, there can be an extremely large number of variations and permutations.

Any biologically active polypeptide can be used as a polypeptide drug. PCT Patent Application No. PCT / US90 / 02923, incorporated herein by reference, PCT Patent Application No. PCT / US91 / 08248, incorporated herein by reference, and US Patent Application Serial No. 07 / 368,231, incorporated herein by reference, and each description of bovine release factor analogs. growth hormone, which can be used in the medical composition as a protein drug of the invention. Any analog described in those applications can be used as the nuclear peptide portion of the fusion protein of the invention. Bound proteins containing such nuclear protein moieties linked to the extension moieties can be prepared by anyone of ordinary skill in the art using well known methods.

Other exemplary embodiments of the invention include hormones, receptors, enzymes, storage proteins, and blood proteins. Specific examples include: vasoactive intestinal peptide (VIP), human β-casomorphine, gastric inhibitory peptide (GIP), gastric releasing peptide (GRP), human HI peptide, human YY peptide, 7-37 glucagon-like peptide 1 fragment, glucagon-like peptide 2, substance P, neuropeptide Y, human pancreatic polypeptide, insulin-like growth factor-1 (IGF-1), human growth hormone (hGH), bovine growth hormone (bGH), pig growth hormone (pGH), prolactin (PRL), human, bovine or porcine growth hormone releasing factor (GRF), interleukin-β (IL-β), EGF, IGF-2, glucagon, corticotropin releasing factor (CRF), dynorphin, somatostatin-14, endothelin, growth transforming factor and ( TGF a), growth transforming factor β (TGF β), interleukin-4, interleukin-6, nerve growth factor (NGF), tumor necrosis factor (TNF), insulin, fibroblast growth factor (FGF), interferon, CD4 and interleukin- 2 (IL-2) or is their synthetic or biosynthetic analogs. These polypeptides can also be used to form the core portion of the fusion proteins of the invention. These polypeptides are intended to serve as examples only and are not intended to limit the scope of the invention.

Smaller coupled proteins of the invention can be synthesized using, for example, solid phase methodology using an Applied Biosvstems 430A peptide synthesizer (Applied Biosystems, Foster City, California) as described in detail in PCT / US90 / 02923 and 07 / 368,231.

For larger molecules, it is preferred to use recombinant DNA in host cells. Here, several methods are available to those skilled in the art who wish to use the recombinant DNA technique to prepare linked proteins. Generally, genes encoding polypeptides are inserted into expression vectors, which are then used to transform or transfect suitable host cells. The inserted gene is then expressed in a host cell and the desired polypeptide is synthesized. To produce the fusion proteins of the invention in a similar manner, an additional DNA strand is inserted into the gene. DNA encoding N-terminal extension residues is specifically linked to the 5 'end of the gene encoding the polypeptide of interest. This additional genetic material must be placed downstream of the promoter of the expression vector so as to be under the control of the promoter. In addition, it must be positioned in a suitable reading frame relative to the gene, such that the expressed protein product comprises N-terminal extension residues covalently linked to the desired polypeptide.

Thus, in order to obtain the fusion proteins of the invention using recombinant DNA techniques, it is necessary to design oligonucleotides that encode the amino acid chains of the desired N-terminal extension and these oligonucleotides must be capable of upstream from the 5 'end of the gene encoding the nuclear protein portion. . Oligonucleotide production techniques and techniques used to produce chimeric genes are well known to those of skill in the art.

In addition to the usefulness of coupled proteins as protein drugs, the present invention relates to the purification and processing of biologically active recombinant polypeptides. Desired biologically active recombinant polypeptides are most preferably prepared in soluble form or secreted from the host. According to the present invention, the expanding portion of the fusion protein can be recognized by detergents. The pooled protein is purified from the material present in the secretion medium or extraction solution and then processed to separate the extension from the core portion of the protein to prepare the desired protein. Therefore, the proteins of interest most useful in the present invention are biologically active polypeptides that are not themselves substrates for cleavage of DPP IV.

In accordance with the present invention, the gene sequence encoding the protein of interest is isolated, synthesized or otherwise obtained and operably linked to a DNA sequence encoding an extension portion. A hybrid gene comprising a gene for a protein of interest operably linked to a DNA sequence encoding an extension portion is termed a chimeric gene.

Methods and materials for preparing chimeric genes and recombinant vectors, transforming or transfecting host cells, replicating vectors in host cells, and expressing biologically active foreign polypeptides and proteins are described in Principles of Gene Manipulation, Old and Primrose, 2nd edition, 1981 and in Sambrook et al. .: Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, NY (1989). Both are incorporated herein by reference.

The present invention relates to recombinant chimeric genes that encode linked proteins, expression vectors of the same function, hosts transformed or transfected with these expression vectors, and methods of obtaining such genes, expression vectors and hosts transformed or transfected with said vectors.

The present invention can be used to purify any prokaryotic or eukaryotic protein that can be expressed as a product of recombinant DNA technology in transformed or transfected host cells. These recombinant protein products include hormones, receptors, enzymes, storage proteins, blood proteins and mutant proteins made by protein engineering techniques, or synthetic proteins. The desired polypeptides produced may include HIV RNase H, tPA, IL-1, IL-1 receptor, CD 4, human nerve growth factor, SCD4-PE40, human respiratory syncytic virus (RSV) chimeric glycoprotein (see US patent application serial number 07 / 543,780, incorporated herein by reference, EGF, IGF-1, IGF-2, glucagon, corticotropin releasing factor (CRF), dynorphin, endothelin, growth transforming factor α (TGF α), Pseudomonus endotoxin 40 (PE40), growth transforming factor β (TGF β), insulin and analogues thereof.

Examples of detergent compositions include IMAC and immunoaffinity. Other detergents that utilize DPP IV removable extended peptides are also within the envisaged scope of the invention.

In one embodiment of the invention, fusion proteins comprising a biologically active polypeptide and an extension that is a metal chelating peptide are useful in immobilized metal affinity chromatography systems.

Immobilized metal affinity chromatography (IMAC) for protein fractionation was first described by Porath et al., Nature 258: 598-599 (1975). Porath described the resin treatment with iminodiacetic acid (IDA) and chelating metal ions to a resin modified with IDA. Porath described that proteins can be immobilized on a column containing immobilized metal ions. This requires the attachment of commonly used iminodiacetic acid (IDA) to a matrix followed by chelating metal ions, to the resin modified by IDA. Proteins bind to the metal ion (s) through functional groups of amino acid residues capable of giving electrons. Potential electron donors from amino acid residues are cysteine, histidines and tryptophan. Proteins react with metal ions via one or more of these amino acids with electrodonor branch chains.

Smith et al., In U.S. Patent No. 4,569,794, incorporated herein by reference, disclose that certain amino acid residues are responsible for binding the protein to immobilized metal ions. Bound protein, however, can be eluted by lowering the pH or by using competitive anti-ligands such as imidazole when they are involved in histidine side chain binding. Histidine-containing di- or tripeptides have been used to demonstrate that IMAC is a selective purification technique. Accordingly, Smith et al. Disclose the use of recombinant DNA techniques to produce a fusion protein comprising a metal chelating peptide covalently linked to a polypeptide of interest. The metal chelating peptide is an expanding moiety that is an effective manipulation lever for the desired polypeptide. This manipulation lever can be used to purify the protein.

The use of IMAC technology with metal chelating peptides having alternating His residues is described in U.S. Patent Application Serial No. 07 / 506,605, incorporated herein by reference. U.S. Patent Application Serial No. 07 / 506,605 discloses specific metal chelating peptides that give unexpectedly excellent results in IMAC purification of the coupled protein when the metal chelating peptide contains three to six alternating His residues. According to the teachings of US Patent Application Serial No. 07 / 506,605 and US Patent No. 4,569,794, it is possible to use the commonly used IDA resin in IMAC for purification of fusion proteins having a metal chelating peptide moiety with at least three alternating histidine residues forming DPP IV recognizable chains. Construction of fusion proteins and use in the IMAC system is the subject of US Patent No. 4,569,794. The construction and use of a metal chelating peptide moiety with alternating His residues are explained in U.S. Patent Application Serial No. 07 / 506,605. By creating a fusion protein with residues recognizable by DPP IV among alternating His residues, the present invention provides a fusion protein that can be purified by IMAC technology and subsequently processed by DPP IV to the desired polypeptide.

According to this embodiment of the present invention, the extension portion is a metal chelating peptide which may be represented by the formula

AXY (X'-Y) _n wherein A and either is not or represents methionine, n represents the number of serially linked X'-Y groups, this number being from 0 to 20 such groups, preferably 0 to 10 groups.

X is selected from the group consisting of all naturally occurring amino acids

Y is selected from the group consisting of proline, alanine, serine, and threonine, except when n = 0, then Y is selected from the group consisting of alanine, serine, and threonine.

X 'is selected from the group consisting of all naturally occurring amino acid residues except proline and hydroxyproline, wherein at least two to three residues designated X' and optionally X are histidines (His). Preferably, Y is Pro and n is at least 3. By the effect of DPP IV, the N-terminal extension is progressively cleaved to form a biologically active polypeptide, although the biologically active polypeptide is not itself a substrate of DPP IV.

