RU2114119C1 - Fused protein and a method of isolation of the fused protein - Google Patents

Abstract

FIELD: biology, medicine, biochemistry. SUBSTANCE: fused protein no occurring in nature and containing elongating peptide fragment covalently bound by its C-end with N-end of protein central part of the general formula (I): A-X-Y-(X¹-Y)_n-protein where A - H, Met and if A - H then protein is an analog of bGRF; if A - Met then protein is HIV-RNAase-4; X - Ala, Ser, Tyr, Val, Arg, Gly, Lys, Pro, Phe; Y - Ala, Ser, Thr, Pro; X′ - Ala, Gly, Ile, His, Lys, Phe, Tyr; n = 0-4; if n = 0 and A - H, Y - Ala, Ser, Thr. Method of isolation of fused protein of the formula (I) where A - Met; n = 4; X - Ala, Gly, Pro; Y - Ala, Pro; X′ - His; "protein" - HIV-RNAase involves column chromatography on Sepharose Pharmacia containing immobilized metal ions. Fused protein can be used for construction of drugs showing the prolonged effect. EFFECT: improved method of isolation. 9 cl, 6 tbl

Description

 The invention relates to fused polypeptides that are not found in nature and contain N-terminal parts cleaved by dipeptidyl peptidase IV (DPP IV).

 The molecular biology technique, in particular the recombinant DNA technique, allows one to obtain relatively large quantities of the desired biologically active polypeptides. In addition, by modifying the genetic information encoding these polypeptides, relatively large amounts of modified polypeptides can be obtained. Modifications of these polypeptides are often used to improve their activity or to facilitate their production and / or synthesis. In accordance with this, many attempts have been made to determine which modifications are necessary to increase, enhance or other changes in the biological activity of the desired polypeptides. In addition, a lot of work was done to modify the desired polypeptides to facilitate their synthesis and purification.

 Natural polypeptides are often first biologically synthesized as larger precursors, which are then truncated by a series of proteolytic cleavages to produce end products. For example, there are several proteases that recognize and cleave specific amino acids and / or amino acid sequences. 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 was first described by Hopsu-Havu, V.K. and G.G. Glenner, Histo. Chenue 3: 197-201 (1966), and has been shown to be present in many tissues and mammals. DPP IV is commercially available and manufactured by Enzyme Systems Products (Dublin, CA). DPP IV recognizes specific amino acid sequences at the N-termini of proteins. In particular, DPP IV will cleave the dipeptide from the N-terminus if the second amino acid from the N-terminus is proline (Pzo), hydroxyproline (Hyp), alanine (Ala), serine (Ser) and threonine (Thr), and in position N the amino acid residue is any amino acid, provided that proline or hydroxyproline is not the third amino acid residue from the N-terminus. DPP IV activity is more effective if proline or alanine is the second amino acid from the N-terminus, and especially effective if proline is in this position. The activity of DPP IV in the stepwise cleavage of the "PRO" portions of the precursors of natural peptides is widely described.

 Modern technologies make it possible to obtain a high level of biologically active protein production. Important polypeptides can be obtained using peptide synthesizers or can be produced in host cells using recombinant DNA techniques. Often biologically active proteins are used as medicines. There are many examples in which active proteins are used as therapeutic or prophylactic agents, or to enhance or suppress any specific signs. Since DPP IV and other proteases break down proteins, these drugs are susceptible to degradation. Thus, the disadvantage of using biologically active polypeptides as medicines is that the time of their presence in the body should be minimized, and therefore they should be administered more often.

 The rapid development of recombinant DNA technology, which allows the production of polypeptides, proteins and their analogues in large quantities and in a very short period of time, requires the development of a highly efficient and predictable method for isolating these proteins from complex mixtures, which include the full amount of the protein produced by the host cell, and protein in the culture medium. Purification of heterologous polypeptides produced by host cells can be a very expensive procedure and can cause denaturation of the protein product itself. An overview of protein purification methods is provided in US Pat. No. 4,782,137 in the prior art chapter, issued November 1, 1988 to Hopp et al. And incorporated herein by reference.

 To eliminate the above limitations of the prior art and to obtain better methods, the recombinant DNA technique can be used to obtain the desired polypeptides in the form of synthetic proteins containing a linker peptide that can be used as a ligand or other target in purification methods. For example, US Pat. No. 4,782,137 describes the synthesis of a non-naturally occurring peptide containing an antigenic linker peptide. This synthetic peptide can be passed through a column containing immobilized antibodies that bind to the antigenic linker and thereby facilitate the release of the synthetic protein. US Pat. No. 4,569,794 discloses a method for purifying synthetic proteins that contain N-terminal extension segments having affinity for immobilized metals. Synthetic proteins bind to immobilized metal ions in a column. These methods have the disadvantage that the use of a linker peptide is often undesirable, and its removal can be difficult.

 The invention relates to synthetic fusion proteins, which consist of a central portion of a protein and an N-terminal portion that is cleaved by DPP IV. In accordance with the present invention, the synthesized protein is a hybrid protein in which the extension portion attached to the central portion of the protein is not a natural N-terminal extension portion associated with the core of the protein, that is, the synthesized protein is not natural. The present invention relates to prodrugs that are unnatural DPP-IV-cleavable proteins, where the central part of the protein is a biologically active protein. The present invention relates to non-naturally occurring DPP-IV-cleavable proteins that can be used in purification methods, since the N-terminal extension segment present in them provides these proteins with a distinctive feature or property that facilitates their purification.