One example of a fusion protein has a spacer portion of the formula

His-Y (His-Y) _n wherein n = 3 to 8 and Y are Pro, Hyp, Ala, Ser or Thr, most preferably Pro. Another example of a fusion protein has an expanding portion of the formula

XY (His-Y) _n wherein n = 3 to 8, X is any naturally occurring amino acid and Y is Pro, Hyp, Ala, Ser or Thr, most preferably Pro.

Because the Met at the N terminus is due to protein synthesis in E. Coli and is known to be processed by the E. Coli enzymatic system when the adjacent amino acid is Pro, Gly, Ala or Ser, the following extension represents chains useful for IMAC purification and cleavage of biologically active peptides or proteins expressed within E. Coli cells by recombinant DNA techniques

Met-XY (His-Y) _n wherein n = 3 to 8, Y is Pro, Gly, Ser or Ala and Y is Pro, Hyp, Ala, Ser or Thr, most preferably Pro.

In another example, if a desired peptide or protein is secreted from a given host after transformation or transfection, the vectors may be designed to secrete the protein or polypeptide using an expanding moiety that facilitates transport, such as

X-Y (His-Y) n wherein n = 3 to 8, X may be any naturally occurring amino acid compatible with the secretion system of a given host and Y is Pro, Hyp, Ala, Ser or Thr.

Another protein purification system that uses fusion proteins and that is well adaptable to DPP IV processing technology is immunoaffinity purification. U.S. Patent No. 4,782,137, issued November 1, 1988 to Hopp et al., Incorporated herein by reference, describes the synthesis of a fusion protein having a highly antigenic N terminal portion and a polypeptide of interest in the C terminal portion. According to Hopp et al., The coupled proteins are purified from the crude supernatant by passing through a column containing immobilized antibodies that recognize the antigenic portion of the coupled protein. Immobilized antibodies retain the protein in the column while unwanted supernatant components elute. The column conditions can then be changed to dissociate the antigen-antibody complex.

According to the present invention, the highly antigenic N terminal portion of the fusion protein is an extension portion that contains residues recognizable by DPP IV. After collection of the fusion protein described in the Hopp ' s patent, the protein is exposed to DPP IV to remove the extension. Those of ordinary skill in the art can operate the Hopp immunoaffinity purification system with the extension portion of the invention at the N terminus.

The embodiments and examples described herein are intended to illustrate the invention and are not intended to limit the scope of the invention. Contemplated equivalents include pooled proteins having an N-terminal extension capable of processing by at least one of the other means so that removal of the extension portion is achieved by a combination of means. Contemplated equivalents also include fused proteins having chemically modified amino acid residues.

Examples

Example 1

Synthetic Protein Drugs that are associated pr-eine drugs having nuclear proteins that are substrates of DPI ’

IV.

The coupled polypeptides, which can be synthesized and administered as protein drugs, have an N-terminal extension of the cleaved DPP IV covalently linked to the N-terminus of the biologically active polypeptide. The structure of these protein drugs can be expressed by the formula:

expanding moiety-core portion of the protein wherein the expanding moiety means expanding the N terminus cleaves by DPP IV, represents a covalent peptide bond, and the nucleus of the protein is the desired peptide that is released from the expanding moiety by Opr IV processing. a DPP IV potential substrate resulting from the removal of the extension portion.

Synthetic protein drugs can be prepared by peptide synthesis techniques well known in the art.

In one embodiment, the core protein moiety is t p; a 'and the growth factor (EGF) and the extension moiety is Gly-Pro-Phe-Ala:

Gly ⁴ -Pro ³ -Phe ² -Ala ^1- (EGF)

In another embodiment, the core protein moiety is a glucagon and the expanding moiety is Ala-Pro-Phe-Ala:

Ala ⁴ -Pro ³ -Phe ² -Ala ¹ - (GLUCAGON)

In another embodiment, the nuclear protein moiety is (Ala ¹⁵ Leu ² β) -bGRF (1-29) NH ₂ (Seq ID 3) and the extension moiety is Tyr-Ala

Tyr ² -Ala ^1- (Ala ¹⁵ Leu ²⁷ ) bGRF (1-29) NH ₂

Example 2

Synthetic protein drugs that are fusion protein drugs having nuclear proteins that are not substrates of DPP IV.

The coupled polypeptides that can be synthesized and administered as protein drugs have an N-terminal extension of the cleavable DPP IV covalently linked to the N-terminus of the biologically active polypeptide. The structure of these protein drugs can be expressed by the formula:

expanding moiety-core portion of the protein wherein the expanding moiety means extending the N terminus of the cleavable DPP IV, represents a covalent peptide bond, and the core portion of the protein is the desired peptide that is released from the expanding moiety by DPP IV processing. In this example, the core protein of the fusion protein is a potential substrate of DPP IV resulting from the removal of the extension.

Synthetic protein drugs can be prepared by peptide synthesis techniques well known in the art.

In one embodiment, the core protein portion is a bGRF analog, ⁽² Val, Ser ^8.28, Leu ²⁷⁾ bGRF (1-33) 0H (Seq ID 1) and the extension portion is Gly-Pro-Phe-Ala:

Gly4-Pro ³ -Phe ² -Alai - ((Val ² , Ser ⁸ , ² sec, Leu ^{2 7} ) bGRF (1-3 3) OH)

In another embodiment, the core protein moiety is corticotropin releasing factor (CRF) and the expanding moiety is Gly-Pro-Phe-Ala:

Gly4-Pro3-Phe ² -Ala ² (CRF)

In another embodiment, the core protein moiety is dynorphin and the expanding moiety is Phe-Pro-Phe-Ala:

Phe * -Pro 3-Phe ² -Ala1- (DYNORFIN)

In another embodiment, the core protein moiety is somatostatin-28 and the expanding moiety is Gly-Pro-Phe-Pro:

Gly 4-Pro ³ -Phe ² -Pro ^3- (SOMATOSTATIN-28)

In another embodiment, the nuclear protein moiety is endothelin and the expanding moiety is Ala-Pro-Phe-Ala:

A1a- * -Pro ³ -Phe ² -A1a1- (ENDOTHELIN)

In another embodiment, the core protein portion is a bGRF analog (Ile Ser ^{and 2} - ²⁸ Ala ¹³ Leu ²⁷ bGRF (l-40) 0H (Seq ID 2) and the extension portion is Phe-Ala:

Phe ² -Ala ^1- (Ile ² Ser ⁸ » ²⁸ Alal ^and Leu ²⁷ bGRF (1-40) OH

In another embodiment, the core protein moiety is (IIe ² Ala ¹⁵ Leu ²⁷ bGRF (1-29) NH ₂ (Seq ID 4) and the extension portion is Tyr-Ser:

Tyr ² -Ser ^1- (Ile ² Ala ^{1 and} Leu ^{2 7} bGRF (1-29) NH ₂₎

Example 3

Extended presence of bGRF analogue Leu ²⁷ bGRF (1-29) NH ₂

The bGRF Leu ¹⁷ bGRF (1-29) NH 2 analogue, whose chain is shown as Seq ID 5, can be administered as a drug containing the nuclear protein shown as Seq ID 5 and a variety of N-terminal protein drugs.

A variety of protein drugs can be prepared using well known methods using Seq ID 5 as the core protein moiety. Expansion portions of protein drugs based on Seq ID 5 are Phe-Ala, Gly-Pro-Ile-Pro, Seq ID 6, Seq ID 7, Tyr-Ala, Gly-Pro-Tyr-Ala, Seq ID 8, Seq ID 9, Seq ID 10, Seq ID 11, Seq ID 12, Seq ID 13, Tyr-Ala-Tyr-Ala, and Val-Ala.

Example 4

Prolonged presence of bGRF analogue (Thr2Ala * -'Leu ^{2 7} ) bGRF (1-29) NH ₂

An analog of bGRF (Thr? AlaiSLeu? 7) bGRF (1-29) NH ₂ , whose chain is shown as Seq ID 14, can be administered as a drug containing the nuclear protein shown as Seq ID 14 and a series of N-terminal protein drugs. Three versions of protein drugs with Tyr-Thr, Tyr-Ser and Tyr-Thr-Tyr-Thr extensions were prepared.

Example 5

Linked proteins containing HIV RNase H and spread

N ends

Disclosed is a strategy for purifying chimeric proteins from recombinant E. coli based on metal chelating peptide domains containing alternating histidines with affinity for immobilized metal ions. Vectors are constructed to direct synthesis of fusion proteins using HIV RNase H as a nuclear protein. As shown below, these fusion proteins are designed to have alternating histidines for purification by affinity chromatography to immobilized metal ions (IMAC) and alternating proline or alternating alanines for digestion by DPP IV to remove the metal chelate peptide33 (mcp). ).

The N-terminal extension of the cleavable DPP IV, which is preferred according to the invention, is developed as follows:

The linked HIVRH / mcp protein # 1 contains as extension of the N terminus Seq ID 15 linked to HIV RNase H:

Met ¹¹ -Pro ¹ ° -Ala ⁹ -His ³ -Pro ⁷ -Pro ³ -His ⁵ -His ⁴ -Pro ³ -His ² -Ala ¹ (HIV RNase H)

The linked HIVRH / mcp protein # 2 contains as extension of the N terminus Seq ID 16 linked to HIV RNase H:

Met ¹¹ -A1a ¹⁰ o-Pro ⁹ -His ³ -A1a ⁷ -His ³ -A1a ³ -His ⁴ -A1a ³ -His ² -A1 a ¹ (HIV RNase H)

The linked HIVRH / mcp protein # 3 contains as extension of the N terminus Seq ID 17 linked to HIV RNase H:

Met ¹¹ -Gly ¹⁰ -Pro ⁹ -His ³ -Pro ⁷ -Pro ³ -His ⁵ -His ⁴ -Pro ³ -Hi with ² -Ala ¹ (HIV RNase H).

These coupled proteins are cloned and expressed in E. Coli and purified by DEAE chromatography and RP-HPLC. N-terminal chaining is used to characterize linked proteins. Application of fusion proteins containing alternating histidines to the purification of recombinant proteins by IMAC and subsequent removal of the N-terminal extension of DPP IV confirms the usefulness of the invention.

Construction of chimeric genes containing the HIV RNase H gene

All recombinant DNA is prepared by standard techniques. The oligonucleotides responsible for the chelating metals / cleavable chains are prepared, purified, cooled, and ligated to the gene encoding IV RNase H to generate chimeric genes.

To prepare expression vectors encoding HIV residues

RNase H, containing alternating histidines and both recognizable and cleavable DPP IV, is inserted into the final expression vector. Expression vectors containing chimeric gene constructs are used to transform E. coli using standard techniques. Expression of genes in E. coli leads to production of linked proteins encoded by chimeric genes. These linked proteins comprise the amino acids HIV RNase H and the N-terminal extension, containing alternating histidines (metal chelating peptide) and alternating proline or alanine.

Preparation of crude E. coli extracts and isolation of coupled proteins for chaining.

About 3 g of E. coli cell paste is suspended in 30 ml of 0.25 mol potassium phosphate, pH 7.2, containing 1 mmol dithiothreitol (DTT), EDTA, phenylmethylsulfonyl fluoride (PMSF) and benzamidine hydrochloride, 10 mg / l aprotinin, leupeptin and bestatin. This suspension was passed through a French press three times to break the cells. The cell lysate was centrifuged at 12,000 rpm for 1 hour. The slurry water was separated and solid ammonium sulfate was added to 70% saturation. After stirring for 1 hour, the suspension was centrifuged at 12,000 rpm for 1 hour. The sludge water was discarded and the cake was redissolved in 2.25 ml of 50 mmol Tris, pH 7.5, 1 mmol of DTT, PMSF and benzamidine. The solution was then dialyzed overnight in 20 mmol Tris, 50 mmol NaCl, 1 mmol DTT, 10% glycerol and 0.1 mmol EDTA, pH 7.5 (buffer A), at 4 ° C. The dialysate was collected, diluted with one volume of buffer A, and loaded onto a 10 ml column of washed DEAE cellulose leveled with buffer A. The flow was collected in portions and the column was further washed with 50 ml buffer A. These solutions were collected, pooled and concentrated by precipitation with 70% sulfate. ammonium chloride and resuspended in 2 ml of buffer A and dialyzed as described above. Concentrated RH analysis is used to characterize the chain at the N terminus.

Purification of pooled proteins by IMAC

The suitability of using a metal chelating peptide to purify recombinant proteins from crude extracts can be demonstrated using the following chimeras expressed in recombinant E. coli with HIV RNase H as a model protein. Purified HIV RNase H / mcp # 1 coupled proteins, HIV RNase H / mcp # 2, HIVRNase H / mcp # 3 purified.

The IMAC column is prepared as follows: The chelating Sefarose Fast Flow from Pharmacia is thoroughly washed with Milli-Q water on a sintered glass filter. The gel is then resuspended in water to form a suspension. The suspension is poured carefully into a glass column (Pharmacia) to a volume of 6 ml (1 * 7 cm). After settling the gel, the column is washed with 5 volumes of 50 mmol EDTA (ethylenediaminotetraacetic acid), pH 8.0. Then the column is washed with 5 volumes of 0.2 N NaOH and 5 volumes of Milli-Q water. The column was then saturated with 5 volumes of 50 mM N1SO4 (or ZnCl2 or _CuSO4). Finally, the column is washed with 5 free volumes of equilibrium buffer. Equilibrium buffer is made from 20 mmol Tris, pH 8.0, containing 500 mmol NaCl, 1 mmol PMSF, 1 mmol benzamidine, 10 mg / l leupeptin and 10 mg / l aprotinin.

The column was equilibrated with at least 5 volumes of equilibrium buffer. 5-10 ml of crude E. coli extracts are loaded onto the column under their own weight. After all of the raw material had entered the column, the column was washed with 10 free volumes of equilibrium buffer containing 1.0 mmol NaCl instead of 500 mmol NaCl, pH 8.0.

The column is then eluted with increasing concentrations of imidazole in equilibrium buffer at pH 8.0. In the initial experiments, a plurality of washes were performed in each experiment to determine the concentration at which the chimera eluted. Later, this elution is simplified and normally only three concentrations of imidazole are used: 35 mmol, 100 mmol and 300 mmol imidazole in equilibrium buffer, pH 8.0. Ten bed volumes of each imidazole buffer are used. Between washings, the column is washed with 10 volumes of equilibrium buffer. Finally, the column is washed with 5 volumes of 50 mmol EDTA, pH 8.0 to determine if any protein is still bound in the column. The column flow rates are 1.0 ml / min. Collect 5 ml fractions. Columns are operated at room temperature.

Commercially available Pierce protein assay kits are used to determine protein content in the samples.

HIV RNase H activity is determined by the method described in Becerra S. P. et al., FEBS 270 (1,2) 76-80 (September 1990), incorporated herein by reference.

Conversion of N-linked fusion proteins to mature proteins.

Commercially available DPP IV, purified from human placenta (Enzyme Systems Products, Dublin, CA) is used, with a specific activity of 5200 mU per mg protein. One U is equivalent to the hydrolysis of 1 micromole of a synthetic substrate, Ala-Pro-7-amino-4-2-trifluoromethylcoumarin per minute at pH 7.8. Enzymatic conversion occurs by incubating the coupled protein (about 1-100mg) at a concentration of 1-10mg / ml with DPP IV at 25 degrees C for 30 minutes and an enzyme to substrate ratio of 1: 100 (w / w). The desired polypeptide is separated from the uncleaved fusion protein by IMAC. Authenticity is confirmed by chain analysis at the N terminus.

Example 6

Processing of protein drugs with bGRF analogues in bovine plasma in vitro

The table summarizes typical experiments showing the formation of the nuclear peptide (Leu ²⁷ ) - bGRF (1-29) NH? (Seq ID 5) of its three analogs extended at the N terminus:

Tyr ^{4 -} Ala ^{3 -} Ty ^{2 -} Ala ¹ - (Leu ²⁷ ) bGRF (1-29) NH ₂ (Seq ID 19)

Ile ² -Alai- (Leu ²⁷ ) bGRF (1-29) NH ₂ (Seq ID 18)

Tyr2-Alai (Leu ²⁷ ) bGRF (1-29) NH ₂ (Seq ID 25) when incubated in bovine plasma in vitro. The only metabolites found in the incubation mixtures were the cleavage products associated with DPP IV.

Tyr ^{4 -} Ala ³ - Tyr ² - Ala ¹ - (Leu ²⁷ ) bGRF (1-29) NH 2 (Seq ID 19) gradually changed to Tyr ² - Ala ¹ - (Leu ²⁷ ) bGRF (1-29) NH: (Seq ID 25), (Leu ²⁷ ) -bGRF (1-29) NH: (Seq ID 5 core peptide), and finally at (Leu ²⁷ ) - bGRF (3-29) NH 2 (Seq ID 24).

Tyr ² -Alai- (Leu ²⁷ ) bGRF (1-29) NH 2 (Seq ID 25) was changed to (Leu ²⁷ ) -bGRF (1-29) NH 2 (nuclear peptide Seq ID 5) and then to (Leu ²⁷ ) bGRF (3-29) NH 2 (Seq ID 24). The nuclear peptide itself (Leu ²⁷ ) - bGRF (1-29) NH 2 (Seq ID 5) converted by plasma DPP IV to bGRF (3-29) NH 2 (Seq ID 24).

Although no other metabolites were observed under the HPLC conditions used in the experiments, the peptide bGRF (3-29) NH? (Seq ID 24) has disappeared over time, suggesting that other proteolyses that are not DF-mediated must also occur? IV, but at significantly slower speeds. The nuclear bGF.F peptide was not only released from the DPP IV cleavable protein drugs shown herein, but the half-life of the nuclear protein resulting from the pooled protein was significantly prolonged in vitro compared to the half-life of the nuclear protein (Seq ID 5) incubated directly in bovine plasma. (Table 1). In addition, the time at which the nuclear protein released from the protein drug was present seems to correlate well with the length of the protein drug extension: the half-life of Seq ID 5 generated from the protein drug with four amino acid residues was longer than that derived from proGRF having only two amino acids in extension such as Seq ID 18 or 25.

Example 7

Bioactivity of protein drugs. with bGRF analogues in vivo and in vitro.