 The present invention relates to synthetic proteins having N-terminal extension segments cleaved by DPP IV, so that under the action of DPP IV, these synthetic proteins are converted to the desired proteins. When using these non-natural proteins as prodrugs, they are subjected to in vivo processing using DPP IV present in the target species, resulting in the formation of a biologically active protein. When such an unnatural protein is used in the purification process, it can be purified using specifically designed N-ends that are used as a ligand and then removed by processing with the participation of DPP IV, which releases the desired protein.

 The invention allows the production of the desired proteins in the form of synthetic proteins, which are then converted into the desired proteins by DPP IV. A prodrug is converted to a drug over a certain period of time using the endogenous DPP IV of the patient, resulting in a prolonged presence of the active drug in the patient's body and, therefore, the frequency of administration of this drug to the patient is reduced. In accordance with the present invention, pure desired proteins can be obtained by producing and purifying the synthesized proteins, followed by their in vitro processing using DPP IV, resulting in the formation of the desired protein.

The invention relates to a fused (hybrid) protein that is not found in nature and contains an extension peptide portion, which is covalently linked to the N-terminus of the central part of the protein with its C-terminus and which has the following formula:
AXY (X'-Y) _n
Where
A is optional, and if present, it is methionine;
n is 0-7;
X is selected from the group Ala, Arg, Gly, Lys, Pro, Phe, Ser, Tyr, Val;
X '= Ala, Gly, Ile, His, Lys, Phe, Tyr;
Y is selected from the group consisting of proline, alanine, serine and threonine, provided that if n = 0, then Y is selected from the group consisting of alanine, serine and threonine.

 The invention also relates to the use of said synthetic proteins in the manufacture of pharmaceutical preparations and to a method for isolating the desired proteins from a mixture containing said unnatural proteins and impurities, which comprises the steps of selectively interacting said unnatural protein with a material immobilizing this protein; removal of these impurities; the selection of these unnatural proteins from the specified material; the interaction of these unnatural proteins with DPP IV and the selection of these desired proteins.

 US patent 4,569794, issued by Smith et al. February 11, 1986, relates to a method for purifying proteins and to the compounds used in this method. This invention describes a method for isolating fusion proteins that have bioactive polypeptides at their C-terminus and N-terminal N-terminal extension linker that forms a chelate complex with a metal ion. This hybrid peptide has an affinity for immobilized metal ions. Impurities can be removed by passing the mixture containing the hybrid protein through a column containing immobilized metal ions. This fusion protein binds to metal ions, and only impurities elute as a result. Then, by changing the conditions, the fusion protein is freed from immobilized metal ions, thereby obtaining a purified fusion protein.

 US Pat. No. 4,782,137, issued to Hopp et al. November 1, 1988, discloses the synthesis of a fusion protein having a highly antigenic N-terminal portion and the desired polypeptide at its C-terminal portion. According to Hopp et al., Fusion proteins are isolated from the crude supernatant by passing the crude supernatant through a column containing immobilized antibodies that recognize the antigenic portion of the fusion protein. These immobilized antibodies hold the protein in the column, while the unwanted components of the supernatant elute. Then, by changing the conditions in the column, the dissociation of the antigen-antibody complex is promoted. After that, the fusion protein is eluted and collected.

 U.S. Patent 4,734,399 to Felix et al., March 29, 1988, relates to growth hormone release factor analogues. This patent discloses several analogs that have terminal Tyr-Ala and His-Ala. However, these molecules are not fusion proteins, but only proteins with a central polypeptide. The N-terminal dipeptides of Felix et al. Are part of the core of the bGRF analog molecule.

 European Patent Application Publication No. 0220958, published May 6, 1987, describes selective chemical removal of N-terminal residues. This invention relates to a method for removing N-terminal residues from desired polypeptides and to the compounds used in this method. The desired polypeptide is present in the form of a fusion protein having the desired polypeptide, which is connected at the N-terminus to a linker having the formula X-Pro. When a specific protein is exposed to a fusion protein, diketopiperazine of the X-Pro portion of the fusion protein is formed, after which the desired polypeptide is produced from the fusion precursor. Hybrid proteins of the aforementioned application EPO 220958 ('958) cannot be included in the present invention, since according to the present invention, if the N-terminal extension fragment is just a dipeptide, i.e. if A is absent, n = 0, and X is a natural amino acid, then Y is either alanine, or serine, or threonine. Thus, whenever the extension moiety is a dipeptide, it has the formula X-Ala, X-Ser or X-Thr. The '958 application describes the chemical, non-enzymatic, cleavage of the X-Pro dipeptide. The dipeptide X-Ala, X-Ser and X-Thr is immune to the cleavage of the chemical type specified in the application '958, where the extension segment of X-Pro is cut from the Central part of the protein.

 Australian Patent Application AU-A-12709/88 discloses fusion proteins that contain affinity peptides used in immobilized metal affinity chromatography (IMAC). The affinity peptides that are disclosed in this document contained at least two adjacent histidine residues. The described method of IMAC purification requires special chemical technology for the synthesis of polymers of nitrilotriacetic acid (NTA).

 Article by Tallon et al. In Biochem. 26: 7767-7774 (1987) refers to elongated analogs of the tridecapeptide α factor from Saccharomyces cerevisiae. These synthesized analogs are elongated α-factors that have sequences of the natural pro-α-factor encoded by the structural gene MPαI.

 In an article by Kriel et al. In Eur. J. Biochem. III: 49-58 (1980) describes the stepwise cleavage of the N-terminal portion of the precursor of melittin (promeltin) by dipeptidyl peptidase IV. Promeltin is the main component of bee venom. In the amino acid sequence of the N-terminal portion of the precursor, every second residue is either proline or alanine. By exposing promeltitin to DPP IV isolated from pig kidney, the N-terminal region of the precursor is cleaved stepwise, thereby obtaining a mature protein. Unlike the fusion proteins of the present invention, promeltin is a natural protein.