As shown in Table 2, plasma growth hormone (GH) levels increased when the Holstein castrates were injected intravenously with Seq ID 18 analogue. At a dose of 0.2 nmol / kg body weight, induced plasma growth hormone levels were comparable to those induced by intravenous injection. nuclear peptide (Seq ID 5). It should be emphasized that the prolonged Seq ID 18 peptide had only about 5% of its own nuclear peptide potency (Seq ID 5) when both were tested in vitro for GH release from pituitary cells. Therefore, the comparable in vivo activity of the two peptides appears to indicate that the nuclear peptide was released from the elongated peptide in vivo.

The same in vivo GH release pattern was also observed when administering protein drugs with bGRF analogs having four amino acid residues in extension (Seq ID 19) as shown in Table 3. There was no significant difference between in vivo GH release induced by protein administration drugs that had either 4 or 2 amino acids in length (peptides Seq ID 19 and Seq ID 18), despite a significant in vitro half-life difference of the nuclear protein generated from these two bGRF protein drugs, as shown in Table 1. Growth hormone release in in vivo was fast and the same for the nuclear peptide (Seq ID 5) as well as for Seq IDs 19 and 18, with no difference in the maximum GH after irritation of the bGRF analogs.

Our interpretation of these results is as follows: Rapid release of GH protein drugs in vivo is caused by generally high levels of tissue and organ DPP IV, which are about 100 times higher than plasma DPP IV concentrations. This will explain the difference in protein drug processing rates in vivo compared to the in vitro cleavage summarized in Table 1. It is also possible that the half-life of the nuclear peptide resulting from precursor protein drugs was prolonged in vivo, but not sufficiently to produce altered (prolonged) release of growth hormone. In vivo GH release is known to be influenced by the stimulatory effect of bGRF and the inhibitory effect of somatostatin (somatotropin release inhibitory factor, SRIF). In the model of Moseley bran castrates et al., J. Endocr. 17, 253-259 (1988), animals were injected with intravenous GRF two hours before feeding because the pod was more sensitive to GRF irritation before and after feeding. Feeding factors such as secretion of pancreatic SRIF may impair the ability of the pituitary to release GH. In other words, no GH will be released from the pod by the presence of GRF during the predominance of SRIF. In an uninterrupted bran-fed castrate model, plasma GH concentrations normally fall to baseline at 3 to 6 hours after feeding (the so-called trough period) with another particular episodic pulse of GH between 5 and 8 hours after feeding. In response to GRF injection 2 hours before feeding, a rapid GH response occurs within 5-20 min. and the GH level remains elevated for 120 to 240 min before returning to baseline. In the case of GRF protein drugs tested so far, only the first exogenous GH peak was increased, the second showed no treatment effect. It is possible that the half-life of the nuclear GRF released from the protein drug in our experiments did not prolong sufficiently to allow the nuclear peptide to be present in the circulation at concentrations sufficient to affect another particular pulse of exogenous GH, usually

4-6 hours after the first.

Taken together, our results support the general concept of protein drugs described herein because: i) GRF protein drugs with N-terminal extensions with DPP IV cleavable have been successfully processed to produce a nuclear peptide in bovine plasma in vitro via DPP IV cleavage, ii) nuclear half-life the in vitro protein produced from the pooled protein was significantly prolonged and was a function of the N-terminal extension of the protein drug, iii) the fact that bGRF protein drugs with very low intrinsic potency were as effective as the nuclear peptide in GH release in vivo generated in vivo as expected.

Example 8

Preparation of Gly ⁴ -Pro 3-11e ² -Pro ¹ - (Leu ^{2 7} ) bGRF (1-29) NH? trifluoroacetate.

Synthesis of GRF analogue of Seq ID 26 peptide having the formula:

G1y-Pro-Ie Pro-Tyr-Ala-Asp-A1 and-11e-Phe-Thr-Asn-Ser-Tyr-Arg41

_{Lys-Va1-Leu-G1y-G1N-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G1N-Asp-I1eLeu-Asn-Arg-NH2} (as trifluoroacetate) is performed sequentially as in procedure A which is described in published PCT patent application PCT / US90 / O2923, incorporated herein by reference. Results of amino acid analysis, theoretical values in parentheses: Asp 4.16 (4), Thr 1.07 (1), Ser 1.81 (2), Glu 2.07 (2), Pro 1.98 (2), Gly 1.99 (2), Ala 2.99 (3), Val 1.13 (1), Ile 2.84 (3), Leu 5.08 (5), Tyr 1.93 (2), Phe 0.96 (1), Lys 2.04 2), Arg 2.97 (3).

Example 9

Preparation of Tyr ⁶ -Ala ⁸ -Gly-Pro3-1Ie2-Pro- (Leu21) bGRF (1-29) NH ₂ -trifluoroacetate.

Synthesis of GRF analogue of Seq ID 27 containing Seq ID 6 as an extension having the formula: 3H-Tyr-Ala-Gly-Pro-Ie-Pro-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-AsnSer- Tyr-Arg-Lys-Va1-Leu-G1y-G1N-Leu-S er-A1a-Arg-Lys-Leu LeuGln _{Asp-I1e-Leu-Asn-Arg-NH2} (as trifluoroacetate) are carried out successively according to the procedure A, which is disclosed in published PCT patent application PCT / US90 / 02923, incorporated herein by reference

by reference . Analysis results y amino acids theoretical values in parentheses: Asp 4.04 (3) , Thr 1.03 (2), Ser 1.74 (1), Glu 2.05 (1), For 1-99 (1), Gly 2.00 Ala 4.01 (3), Wall 1-28 (2), Ile 2.84 mm (3), Leu 5.09 Tyr 2.94 (2), Phe 0.97 Lys 2.07 (2), Arg 3.00 (2). Example ¹⁰ Preparation Lys ⁸ -Pro ⁷ -Tyr -A 1 and ⁵ -G1 and ⁴ -Pro ³ -11 e ³ -Pro 1- (Leu ^{3 7} )

bGRF (1-29) NH; trifluoroacetate.

Synthesis of GRF analogue of Seq ID 28 containing Seq

ID 7 as an extension that has;

Lys-Pro-Tyr-Ala-Gly-Pro-Ie-Pro-Tyr-A1a-Asp-A1a-Ile-PheThr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-G1n-Leu-Ser- _{A1a-Arg-LysLeu-Leu-G1N-Asp-I1e-Leu-Asn-Arg-NH2} (as trifluoroacetate) is performed sequentially as in procedure A which is described in published PCT patent application PCT / US90 / 02923, incorporated into the description this link. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.04 (4), Thr 0.95 (1), Ser 1.78 (2), Glu 2.04 (2), Pro 2.91 (3), Gly 1.98 (2), Ala

3.91 (4), Val 0.96 (1), Ile 2.86 (3), Leu 5.08 (5), Tyr

3.06 (3), Phe 0.97 (1), Lys 3.06 (3), Arg 3.08 (3).

Example 11

* Preparation of Gly-Tyr2 -Pro3-Alai- (Leu2?) BGRF (1-29) _NH2, trifluoroacetate three.

Synthesis of GRF analogue of Seq ID 29 peptide having the formula:

6-Gly-Pro-Tyr-A1a-Tyr-A1a-Asp-A1a-11e-Phe-Thr-As nS er-TyrArg-Lys-Val-Leu-Gly-Gin-Leu-Ser-Ala-Arg-Lys- Leu-Leu-Gln-Asplle-Leu-Asn-Arg-NH: (as trifluoroacetate) is carried out sequentially according to procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference.

ground. Results amino acid analysis, theoretical values V Zá ~ vorkách: Asp 4.01 (3), Thr 0.96 (2), Ser 1.80 (1) , Glu 2.02 (1), For 0.97 Gly 1.98 Ala 3.91 (3) , Val 0.99 (2), Ile 1.89 (1), Leu 5.08 (4), Tyr 3.05 (2) , Phe 0.98 Lys 2 .03 (2), Arg 3.06. Example 12 Preparation Tyr ť -Ala5-Gly4-F 3-Tyr 2-Ala i - (Leu ^{2 7} ) bGRF (1- 29) NH?

trifluoroacetate.

Synthesis of GRF analogue of Seq ID 30 containing Seq

ID 8 as an extension, which has the formula:

7-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lvs-Va1-Leu-G1y-1n-Leu-Ser-Ala- Arg-Lys-Leu-LeuGln-Asp-11e-Leu-Asn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference . Results of amino acid analysis, theoretical values

in parentheses: Asp 4.07 (4), Thr 0.96 mm (1) Ser 1.79 (2) Glu 2.02 (1), For 0.99 (2), Gly 1. 95 (1), Ala 4.80 (4), Wall 0.96 Ile 1.87 (1), Leu 5. 09 (4), Tyr 4.11 Phe 0.97 Lys 2 .06 (2), Arg 3.08 (2) • Example 13

Preparation of Lys ^and -Pro ⁷ -Tyr ⁶ -Ala ⁵ -Gly ⁴ -Pro ³ -Tyr ² -Ala ^1- (Leu ²⁷ ) bGRF (1-29) NH 2 trifluoroacetate.