 An article by Julius, D. et al. In Cell, v. 32: 838-852 (March 1983) relates to the role of membrane-bound DPP IV in the processing of yeast α-factor from a larger precursor polypeptide. Unlike the fusion proteins of the present invention, the yeast α-factor is a natural protein.

 Mollay, C. et al. In Eur. J. Biochem. 160: 31-35 (1986) describes the isolation of DPP IV from Xenopus laevis dermal skinnet. The activity of DPP IV is also discussed.

 In an article by Mentlein, R. in FEB vol. 234, 2, p. 251-256 (July 1988), proline residues during the maturation and degradation of peptide hormones and neuropeptides are discussed. In mammals, proline-specific proteases, such as DPP IV, have been reported not to be involved in the biosynthesis of regulatory peptides, but may play an important role in their cleavage. So, for example, it is concluded that although in vertebrates and lower vertebrates, the conversion of precursor proteins to mature forms occurs mainly using DPP IV, however, in the processing of regulatory proteins in mammals, DPP IV is mainly used as proteolytic enzymes for protein degradation.

 Frohman L. A. et al. In J. Clin. Invest. 78: 906-913 (1986) indicate that the human growth hormone release factor (hGRF) and their analogues are rapidly degraded in vivo in humans and in vitro by plasma DPP IV.

 Frohman L.A. et al. in J. Clin. Invest. 83: 1533-1540 (1989) indicate that human growth hormone release factor (hGRF) and their analogs rapidly degrade in vivo in humans and in vitro by plasma DPP IV.

 Article Kubiak T.M. et al. Drug Metabolism and Disposition, vol. 17, No. 4, p. 393-397 (1989) relates to the metabolic degradation of an analogue of bovine and porcine plasma growth hormone release factor and to the correlation of this degradation by plasma DPP IV activity. Consider bGRF analogues had an AIa residue at the 2-position of the N-terminus. It is indicated that the metabolic degradation of bGRF in plasma occurs due to the presence of DPP IV in the plasma.

 Hong W. et al. Biochemistry, 28: 8474-8479 (1989) describe the expression of enzyme-active DPP IV in Chinese hamster ovary cells after transfection.

 Kreil G., TIBS 15: 23-26 (January 1990) describes stepwise digestion of dipeptides by DPP in the conversion of precursors to final products. The precursors described by Kreil are natural proteins. The fusion proteins of the present invention are unnatural fusion proteins.

 Boman et al. J. Biol. Chem. 264: 5852-5860 (1989) showed that dipeptidyl peptidase isolated from cecropia pupae with similar specificity for DPP IV has the ability to cleave the natural N-terminal sequences of Ala-Pro-GIu-Pro from N-terminal synthetic copies of natural cecropia A precursors and B. The preprocecropin disclosed by Boman is a natural protein.

 Dalboge H. et al. Bio / technology 5: 161-164 (February 1987) disclose the conversion of E. coIi, the produced human growth hormone precursor (hGH) precursor, into authentic hGH in vitro. The N-terminal extension of the precursor segment is removed using dipeptidyl peptidase I.

 Dalboge H et al. FEBS, Vol. 246 (1,2): 89-93 (March 1989) disclose the cloning and expression of an IL-I β precursor and its conversion to IL-I β by removing the N-terminal segment of said precursor using dipeptidyl peptidase I.

 Dalboge H. et al. FEBS, Vol. 266 (1,2): 1-3 (June 1990) describe in vivo processing of the N-terminal methionine in E. coli. It is indicated that the removal of the N-terminal methionine on an extended human growth hormone depends on which amino acid is adjacent to methionine.

 Hopp T. P. et al. Bio / Technol. 6: 1204-1210 (October 1988) disclose the addition of an eight amino acid peptide to the N-terminus of a desired recombinant lymphokine in order to impart to it an antigenic N-terminus that can be used in immunogenic purification. This publication relates to US Pat. No. 4,782,137, described above.

Smith MC et al. J. Biol. Chem. Vol. 263, 15: 7211-7215 (1988) disclose experimental results supporting the hypothesis that peptides forming specific chelate complexes with metals at the NH ₂ end of a small peptide can be used to purify the protein using affinity chromatography on metal ions. This work presents specific data related to the examples in US Pat. No. 4,569,794 described above. In particular, the use of a metal chelate complex with His-Trp peptide associated with either the luteinizing hormone releasing hormone or proinsulin allows the chimeric peptide to be purified using IMAC, whereas control molecules lacking a His-Trp linker could not be isolated in a similar way.

Hochuli E. et al. J. Chromat. 411: 177-184 (1987) disclose a nitrilotriacetic acid absorbent used for affinity chromatography of a metal chelate complex. It is indicated that the disclosed absorbent charged with a Ni ²⁺ ion can be used to bind peptides and proteins containing neighboring histidine residues.

Ljungquist C. et al. Eur. J. Biochem. 186: 563-569 (1989) disclose the use of the Ala-His-Gly-His-Arg-Pro peptide, which forms a chelate complex with a metal in a series of columns consisting of two, four and eight columns, containing immobilized Zn ^{2+ ions} . According to Ljungquist C., the use of this peptide, which forms a chelate complex with zinc in columns, gives unexpectedly good results in the purification of fusion proteins.