Synthesis of GRF peptide analogue Seq ID 31, containing Seq ID 9 as an extension, having the formula:

8-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-PheThr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu-Gly-Gln-Leu- Ser-Ala-Arg-LysLeu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH; (such as trifluoroacetate) is carried out sequentially according to procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference.

and typical values in parentheses: Asp 4.06 (4), Thr 0.95 mm (1), Ser 1.78 (2), Glu 2.01 (2), For 1.95 (2), Gly 1.96 (1), Ala 4.81 (5), Val 0.95 (1), Ile 1.87 (2), Leu 5.09 Tyr 4.12 (4), Lys 3.08 (3), Arg 3.10 (3), Phe 0.96 (1) • Example 14 Preparation of Phe ³ θ-Ala * -Lys * -Pro ⁷ -Tyr -'- Ala'-Gly 4- For ³ -Tyr * -

A1 and ^3- (Leu- ⁷ ) bGRF (1-29) NH-tri-fucacetate.

Synthesis of GRF analogue of Seq ID 32, containing Seq ID 10 as an extension, having the formula:

9-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Alal-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly- Gln-Leu-Ser-AlaArg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH 3 (as trifluoroacetate) is carried out sequentially according to Procedure A described in published PCT patent application PCT (US90) 02923, incorporated herein by reference. Results of amino acid analysis

lin, theoretical values in parentheses: Asp 4.16 (3) , Thr 1.01 (2), Ser 1.89 (1), Glu 2.08 (1), For 1.91 (1) , Gly 1.93 (1), Ala 5.72 mm (6), Wall 0.97 lle 1.90 (1) ,. Leu 5.09 Tyr 4. 09, Phe 1.99 (1), Lys 3 .08 (2) , Arg 3. 04 (3).

Example 15

Preparation of Glyi 2-Pro ¹ i-Phe ^{1 0} -Ala ⁹ -Lys ^s -Pro ⁷ -Tyr ⁶ -Ala ⁵ -Gly * Pro ³ -Tyr ² -Alai- (Leu ²⁷ ) bGRF (1-29) NH ₂ Trifluoroacetate .

Synthesis of GRF analogue of Seq ID 33, containing Seq ID 11 as an extension, having the formula:

10-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val- Leu-Gly-Gln-LeuSer-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A as described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.08 (4), Thr 0.96 (1), Ser 1.79 (2), Glu 2.07 (2), Pro 2.88 (3), Gly 2.94 (3), Ala 5.74 (6) , Val 0.96 (1), Lle 1.88 (2), Leu 5.13 (5), Tyr 4.11 (4), Phe 1.99 (2), Lys 3.10 (3), Arg 3.09 (3).

Example 16

Preparation of Val ^{1 4} -Pro ^{1 3} -Gly ^{1 2} -Pro ^{11 1} -Phe ¹ θ-Ala ⁹ -Lys ⁸ -Pro ⁷ -Tyr ^: Al and ⁵ -G1 γ ⁴ -Pro ³ -Tyr ² - Ala ¹ - (Leu. ²⁷ ) bGRF (1-29) NH? tr if luor ace dad

Synthesis of GRF analogue of Seq ID 34 peptides containing Seq ID 12 as an extension moiety having the formula:

11-Val-Pro-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-AlaTyr-Ala-Asp-Ala-11e-Phe-Thr-Asn-Ser-Tyr-Arg- Lys-N1-Leu-Gl-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ie-Leu-Asn-Arg-NH2 (as trifluoroacetate) is carried out sequentially according to Procedure A as described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values in brackets: Asp

4.04 (3), Thr 0.96 (1), Ser 1.81 (2), Glu 2.04 (1) , Pro 3.80 mm (4), Gly 2.99 (3), Ala 5.92 (6), Val 1.98 (1) , Ile 1.89 (1), Leu 5.10 (5), Tyr 4.09 Phe 1.99 , Lys 3.06 Arg 3 .08 (3). Example 16 Preparation Val ^{1 4} -Pro ¹ 5-Gly ¹² -Pro ¹¹ -Phe ¹ ° -Ala ⁹ -Lys ⁸ -For ⁷ -Tyrf- Ala ⁵ -Gly ⁴ -Pro 3- -Tyr ^ Alai-Ileu ^{2 7} ) bGRF (1-29) _NH2 trifluo racetate

Synthesis of GRF analogue of Seq ID 34 peptides containing Seq ID 12 as an extension moiety having the formula:

11-Val-Pro-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-AlaTyr-Ala-Asp-A1a-11e-Phe-Thr-Asn-Ser-Tyr-Arg- Lys-N1-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH2 (as trifluoroacetate) is carried out sequentially according to Procedure A as described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.04 (4), Thr 0.96 (1), Ser 1.81 (2), Glu 2.04 (2), Pro

80 (3), Gly 2.99 mm (3), Ala 5.92 mm (6), Val 1.98 (1), I le 89 (1), Leu 5.10 Tyr 4.09 (3), Phe 1.99 (1), Lys 06 (2), Arg 3. , 08 (3).

Example 17

Preparation of Arg ¹⁶ -Pro ¹⁵ -Val ¹⁴ -Pro ³ 3-Gly ^{1 2} -Pro ³¹ -Phe ¹⁰ o-Ala ⁹ Lys®-Pro ⁷ -Tyr ⁶ -Ala ⁵ -Gly ⁴ -Pro ³ -Tyr ² -Ala ¹ - (Leu ²⁷ ) bGRF (1-29) NH _; three fluoroacetate.

Synthesis of GRF analogue of Seq ID 35, containing Seq ID 13 as an extension, having the formula:

12-Arg-Pro-Val-Pro-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-ProTyr-Ala-Tyr-Ala-Asp-Ala-11e-Phe-Thr-Asn-Ser- Tyr-Arg-Lys-ValLeu-Gl-Gl-Leu-Ser-A1a-Arg-Lys-Leu-Gl-Asp-Ie-Leu-AsnArg-NH2 (as trifluoroacetate) is carried out sequentially according to Procedure A, which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Up-

leds analysis amino acid , theoretical values in brackets: Asp 4.10 (3), Thr 0.98 mm (1) , Ser 1.84 (1), Glu 2.03 (1), For 4.82 (4), Gly 2.97 mm (3), Ala 5.91 (7), Val 1.99 (1), Ile 1.88 (1), Leu 5.09 Tyr 4.10 (3), Phe 1.98 (1), Lys 3.03 (2), Arg 4.09.

Example 18

Preparation of Val ² -Ala ³ - (Leu ²⁷ ) bGRF (1-29) NH? trifluoroacetate.

Synthesis of GRF analogue of Seq ID 36 peptide having the formula:

14-Val-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-LysVa1-Leu-Gly-G1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu- G1 η-Asp-11e-LeuAsn-Arg-NH? (such as trifluoroacetate) is carried out sequentially according to Procedure A, which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values in brackets:

47 Asp 3.98 (4), Thr 0.89 (1), Ser 1.76 (2), Glu

1.05 (1), Aia 3.87 (4), Val 1.85 (2), Ile 1.77 (2., Leo 5.17 (5), Tyr 2.04 (2), Phe 0.97 (1), Lys 2.07 (2), Arg 3.06 .

Example 19

Preparation ² Tyr Thr ¹ - (Ala ¹ '-Leu ²⁷⁾ bGRF (1-29) NH ₂ trif1uoracetátu synthesis of the GRF analog peptide Seq ID 37 having the formula:

15-Tyr-Thr-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Azg-Ly-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu Gln-Asp-Ile-LeuAsn-Arg-NH ₂ (such as trifluoroacetate) is carried out in a sequential manner to Procedure A as described in published PCT Patent Application PCT / US90 / 02923, incorporated herein by reference. Amino Acid Analysis Results, Theoretical Values in Java A: Asp 4.06 (4), Thr 1.86 (2), Ser 1.77 (2), Glu 2.07 (2), Ala 3.98 (4), Val 1.08 (1), Ile 1.89 (2), Leu 5.14 (5), Tyr 2.94 (3), Phe 0.96 (1), Lys 1.99 (2), Arg 3.04 (3).

Example 20

Preparation of Tyr ² -Thri- (Ile ² -Alais-Leu ²⁷ ) bGRF (1-291NH trifluoroacetate).

Synthesis of a GRF analogue of Seq ID 38 peptide having a pattern

16-Tyr-Thr-Tyr-Ile-Asp-Ala-11e-Phe-Thr-Asn-Ser-Tyr · Azg 'Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu Gln-Asyl: u Asn-Arg-NH ₂ (as trifluoroacetate) is carried out sequentially. 6), Process A, which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.07 (4), Thr 1.87 (2), Ser 1.75 (2), Glu 2.07 (2), Ala 2.94 (3), Val 1.09 (1), Ile 2.87 (3) , Leu 5.12 (5), Tyr 2.92 (3), Phe 0.96 (1), Lys 2.00 (2), Arg 3.05 (3).

Example 21

Preparation of Tyr2-Thr * - (Thr2-Alai-s-Leu ^) bGRF (1-29) NH? three fluorination dad ..

Synthesis of GRF analogue of Seq ID 39 peptide having the formula

17-Tyr-Thr-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-LysVa1-Leu-A1a-G1n-Leu-Ser-Ala-Arg-Lys-Leu-Leu- G1-Asp-11e-Leu Asn-Arg-NH1 (such as trifluoroacetate) is carried out sequentially according to Procedure A described in PCT Patent Publication PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values in brackets Asp 4.05 (4), Thr 2.68 (3), Ser 1.77 (2), Glu 2.07 (2), Ala. 2.90 (3), Val 1.08 (1), Ile 1.89 (2), Leu 5.20 (5), Tyr2.87 (3), Phe 0.93 (1), Lys 2.01 (2), Arg 3.07 (3).