 Used in the present description, the terms "unnatural fusion protein", "unnatural fusion polypeptide", "fusion polypeptides" and "fusion proteins" refer to proteins and polypeptides, which are usually not found in nature and which include a central fragment of the protein and its extension a fragment (or an extension of the protein).

 As used herein, the terms “protein core”, “central protein fragment” and “polypeptide fragment” refer to a region of a hybrid polypeptide that is located at the C-terminus of the molecule and does not include an extension portion that is the desired polypeptide and / or biologically active protein, including natural biological active proteins and polypeptides, as well as their analogues and mutants.

 As used herein, the term “N-terminal extension segment” refers to the first from the N-terminus of approximately 45 amino acids that are not part of the central region of the protein.

 Used in the present description, the term "prodrug" refers to hybrid proteins, where the desired part is a biologically active protein used as a medicine.

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

 As used herein, the terms “desired protein” and “desired protein” refer to proteins or polypeptides that need to be obtained in pure form.

 As used herein, the term “extension fragment” refers to that portion of the fusion protein that is the N-terminal extension segment and which is not part of the biologically desired region of the protein.

 As used herein, the term “DPP IV-cleavable N-terminal extension” refers to an extension of a fusion protein having an amino acid sequence that can be removed by stepwise cleavage using DPP IV.

 In the sequence listing presented at the end of the present description, some amino acid residues of the SegID sequences are designated Xaa. This designation means the following.

In SegID N 3 Xaa ²⁹ means a C-terminally amidated argininyl residue.

In SegID N 4, Xaa ²⁹ means a C-terminally amidated argininyl residue.

In SegID N 5, Xaa ²⁹ means a C-terminally amidated argininyl residue.

In SegID N 14, Xaa ²⁹ means a C-terminally amidated argininyl residue.

In SegID N 18, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 19, Xaa ³³ means a C-terminally amidated argininyl residue.

In SegID N 20 Xaa ³⁹ means a C-terminally amidated arginine residue.

In SegID N 21 Xaa ⁴⁵ means a C-terminally amidated arginine residue.

In SegID N 24, Xaa ²⁷ is a C-terminally amidated argininyl residue.

In SegID N 25, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 26, Xaa ³³ means a C-terminally amidated argininyl residue.

In SegID N 27, Xaa ³⁵ means a C-terminally amidated argininyl residue.

In SegID N 28, Xaa ³⁷ means a C-terminally amidated argininyl residue.

In SegID N 29, Xaa ³³ means a C-terminally amidated argininyl residue.

In SegID N 30, Xaa ³⁵ means a C-terminally amidated argininyl residue.

In SegID N 31, Xaa ³⁷ means a C-terminally amidated argininyl residue.

In SegID N 32, Xaa ³⁹ means a C-terminally amidated argininyl residue.

In SegID N 33, Xaa ⁴⁵ means a C-terminally amidated argininyl residue.

In SegID N 34, Xaa ⁴³ means a C-terminally amidated argininyl residue.

In SegID N 35, Xaa ⁴⁵ means a C-terminally amidated argininyl residue.

In SegID N 36, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 37, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 38, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 39, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 40, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 41, Xaa ³³ means a C-terminally amidated argininyl residue.

In SegID N 43, Xaa ³¹ means a C-terminally amidated argininyl residue.

In SegID N 43, Xaa ³³ means a C-terminally amidated argininyl residue.

 The invention relates to improved proteins and polypeptides. According to the present invention, biologically active polypeptides are first obtained in the form of hybrid proteins containing two parts: the first part, which is the central part of the protein; and the second part, which is an N-terminal extension fragment, which at its carboxy terminus is covalently linked to the amino terminus of the first part. The N-terminal extension of the hybrid polypeptide has an amino acid sequence, which is then cleaved by dipeptidyl peptidase IV (DPP IV).

The fusion protein of the present invention has the following formula:
Extension part - Central part of the protein
where the "Extension part" is a DPP IV-cleavable N-terminal extension part ";" - "represents a covalent peptide bond; and the" central part of the protein "is any desired protein that is separated from the" extension part "by processing with using DPP IV.

The extension fragment has the amino acid sequence of the formula
AXY- (X'-Y) _n ,
Where
A is optional, and if present, is methionine;
n represents the number of consecutively linked X '- Y - groups, the number of which is from 0 to 20, and preferably from 0 to 10;
X is selected from the group including all naturally occurring amino acids;
Y is selected from the group consisting of proline, alanine, serine and threonine, except that if 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 acids except proline and hydroxyproline.

 According to the above formula, if n = 1, then there are two residues Y. In addition, up to twenty-one residues Y and twenty residues X ′ may be present in one embodiment of the invention. The individual Y-residues and X'-residues may be respectively any residues of the group from which they are selected. That is, all of the individual Y residues need not be the same in this embodiment. Similarly, in any embodiment with more than one X ′ residue: each individual X ′ residue present may be any amino acid residue except proline and hydroxyproline, regardless of which residue the other X ′ residue will be. Each individual Y and X '- residue, respectively, must satisfy the rules for this particular group, that is, various individual residues in specific provisions must obey the rules defined above.