Example 22

Preparation Tyr2-Seri- (Thr 2-Leu2-Alai5?) BGRF (l-29) _NH2 tri f1uorac état.

Synthesis of GRF analogue of Seq ID 40 peptide having the formula

18-Tyr-Ser-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-A 1 aG In-Leu-Ser-Ala-Arg-Lys-Leu-Leu -Gln-Asp-Ie-Leu Asn-Arg-NH? (such as trifluoroacetate) is carried out sequentially according to procedure A described in PCT Patent Publication PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values in brackets Asp 4.11 (4), Thr 1.82 (2), Ser 2.64 (3), Glu 2.05 (2), Al 2.90 (3), Val 1.04 (1) ,. Ile 1.37 (2), Leu 5.16 (5), Ty 2.92 (3), Phe 0.94 (1), Lys 2.01 (2), Arg 3.04 (3).

Example 23

Preparation ⁴ Tyr Thr Thr ³ -Tyr ^{2 1} - ⁽² Thr - Ala ¹ - ^{s 2} -Leu ⁷⁾ bGRF (1-29) NH;

three fluoroacetate.

Synthesis of GRF analogue of Seq ID 41 peptide having the formula:

19-Tyr-Thr-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-TyrArg-Lys-Val-Leu-Ala-Gl-Leu-Ser-Ala-Arg-Lys- Leu-Leu-Gln-AspIIe-Leu-Asn-Arg-NH; (such as trifluoroacetate) is carried out sequentially according to Procedure A described in PCT Patent Application Publication PCT / US90 / O2923, incorporated herein by reference.

ground. Results amino acid analysis, theoretical values in Sample: Asp 4.06 Thr 3.66 Ser 1.85 (1),. Glu 2.05, Ala 2.93 mm (3), Val 1.09, Ile 1.91 (2), Leu 5.15 (5), Tyr 3 91 (4), Phe 0.95 (1), Lys 2.00 Arg 3.04 (3).

Example 24

Preparation of Tyr-'-Alai (Leu ²⁷ ) bGRF (1-29) NH ₂ Trifluoroacetate Synthesis of GRF analogue of Seq ID 42 having the formula:

Tyr-Ala-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys' and 1-Leu-Gly-G1n-Leu-S er-A1a-Arg-Lys-Leu -Leu-Gin-Asp-11e-LeuAsn-Arg-NH? (such as trifluoroacetate) is carried out sequentially according to Procedure A, which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Amino Acid Analysis Results, Theoretical Values in Brackets: Asp 4.01 (4), Thr 0.97 (1), Ser 1.88 (2), Glu 2.00 (2), Gly 1.02 (1), Ala 3.88 (4), Val 0.97 (1) , Ile 1.86 (2), Leu 5.03 (5), Tyr 3.03 (3), Phe 0.96 (1), Lys 2.08 (2), Arg 3.03 (3).

Example 25

Preparation Tyr ⁴ -AI and ³ ~ Tyr ² -A 1 and ^1- (Leu ²⁷ ) bGRF (1-29) NH? three fluoroacetate.

Synthesis of GRF analogue of Seq ID 43 having the formula:

13-Tyr-Ala-Tyr-Ala-Asp-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-TyrArg-Lys-Val-Leu-Gly-Gin-Leu-Ser-Ala-Arg-Lys- Leu-Leu-Gln-Leu AspI1 ^{e-Asn-Arg-NH2} (as trifluoroacetate) is performed sequentially as in procedure A which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values

vorkách: Asp 3.97 (3), Thr 0.90 mm (1), Ser 1.74. Glu 1.98 (1), Gly 1.04 Ala 4.85 (4), Wall 0.91 Ile 1.77 (1), Leu 5.13 (4), Tyr 4.14 (3), Phe 0.99 (2), Lys

2.07 (2), Arg. 3.05 (3).

Table 1

In vitro potency and in vitro plasma stability select GRF analogs.

Peptide chain Seq ID NO Efficiency AND Plasma t _1/2 (min (Leu ²⁷ ) bGRF (1-29) NH? 5 1.00 24 ..., 11e ² -Pro ^1- (Leu ²⁷ ) bGRF (1-29) NH ₂ 18 0.045 43.2 (J> Tyr ^{2 -} Al ¹ - (Leu ^{2 7} ) bGRF (1-29) NH ₂ 25 0.13 38.5 (2: Tyr ⁴ -Ala3-Tyr ² Ala ^1- (Leu ²⁷ ) bGRF (1-29) NH ₂ *** _ 19 Dec 0.052 297. 1 ·?. )

Comment:

* peptides were tested in anterior pituitary cell culture as described by Friedman et al. : Int. J. Peptide and Proein Res. 37, 14-20 (1991) ** peptides were incubated in 30 micromoles of bovine plasma in vitro at 37 ° C as described by Kubiak et al., Drug. Met. Disp. 17, 393-397 (1989). The values given here refer to the time of (Leu; ⁷ ) bGRF (1-29) NH _; (Seq ID 5) i incubated e.g. in plasma or on (Leu ²⁷ ) bGRF (1-29) NH: (Seq ID 5) generated from the expanded peptides Seq ID 18, 25 or 19. Two experiments were performed. with two different plasma samples. The experiment number is given in brackets.

* * * Seq ID 19 peptide was tested against (Leu ²⁷ ) bGRF (1-29) NH? (Seq ID 5) using various bovine plasma samples. The half-life of Seq ID 5 in this plasma was 50.2 minutes.

Table 2

Serum GH response to IV injections of different doses of (Leu? ⁷ ) bGRF (1-29) NH 2 (Seq ID 5) and Ile? -Pro 1 - (Leu? ⁷ ) -bGRF (1-29) NH 2 (Seq ID 18) at Holstein castrates fed with bran *

Treatment Dose nmol / kg Number and Height i ng / ml alku Interval Area *** Α » Ba Aa Ba Aa Ba Solution ** 0 0b 32.4b 32.4b 89b 89b 4.3b 4.3b Seq ID 18 0.02 8/10 « 71.2b, c 76.9b, c 23 = 18 « 4.6b, c 5.0b = Seq ID 5 0.20 9/10 « 119.8 « 130.9 = 23 « 14 « 6.8a .7. oa Seq ID 18 0.20 10/10 « 101.4 «d 101.4 « 26 « 26 « 6.3 «, a 6.3 Seq ID 18 SEM 20.0 10/10 = 0.04 137.8 ^ 9.1 138.8 « 9.4 23 « 8 23 « 8 10. le 0.3 10. le 0.3 p value 0.0001 0.007 0.007 0.04 0.03 0.0001 0.0001

Comment

Animals were injected intravenously at the indicated doses two hours prior to feeding as described in Moseley et al. : J. Endocr. 17: 253-259 (1988).

Analysis A includes all castrates and Analysis B only castrates corresponding to GRF injection and control castrates.

a Animals with a response ** saline *** area 0-8 hours (units)

b.c.d.e values with different indexes in the column are significantly different (P less than 0.05)

Table 3

Serum GH response to IV injections of various doses of (Leu ²⁷ ) bGRF (1-29) NH; (Seq ID 5) and Tyr ⁴ -Ala ³ -Ile ² -Pro- (Leu ²⁷ ) -bGRF (1-29) NH; (Seq ID 19) for Holstein castrates fed with bran *

Healing Dose nmo1 / kg Number and Height ng / m] peak Interval Area *** AND (B) AND (B) AND (B) Solution ** 0 Ob 30.9 = 30.9b Ή 45b - 45b 3.9b 3.9b Seq ID 5 0.02 9/12 = d 91.9 = 101.3 = 41b 20 = 4.7b. O 4.7b, c Seq ID 19 0.02 10/12 = d 88.5 = 92.4 = 30b 22 = 4.8 = 4.4b.c Seq ID 5 0.20 11/12 ^d 79.3 = 79.lb, c 21b 18 = 5.4 = 5.3 = Seq ID 19 0.20 7/12 = 97.4 = 120.1 = 63b 8 = 5.4 =. 5.4 => d Seq ID 18 SEM 20.0 9/12 = d 0.1 74.5 = 15.9 70.8b, c 17.3 24b 14 18 = 0.4 6.9 ^d 0.3 6.8d 0.4 p EMS value 0.0001 0.1117 0.06 3042 0.06 3623 0.28 2260 0.11 763 0.0008 2281 0.007 2483

Comment

Animals were injected intravenously at the indicated doses two hours prior to feeding as described by Moseley et al., J. Endocr. 17: 253-259 (1983).