 Fusion proteins in which (A) is present as methionine (Met) are sequences used to produce biologically active proteins in E. coli using the recombinant DNA technique. The Met sequence present in these precursors is typically processed by the E. coli enzyme system or by any other method that can be carried out by any person skilled in the art. Protein synthesis in E. coli under normal conditions begins with the AUG translation initiation codon encoding Met. As a result, the newly synthesized polypeptides have a methionine residue in their N-terminal amino acid. E. coli has enzymatic activity capable of efficiently removing the N-terminal Met if the methionine N-terminal residue is adjacent to an amino acid having a relatively small side chain, such as Gly, Ala or Ser, as well as Pro. Highly specific removal of the N-terminal Met can be accomplished by bromine-cyan-mediated Met cleavage. However, for the successful implementation of this procedure, it is necessary that the N-terminal Met was only one Met in the entire protein sequence; otherwise, cleavage will occur after each Met in the sequence. Accordingly, in fusion proteins containing internal Met sequences, the second amino acid from the N-terminus must be Pro, Gly or Ser, if Met is to be removed by the E. coli enzyme system.

 In addition to fusion polypeptides, the present invention relates to recombinant DNA molecules containing DNA sequences encoding a hybrid polypeptide; to methods of using recombinant DNA molecules; methods of using hybrid polypeptides, including methods of purifying the desired polypeptides, and methods of drug delivery to the body, which consist of administering to the patient a precursor of said drug, which is converted into a biologically active form by sequential proteolytic cleavage in vivo of the N-terminal extension.

 The production of fusion polypeptides can be carried out using standard peptide synthesis or using the recombinant DNA technique well known to any person skilled in the art. Peptide synthesis is the preferred method for producing polypeptides that are composed of approximately 50 amino acids or less. For larger molecules, production in host cells is preferred using recombinant DNA techniques.

 Fusion polypeptides containing N-terminal parts that are recognizable and cleavable by DPP IV have greater advantages than unmodified polypeptides containing only one central part of the protein. The present invention describes two applications of these polypeptides. One such use relates to hybrid polypeptides, the so-called "prodrugs", which include biologically active polypeptides that are the desired drug and which are covalently linked to DPP IV-cleavable N-terminal extension segments. These prodrugs, i.e. drug precursors can be converted into biologically active forms by splitting DPP IV in the human body or another animal to which the indicated prodrug has been administered. Accordingly, the present invention relates to hybrid polypeptides used as prodrugs; to the use of hybrid polypeptides in the manufacture of drugs and to a method for delivering biologically active polypeptides to the patient. A second application of the hybrid polypeptides of the present invention is a protein purification method in which the N-terminal extension portion which is a component of a polypeptide, due to its ability to cleave by DPP IV, facilitates efficient purification of the desired polypeptide. Accordingly, the present invention relates to hybrid polypeptides used in purification procedures, and to methods for purifying desired polypeptides. These application examples of the present invention are for illustrative purposes only and should not be construed as limiting the present invention.

 In the two indicated uses of the present invention, the central portion of the protein is separated from the extension portion by DPP IV activity. In the case where fusion proteins are used in purification methods, it is undesirable for the central part of the protein to serve as a substrate for DPP IV cleavage. That is, it is preferred that DPP IV does not have the ability to cleave the central portion of the protein after the extension portion has been removed. Often it is more preferable, when the central part of the protein is a substrate for DPP IV, to use the fusion protein as a prodrug. In this case, the indicated drug precursor may contribute to the continued presence of the core of the protein in the body, since the DPP IV present in vivo (for example, in plasma, kidney and liver tissues) will be used to process the N-terminal extension sites, and thereby contribute to the delay decomposition of the central part of the protein. That is, the extension portion of the fusion protein can act as a substrate for DPP IV and a competitive inhibitor that slows down the effects of DPP IV on the central part of the protein, thereby protecting the specified central part of the protein.

 As used herein, the term “prodrug” means a fusion protein comprising a DPP IV-cleavable N-terminal extension portion covalently linked to a central portion of a protein that is a biologically active polypeptide used as a medicament. According to the present invention, the prodrug can be introduced as a separate prior form of the drug or in combination with other compounds. In a preferred embodiment of the present invention, a separate form is used as a prodrug. In any case, the aforementioned prior form of the drug is processed by the natural DPP IV normally present in the patient.

 The advantage of administering a drug containing the previous dosage form is that the drug helps to delay activity and / or ensures the presence of a biologically active protein in the body for a longer period of time. Said drug precursor may remain active longer than unmodified molecules. A prodrug can exist in an inactive state for a certain period of time until the extension part is cleaved, after which the molecule becomes active. Therefore, the prodrug can serve as a sustained release drug delivery system. In addition, various N-terminal extension segments are cleaved at different rates depending on their length and the specific residues present in their amino acid sequence. Various forms of drug precursors may also be used, having various N-terminal extension segments that can provide a sustained, constant level of active drug over a period of time in the patient. Therefore, the prodrug can serve as a sustained release drug delivery system.

 As indicated above, DPP IV cleaves the dipeptide from the N-terminus of the polypeptide, provided that certain residues occupy certain positions. As used herein, the term “position one” refers to the position of an amino acid residue at the N-terminus. The term “position two” refers to the position of an amino acid residue that is directly adjacent to the amino acid residue at position one, i.e. is the second residue from the N-terminus. The term “position three” refers to the position of an amino acid residue that is directly adjacent to the amino acid residue at position two, i.e. the third residue from the N-terminus. Cleavage of the N-terminal peptide occurs between position two and position three, provided that the amino acid at position three is not proline or hydroxyproline, and the amino acid at position two is one of the following five amino acids: proline (Pro), hydroxyproline (Hyp), alanine ( Ala), serine (Ser) or threonine (Thr).

 DPP IV cleaves N-terminal residues at different rates depending on which of these four amino acid residues is present at position 2. In most cases, the highest efficiency of DPP IV cleavage is achieved when Pro is in the 2-position, and after it the highest efficiency is achieved when position two is occupied by Ala. If tyrosine, phenylalanine or histidine is in position one, then DPP IV acts at almost the same speed as when Pro or Ala is in position two. Then, in relation to the greatest efficiency, there comes a case when Ser is in position two. And the case when Thr is in the 2-position is already the least effective.