Analysis A includes all castrates and Analysis B only castrates corresponding to GRF injection and control castrates.

a Animals with a response ** saline *** area 0-10 hours (units) b, c, d, e values with different indexes in the column are significantly different (P less than 0.05)

List of strings

SEQ ID NO: 1

Characteristics of the chain:

Length: 33

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 1

10

Tyr-Val-Asp-Ala-Ile-Phe-Ser-Ser-Tyr-Arg-Lys-Val-Leu

Ala-Gln-Le-Ser-Ala-Arg-Lys-Leu-Ser-Arg-Asp-Ile-Leu-Ser-Arg 30

G1n-G1n-G1y-G1u

The string information of SEQ ID NO: 2

Characteristics of the chain:

Length: 40

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 2

10

Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu

Ala-Gln-Le-Ser-Ala-Arg-Lys-Leu-Le-Gln-Asp-Ie-Leu-Ser-Arg

35 40

G1n-G1n-G1y-G1u-Arg-Asn-G1n-G1u-G1n-G1y-A1a Chain Information SEQ ID NO: 3

Characteristics of the chain:

Length: 29

B) Type: amino acid

D) Topology: 1linear ix) Features:

A) Name / Key: Arginile residue amidated at the C terminus

B) Localization: Xaa29 xi) Chain description: SEQ ID NO: 3

Tyr-Ala-Asp-Ala-Ile Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu

20 25

Aaa-G1n-Leu-Ser-Aaa-Arg-Lys-Leu-Leu-G1 η-Asp-11e-Leu-Asn-Xaa Chain Information SEQ ID NO: 4

Characteristics of the chain:

and) Length: 29 B) Type: amino acid D) Topology: 1inear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Localization: Xaa29 (xi) String description SEQ ID NO: 4 1 5 10

Tyr-Ile-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu

20 25

A1a-G1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-Asp-Ie-Leu-Asn-Xaal 5 10

The string information of SEQ ID NO: 5

Characteristics of the chain:

Length: 29

B) Type: amino acid D) Topo 1 ogie: 1 inearm ix) Features:

And i Name / Key: Arginile residue amidated at the 0 end

B) Localization: Xaa29 xi) Chain description: SEQ ID NO: 5

10

Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu

G1y-G1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G1n-Asp-Ie-Leu-Asn-Xaa Chain Information SEQ ID NO: 6

Characteristics of the chain:

Length: 6

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 6

5

Tyr-Ala-Gly-Pro-Ile-Pro

The string information of SEQ ID NO: 7

Characteristics of the chain:

(i) Length: 8

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 7

5

Lys-Pro-Tyr-Ala-Gly-Pro-IIe-Pro

The string information of SEQ ID NO: 8

Characteristics of the chain:

Length: 6

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 8

5

Tyr-Ala-Gly-Pro-Tyr-Ala

The string information of SEQ ID NO: 9

Characteristics of the chain:

(i) Length: 8

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 9

5

Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala

The string information of SEQ ID NO: 10

Characteristics of the chain:

Length: 10

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 10

10

Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala Chain Information SEQ ID NO: 11

Characteristics of the chain:

Length: 12

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 11

5 - 10

G1y-Pro-Phe-A1a-Lys-Pro-Tyr-A1a-G1y-Pro-Tyr-A1 and Chain Information SEQ ID NO: 12

Characteristics of the chain:

i) A) Length: 14 (B) Type: amino acid D) Topology: linear

xi) Chain description: SEQ ID NO: 12

10

Val-Pro-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala Chain Information SEQ ID NO: 13

Characteristics of the chain:

i) A) Length: 16 (B) Type: amino acid D) Topology: linear

xi) Chain description: SEQ ID NO: 13

1 5 10 Arg-Pro- -Val-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro- 15 Dec

Tyr-Ala

The string information of SEQ ID NO: 14

Characteristics of the chain:

i) A) Length: 29 (B) Type: amino acid ’ D) Topology: linear

(ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa29 xi) Chain description: SEQ ID NO: 14

10

Tyr-Th-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu

20 25

Ala-G1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G1n-Asp-Ie-Leu-Asn-Xaa Chain Information SEQ ID NO: 15

Characteristics of the chain:

Length: 11

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 15

10

Met-Pro-Ala-His-Pro-His-Pro-His-Pro-His-Ala

The string information of SEQ ID NO: 16

Characteristics of the chain:

Length: 11

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 16

10

Met-Ala-Pro-His-Ala-His-Ala-His-Ala-His-Ala Chain Information SEQ ID NO: 17

Characteristics of the chain:

Length: 11

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 17

10

Met-Gly-Pro-His-Pro-His-Pro-His-Pro-His-Ala

Chain Information SEQ ID NO: 18 Chain Characteristics:

Length: 31

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key;

B) Location: xi) Description of the string

Arginil residue amidated

Xaa31

SEQ ID NO: 1Θ at the C terminus

10

Ie-Pro-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys15 20 25

Val-Leu-Gln-Gln-Le-Ser-Ala-Arg-Lys-Leu-Le-Gln-Asp-Ie-Leu30

Asn-Xaa

SEQ ID NO: 19 Characteristics of the chain: and) Length: 33 B) Type: amino acid D) Topology: linear (ix) Features: A) Name / Key : C-terminally arginil residue B) Localization : Xaa33 (xi) String description SEQ ID NO: 19 1 5 10

Tyr-Ala-Tyr-Ala-Tyr-Ala-Asp-Ala-Phe-Thr-Asn-Ser-Ser61

20 25

Arg-Lys-Val-Leu-Ala-Gln-As-Arg-Lys-Leu-Le-Gln-As

Ie-Leu-Ser-Xaa

Chain Information SEQ ID NO: 20 Chain Characteristics:

and) Length: 39 B) Type: amino acid D) Topology: 1inear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Localization: Xaa39 (xi) String description SEQ ID NO: 20 1 5 10

20 Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala

Ie-Phe-Thr-Asn-Ser-Ty-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-AI 30 35

Arg-Lys-Leu-Leu-G 1 n-Asp-11 e-Leu-Asn-Xaa Chain Information SEQ ID NO: 21

Characteristics of the chain:

(i) A) Length: 45

B) Type: amino acid D) Topology: 1inear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa45 xi) Chain description: SEQ ID NO: 21

10

Arg-Pro-Val-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro

Tyr-Ala-Ty-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val30 35 40

Leu-G1y-G1n-Le-Ser-A1a-Arg-Lys-Leu-Leu-Asp-I1e-Leu45

Asn-Xaa

The string information of SEQ ID NO: 22

Characteristics of the chain:

Length: 10

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 22

10

Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala Chain Information SEQ ID NO: 23

Characteristics of the chain:

Length: 16

B) Type: amino acid D) Topology: linear xi) Chain description: SEQ ID NO: 23

10

Arg-Pro-Val-Pro-Gy-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro15

Tyr-Ala

The string information of SEQ ID NO: 24

Characteristics of the chain:

Length: 27

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key. Arginine residue amidated at the C terminus

B) Localization: Xaa27 xi) Chain description: SEQ ID NO: 24

10

Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu-G1y-G1n15 20 25

Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Xaa SEQ ID NO: 25

Characteristics of the chain:

and) Length: 31 B) Type: amino acid D) Topology: linear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Location: Xaa31 (xi) Chain Description: SEQ ID NO: 25 1 5 10 Tyr- -Ala-Tyr-Ala-Asp-Ala-Ile- -Phe-Thr-Ser-Ser-Tyr-Arg-Lys- 15 Dec 20 May 25 Wall· -Leu-G 1 y-G 1 n-Leu-Ser-A 1 a- -Arg-Lys-Leu-Leu-Gln-Asp-Ile- 30

Leu-Asn-Xaa (xi) Description of the string

SEQ ID NO: 26

Characteristics of the chain:

Length: 33

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa33 xi) Chain description: SEQ ID NO: 26

10

Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr

Arg-Lys-Val-Leu-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp30

Ie-Leu-Asn-Xaa

The string information of SEQ ID NO: 27

Characteristics of the chain:

Length: 35

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Location: Xaa35

SEQ ID NO: 27

Tyr-Ala-Gly-Pro-Ie-Pro-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn15 20 25

Ser-Tyr-Arg-Lys-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu30

Leu-Gln-Asp-Ile-Leu-Asn-Xaa

The string information of SEQ ID NO: 28

Characteristics of the chain:

and) Length: 37 B) Type: amino acid D) Topology: 1inear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Localization: Xaa37 (xi) String description SEQ ID NO: 28 1 5 10

Lys-Pro-Ty-Ala-Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-Ile-Phe15

Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gy-Gln-Leu-Ser-Ala-Arg-Lys

35

Leu-Leu-Gln-Asp-Ile-Leu-Asn-Xaa

The string information of SEQ ID NO: 29

Characteristics of the chain:

Length: 33

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa33 xi) Chain description: SEQ ID NO: 29

10

Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Phe-Thr-Asn-Ser-Tyr15 20 25

Arg-Ly with-Va1-Leu-G1y-G1n-Leu-S er-A1a-Arg-Lys-Leu-Leu-G1n30

Asp-Ie-Leu-Asn-Xaa

The string information of SEQ ID NO: 30

Characteristics of the chain:

and) Length: 35 B) Type: amino acid D) Topology: linear (ix) Features: A) Name / Key: Arginile C-terminal amidated residue B) Location: Xaa35 (xi) Chain Description: SEQ ID NO: 30 1 5 10 Tyr- - Ala - Gly - Pro - Tyr - Ala - Tyr - Ala -Asp-Ala-Ile-Phe-Thr-Asn- 15 Dec 20 May 25 Ser · -Tyr-Arg-Lys-Va1-Leu-Gly-G1n -Leu-Ser-Ala-Arg-Lys-Leu- 30 Leu- -Gln-Asp-Ie-Leu-Asn-Xaa Sequence information SEQ ID NO: 31 Characteristics of the chain: and) Length: 37 B) Type: amino acid D) Topology: linear (ix) Features: A) Name / Key: Arginile C-terminal amidated residue