 Based on this information, a series of N-terminal extension segments can be constructed that are processed at different speeds. So, for example, a drug may be administered that consists of either a particular drug precursor or a combination of precursors. These precursors with suitably selected N-terminal extension segments will each be processed at a rate dependent on their amino acid sequences. The combination of these previous dosage forms can be made up of a series of precursors processed into active polypeptides over various time periods.

 The length and composition of the amino acid sequence are regulated factors regarding the rate of DPP-IV cleavage. Extension parts containing all or almost all of the alternating Y = Pro will process faster than those containing Y = Thr. In addition, it is known that X - Pro dipeptidyl units, where X is either GIu or Asp, are cleaved much more slowly than their counterparts, where X is a neutral or basic amino acid residue. Since the extension parts may contain different residues (of the four indicated) at each cleavage position, a very large number of variations and permutations can exist.

 As the polypeptide drug, any active polypeptide can be used. In patent application PCT N PCT / US 90/02923, in patent application PCT N PCT / US91 / 08248 and in patent application US reg. N 07/368231 (which are incorporated herein by reference) discloses bovine growth hormone release factor analogues that can be used in a medicament as a prodrug of the present invention. Any of the analogs described in these applications can be used as the central part of a fusion protein peptide in accordance with the present invention. Hybrid proteins containing these central parts associated with lengthening parts can be produced by standard methods known to those skilled in the art.

 As other embodiments of the present invention, hormones, receptors, enzymes, storage proteins and blood proteins can be used. Specific examples are: vasoactive intestinal peptide (GIP); human β-casomorphine; gastric inhibitory peptide (GIP); gastro-releasing peptide (GRP), human HI peptide, human YY peptide; fragment 7-37 glucagon-like peptide 1; glucagon-like peptide 2; substance P; neuropeptide Y; human pancreas polypeptide; insulin-like growth factor 1 (IGF-I); human growth hormone (hGH); bovine growth hormone (bGH); porcine growth hormone (pGH); prolactin (PPL); human, bovine, swine or sheep growth hormone (GPF) release factor; interleukin-I β (IL-I β); EGF; IGF-2; glucagon; corticotropin-releasing factor (CPF); dynorphin; somatostatin-14; endothelin; growth transformation factor alpha (TGF-α); growth transformation factor beta (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 their synthetic or biosynthetic analogues. These polypeptides can also be used to form the central portion of the fusion proteins of the present invention. These polypeptides serve only as an example of implementation of the present invention and should not be construed as a limitation of the scope of the present invention.

 According to the present invention, smaller fusion proteins can be synthesized, for example, by solid phase technology using a peptide synthesizer (Applied Biosystems 430 A, Foster City, California), which is described in detail in PCT / US90 / 02923 and 07/368231.

 Larger molecules are preferably produced in host cells using recombinant DNA techniques. There are several different methods that any person can use to produce hybrid proteins using the recombinant DNA technique. Typically, genes encoding the desired polypeptides are inserted into expression vectors, which are then used to transform or transfect the corresponding host cells. The inserted gene is then expressed in the host cell and the desired polypeptide is produced. To produce the hybrid polypeptides of the present invention by this method, an additional DNA sequence is included in the insert gene. In particular, DNA encoding the residues of the N-terminal extension is suitably cross-linked to the 5'-end of the gene encoding the desired polypeptide. This additional gene material should be located below (5 '-> 3') from the promoter of the expression vector so that it is under the control of the promoter. In addition, it must be in the proper reading frame with the gene so that the expressed protein product contains residues of the N-terminal extension portion covalently linked to the desired polypeptide.

 Therefore, for the production of the fusion proteins of the present invention using recombinant DNA techniques, oligonucleotides that encode the amino acid sequence of the desired N-terminal extension part and which must be properly inserted, i.e. they should be located above the 5'-end of the gene encoding the central part of the protein so that they generate a chimeric gene. The technique for constructing oligonucleotides and the technique for producing a chimeric gene are well known to every person skilled in the art.

 In addition to the use of fusion proteins as a prodrug, the present invention can be used for the purification and processing of biologically active recombinant polypeptides. The desired biologically active recombinant polypeptides are most preferably obtained in soluble form or secreted from host cells. According to the present invention, an extension of the fusion protein can be detected by purification. The fusion protein is purified from the material present in the secreting medium or extraction solution in which it is contained, and then processed to remove the extension portion from the central portion of the protein, thereby obtaining the desired purified protein. Accordingly, the desired proteins that are most suitable for processing as the fusion proteins of the present invention are biologically active polypeptides that are not substrates for DPP IV cleavage per se.

 In accordance with the present invention, the gene sequence encoding the desired protein is isolated, synthesized or obtained in any other way and is properly crosslinked with the DNA sequence encoding the extension part. A hybrid gene containing the desired protein gene and properly crosslinked with the DNA sequence encoding the extension portion refers to chimeric genes.

 Methods and materials for producing chimeric genes and recombinant vectors, transformation or transfection of host cells using these materials; replication of host vectors and expression of biologically active foreign polypeptides and proteins are described in Principles of Gene Manipulation, by Old and Primrose, 2nd ed., 1981 and Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, N 4, 1989 (both of these works are introduced into the present description by reference).

 The invention relates to recombinant chimeric genes encoding fusion proteins; expression vectors containing the indicated chimeric genes; to hosts transformed or transfected with the indicated expression vectors; and to methods for producing said genes expressing vectors and host cells transformed or transfected with these vectors.