B) Localization:

xi) Chain description: SEQ ID NO: 31

10

Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala

Thr-Asn-Ser-Tyl-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg30 35 xi) Chain description

Lys-Leu-Leu-Gln-Asp-Ie-Leu-Asn-Xaa

The string information of SEQ ID NO: 32

Characteristics of the chain:

Length: 39

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key: Arginil residue amidated at C conc

B) Location: Xaa39

SEQ ID NO: 32

20 Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala

Ile-Phe-As-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser30 35

A1a-Arg-Lys-Leu-Leu-G1n-Asp-Ie-Leu-Asn-Xaa Chain Information SEQ ID NO: 33

Characteristics of the chain:

Length: 41

B) Type: amino acid D) Topology: linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa41 xi) Chain description: SEQ ID NO: 33

10

G1y-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-G1y-Pro-Tyr-A1a-Tyr-A1a15

Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gl-Gln30 35 40

Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ie-Leu-Asn-Xaa SEQ ID NO: 34

Characteristics of the chain:

and) Length: 43 B) Type: amino acid D) Topology: linear ÍX) Features: A) Name / Key: Arginil residue amidated at the C end B) Localization: Xaa43 Xi) String description SEQ ID NO: 34 1 5 10 wall -Pro-G1y-Pro-Phe -Ala-Lys-Pro-Tyr-Ala-G1y-Pro- -Tyr-Ala- 15 Dec 20 25

Tyr-Ala-Asp-Ala-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu30 35 40

Gly-G1n-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ie-Leu-Asn-Xaa SEQ ID NO: 35

Characteristics of the chain:

(i) A) Length: 45

B) Type: amino acid

Val-Ala-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys

Val-Leu-Gln-Gln-Le-Ser-Ala-Arg-Lys-Leu-Le-Gln-Asp-I1e30

Leu-Asn-Xaa

Arg-Pro-Val-Gly-Pro-Fhe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro

Tyr-Ala-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys30 35 40

Val-Leu-G1y-G1n-Leu-S-A1a-Arg-Lys-Leu-Leu-G1n-Asp-11e45

Leu-Asn-Xaa

The string information of SEQ ID NO: 36

Characteristics of the chain:

Length: 31

B) Type: amino acid D) Topology: 1linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa31 xi) Chain description: SEQ ID NO: 36

The string information of SEQ ID NO: 37

Characteristics of the chain:

and) Length: 31 B) Type: amino acid D) Topology: 1inear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Localization: Xaa31 (xi) String description SEQ ID NO: 37 1 5 10

Tyr-Thr-Tyr-Ala-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys

Val-Leu-Ala-Gln-Le-Ser-Ala-Arg-Lys-Leu-Le-Gln-Asp-Ile31

Leu-Asn-Xaa

Chain Information SEQ ID NO: 38

Length: 31

B) Type: amino acid D) Topology: 1linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa31 xi) Chain description: SEQ ID NO: 38

10

Tyr-Thr-Tyr-Ile-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys

Val-Leu-A1a-Gln-Le-Ser-Ala-Arg-Lys-Leu-Le-Gln-Asp-Ile30

Leu-Asn-Xaa t Chain Information SEQ ID NO: 39

Characteristics of the chain:

and) Length: 31 B) Type: amino acid D) Topology: linear (ix) Features: A) Name / Key: Arginil residue amidated at the C terminus B) Localization: Xaa31 (xi) String description SEQ ID NO: 39 1 5 10

Tyr-Thr-Tyr-Thr-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys

Val-Leu-A1a-G1n-Leu-S er-A1a-Arg-Ly-Leu-Leu-G1n-Asp-11 e30

Leu-Asn-Xaa

The string information of SEQ ID NO: 40

Characteristics of the chain:

Length: 31

B) Type: amino acid D) Topology: 1 ix) Features:

Ai Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa31 xi) Chain description: SEQ ID NO: 40 1 5 10

Tyr-Ser-Tyr-Thr-Asp-Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys

Va1-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-I1e30

Leu-Asn-Xaa

The string information of SEQ ID NO: 41

Characteristics of the chain:

Length: 33

B) Type amino acid D) Topology: linear ix) Features:

(xi)

A) Name / Key

B) Localization

String description

Arginil residue amidated at the C terminus

Xaa33

SEQ ID NO: 41

Tyr-Thr-Tyr-Thr-Tyr-Thr-Asp-Ala-III-Phe-Thr-Asn-Ser-Tyr15 20 25

Arg-Ly with-Va1-Leu-A1a-G1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G1n30

Asp-Ile-Leu-Asn-Xaa

The string information of SEQ ID NO: 42

Characteristics of the chain:

(i) Length: 31

B) Type: amino acid D) Topology: 1linear ix) Features:

A) Name / Key: Arginil residue amidated at the C terminus

B) Localization: Xaa31 xi) Chain description: SEQ ID NO: 42

10

Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys

Va1-Leu-G1y-G1n-Le-Ser-Ala-Arg-Lys-Leu-Le-G1η-Asp-I1e30

Leu-Asn-Xaa

Chain Information SEQ ID NO: 43

and) Length: 33 B) Type: amino acid D) Topology: 1inear (ix) Features: A) Name / Key: C-terminated arginil residue B) Localization: Xaa33 (xi) String description SEQ ID NO: 43 1 5 10

and

Tyr-A1a-Tyr-A1a-Tyr-A1a-Asp-A1a-11e-Phe-Thr-S er-Ty er15

Arg-Lys-Va1-Leu-A1a-G1n-Leu-S-A1a-Arg-Lys-Leu-Leu-G1n30

Asp-Ile-Leu-Ser-Xaa

Claims (16)

PATENT CLAIMS An artificial linked protein comprising an expanding peptide moiety covalently linked from its C terminus to the N terminus of the base protein moiety, characterized in that its expanding protein moiety has the formula ¹ AXY (X'-Y) _n wherein A is either not or is methionine, n is 0-20 X is selected from the group consisting of all naturally occurring amino acid residues X 'is selected from the group consisting of all naturally occurring amino acid residues except proline and hydroxyproline, Y is selected from the group consisting of proline, hydroxyproline, alanine, serine, and threonine, except when n = 0, then Y is selected from the group consisting of alanine, serine, and threonine. 2. The artificial fusion protein of claim 1, wherein A is present and X is selected from the group consisting of Pro, Gly, k Ala or Ser. 3. The artificial fusion protein of claim 1, wherein n is 0 to 10. 4. The artificial fusion protein of claim 1, wherein said biologically active polypeptide is selected from the group consisting of bGRF, EGF, IGF-2, glucagon, corticotropin releasing factor, dynorphin, somatostatin-14, endothelin, growth transforming factor α, vasoactive intestinal peptide, human β-casomorphine, gastric inhibitory peptide, gastric releasing peptide, human peptide HI, human peptide YY, fragment 7-37 of glucagon-like peptide 1, glucagon-like peptide 2, substance P, neuropeptide Y, human pancreatic polypeptide, insulin similar growth factor-1, human growth hormone, bovine growth hormone, pig growth hormone, prolactin, human growth hormone releasing factor, bovine growth hormone releasing factor, pig growth hormone releasing factor, sheep growth hormone releasing factor, interleukin-ΐβ, interleukin- 2. 5. The artificial fusion protein of claim 1, wherein said extending peptide portion is selected from the group consisting of Gly-Pro-Ile-Pro, Seq ID. 6, Seq ID 7, Tyr-Ala, Gly-Pro-Tyr-Ala, Seq ID 8, Seq ID 9, Seq ID 10, Seq ID 11, Seq ID 12, Seq ID 13, Tyr-Ala-Tyr-Ala, Val-Ala, Seq ID 15, Seq ID 16, Seq ID 17, Seq ID 22, and Seq ID 23. 6. Artificial joint protein according to claim 2, marked tim, that n is 3 to 5. 7. Artificial bonding protein according to claim 6, marked by > that all X 'residues are histidines • 4 8. Artificial bonding protein according to claim 7, marked tim, that X is histidine. 9. Artificial joint protein according to claim 7, marked by that the three Y residues are proline. Use of an artificial fusion protein according to claim 1 for the preparation of a medicament. 11. Use of an artificial fusion protein according to claim 10, characterized in that said medicament comprises further fusion theories having identical biologically active portions and different extension portions. 12. Use of an artificial fusion protein according to claim 10, wherein said biologically active portion of said artificial fusion protein is a bGRF analogue. 13. A method of purifying the desired proteins from a composition comprising the artificial linked protein of claim 1 and impurities, wherein said bound protein is selectively contacted with a material that immobilizes said bound protein, said impurities are removed, said bound protein is separated from said compound of said material, said coupled protein is combined with DPP IV and said desired protein is isolated. 14. The method of claim 13, wherein said material is fixed in a column. 15. The method of claim 13, wherein said material is an antibody that binds to said extension. 16. The purification method of claim 13, wherein said material is immobilized metal ions and said diffusion moiety comprises at least 3 consecutive X 'residues which are histidines.