 The invention can be used to purify any prokaryotic or eukaryotic protein that can be expressed as a recombinant DNA product in transformed or transfected host cells. These recombinant protein products may include hormones, receptors, enzymes, storage proteins, blood proteins, mutant proteins obtained using protein engineering techniques, or synthetic proteins. The polypeptides desirable for production may be HIV-RNase-H, tPA, IL-I, IL-I receptor, CD4, human nerve cell growth factor, SCD4-PE40, FG-chimeric respiratory syncytial virus P (see U.S. Patent Application No. 07/543780 (incorporated herein by reference), EGF, IGF-1, IGF-2, glucagon, corticotropin-releasing factor (CRF), dynorphin, endothelin, growth transformation factor alpha (TGF) - α), endotoxin 40 Pseudomonas, growth transformation factor-β (TGF-β), insulin and its analogues.

 Examples of purification methods are IMAC or immuno-affinity. Other purification methods are included within the scope of the present invention, which involve the use of peptides with extension parts that can be removed by DPP IV.

 In one embodiment of the present invention, fusion proteins comprising a biologically active polypeptide moiety and an extension moiety, which is a metal-peptide chelate complex, can be used in an affinity to immobilized metal chromatographic system.

 IMAC immobilized metal affinity chromatography for protein fractionation was first described by Porath J. et al., Nature 258: 598-599 (1975). Porath discloses the derivatization of an iminodiacetic acid resin (IDA) and the formation of a chelate complex of metal ions with an IDA-derivatized resin. Porath also discloses proteins that can be immobilized in a column containing immobilized metal ions. This method involves binding the commonly used iminodiacetic acid (IDA) to a matrix, followed by the formation of a chelate complex of metal ions with an IDA-containing resin. Proteins bind to metal ions through functional groups of amino acid residues capable of electron transfer. Potential electron donors include amino acid residues such as cysteine, histidines, and tryptophan. Proteins interact with metal ions through one or more of these amino acids having electron-donating side chains.

 Smith et al. In US Pat. No. 4,569,794, incorporated herein by reference, have shown that certain amino acid residues are responsible for binding the protein to immobilized metal ions. However, these bound proteins can be eluted by lowering the pH or by using competitive anti-ligands, such as imidazole, if histidine side chains are involved. In order to demonstrate that IMAC is a selective purification method, histidine-containing di- or tri-peptides in proteins were used. Accordingly, Smith et al. Have described the use of recombinant DNA technology to produce a fusion protein containing a peptide that forms a chelate complex with a metal and is covalently linked to the desired polypeptide. The peptide forming the chelate complex with the metal is an extension that is effective for the desired polypeptide. This fact can be used to purify protein.

 The use of the IMAC technique with chelated metal-peptide complexes having alternating histidine residues is disclosed in US Pat. N 07/506605, which is introduced into the present description by reference. In US Patent Application Reg. N 07/506605 describes specific peptides that form chelate complexes with a metal, the use of which in IMAC purification of a fusion protein gives unexpectedly high results when the indicated metal-peptide negligent complex contains from three to six alternating His residues. In accordance with the description of the patent application US reg. 07/507605 and US Pat. No. 4,569,794 can be used with the usual IDA resin in IMAC for the purification of fusion proteins having a chelated metal-peptide moiety with at least three alternating histidine residues that are an integral part of DPP-IV recognized sequences. The construction of fusion proteins and their use in the IMAC system are described in US Pat. No. 4,569,794. The construction and use of a chelated metal-peptide moiety containing alternating histidine residues is described in US Patent Application Reg. N 07/506605. In accordance with the present invention, a fusion protein provided with DPP-IV-recognizable residues located between alternating histidine residues can be purified using IMAC technology followed by processing using DPP-IV to form the desired polypeptide.

 Another protein purification system that uses fusion proteins, and which is well suited to the DPP IV processing technique, is immunoaffinity purification. US Pat. No. 4,782,137, issued November 1, 1988 to Hopp et al., Which is incorporated herein by reference, discloses the synthesis of a fusion protein having a highly antigenic N-terminal portion and the desired polypeptide at its C-terminal portion. According to Hopp et al., Fusion proteins are purified from the supernatant by passing this supernatant through a column containing immobilized antibodies that recognize the antigenic portion of the fusion protein. These immobilized antibodies hold the protein in the column, while unnecessary components of the supernatant elute. Then the conditions in the column are changed and thereby stimulate the dissociation of the antigen-antibody complex.

 In accordance with the present invention, such a highly antigenic N-terminal portion of the fusion protein is an extension portion that contains DPP-IV-recognized residues. After harvesting in accordance with the procedure described in the Hopp patent, the fusion protein of the present invention can be exposed to the DPP IV enzyme, whereby the extension portion is removed. Thus, any person skilled in the art can use the Hopp immunoaffinity purification system using the N-terminal extension parts of the present invention.

 Presented in the present description, the options and embodiments of the present invention serve only to illustrate the invention and should not be construed as a limitation thereof. As equivalents, hybrid proteins having N-terminal extension parts and processing ability in at least one other way can be considered, so that the removal of the extension parts is due to a combination of these methods. Hybrid polypeptides containing chemically modified amino acid residues may also be considered as possible equivalents.

 Example 1. Synthetic drug precursors, which are fusion prodrugs containing the Central parts of the proteins, which are substrates for DPP IV.

Fusion polypeptides that can be synthesized and introduced as prodrugs have a DPP IV cleavage N-terminal extension portion covalently linked to the N-terminus of the biologically active polypeptide. These drug precursors can be represented by the following formula:
The extension part is the central part of the protein drug
where the “extension portion” is a DPP IV-cleavable N-terminal extension portion; "-" represents a covalent peptide bond; and the "central portion of the protein" is any desired peptide that is cleaved from the extension portion by DPP-IV processing. In this example, the central portion of the fusion protein is a potential substrate for DPP IV after removal of this extension portion.

 Synthetic drug precursors can be prepared using a peptide synthesis technique well known in the art.

In one embodiment of the present invention, the central part of the protein is epidermal growth factor (EGF), and the elongated part of the protein is Gly-Pro-Phe-Ala
Gly ⁴ -Pro ^-3 -Phe ^-2 -Ala ^-1 - [EGF].

In another embodiment, the central part of the protein is glucagon and the extension is Ala-Pro-Phe-Ala
Ala ^-4 -Pro ^-3 -Phe ^-2 -Ala ^-1 - (GLUGAGON).

In a further embodiment of the present invention, the central portion of the protein is [Ala ¹⁵ Leu ²⁷ ] -bGRF (1 - 29) NH ₂ (Seg. ID N3), and the extension portion is Tyr-Ala
Tyr ^-2 -Ala ^-1 {[Ala ¹⁵ Leu ²⁷ ] - bGRF (1 - 29) NH ₂ }.

 Example 2. Synthetic drug precursors, which are hybrid prodrugs containing central parts of proteins that are not substrates for DPP IV.

Hybrid polypeptides that can be synthesized and introduced as prodrugs have a DPP IV-cleavable N-terminal extension portion covalently linked to the N-terminus of the biologically active polypeptide. These drug precursors can be represented by the following formula:
The extension part is the central part of the protein drug
where the "extension portion" is a DPP IV-cleavable N-terminal extension portion; "-" represents a covalent peptide bond; and the "central portion of the protein" is any desired peptide that is cleaved from the extension portion by DPP IV processing.

 Synthetic drug precursors can be prepared using a peptide synthesis technique well known in the art.

In one embodiment of the present invention, the central part of the protein is an analog of bGRF, [Val ² , Ser ^8.28 , Leu ²⁷ ] - bGRF (1 - 33) OH (Last II NI), and the extension is Gly-Pro-Tyr -Ala
Gly ^-4 -Pro ^-3- Tyr ^-2 -Ala ^-1 - {[Val ² Ser ^8.28 Ala ¹⁵ Leu ²⁷ ] bGRF (1 - 33) OH}.

In another embodiment, the central portion of the protein is corticotropnin release factor CRF, and the extension portion is Gly-Pro-Phe-Ala
Gly ^-4 -Pro ^-3 -Phe ^-2 -Ala ^-1 - [CRF].

In a further embodiment of the present invention, the central part of the protein is dynorphin and the extension part is Phe-Pro-Phe-Ala
Phe ^-4 -Pro ^-3 -Phe ^-2 -Ala ^-1 - [DYNORFIN].

In yet another embodiment of the present invention, the central part of the protein is somatostatin-28, and the extension is Gly-Pro-Phe-Pro
Gly ^-4 -Pro ^-3 -Phe ^-2 -Pro ^-1 - [SOMATOSTATIN-28].

In another embodiment of the present invention, the central part of the protein is endothelin, and the extension is Ala-Pro-Phe-Ala
Ala ^-4 -Pro ^-3 -Phe ^-2 -Ala ^-1 - [ENDOTHELIN].

In a further embodiment of the present invention, the central part of the protein is the bGRF analogue of [Ile ² Ser ^8.28 Ala ¹⁵ Leu ²⁷ ] - bGRF (1 - 40) OH (SerID2), the extension part is Phe-Ala
Phe ^-2 -Ala ^-1 - {[Ile ² Ser ^8.28 Ala ¹⁵ Leu ²⁷ ] -bGRF (1-40) OH}.

In another embodiment of the present invention, the central part of the protein is [Ile ² Ala ¹⁵ Leu ²⁷ ] -bGRF (1 - 29) NH ₂ (SeqID 4), and the extension part is Tyr-Ser
Tyr ^-2 -Ser ^-1 - {[Ile ² Ala ¹⁵ Leu ²⁷ ] -bGRF (1 - 29) NH ₂ }.

Example 3. The prolonged presence of the analog bGRE Leu ²⁷ - bGRF (1 - 29) NH ₂ .

bGRF - Analog, Leu ²⁷ - bGRF - (1 - 29) NH ₂ (its sequence is shown as SeqID N 5) can be introduced as a drug containing the Central part of the protein shown in SeqID 5, and a number of precursors with N-terminal extending parts.

Several variants of the precursors can be obtained by well known methods using SeqID 5 as the Central part of the protein. The extension parts for these SeqID 5 derived precursors are
Ile-Ala, Gly-Pro-Ile-Pro, SeqID 6, SeqID 7, Tyr-Ala, Gly-Pro-Tyr-Ala, SeqID 8, SeqID 9, SegID 10, SegID 11, SeqID 12, SegID 13, Tyr- Ala-Tyr-Ala and Val-Ala.

Example 4. The prolonged presence of bGRF analogue [Thr ² Ala ¹⁵ Leu ²⁷ ] -bGRF (1 - 29) NH ₂ .

bGRF analog, [Thr ² Ala ¹⁵ Leu ²⁷ ] - bGRF (1 - 29) NH ₂ (its sequences are shown as SeqID 14) can be introduced as a drug containing the Central part of the protein shown in SegID 14, and a number of precursors with N-terminal extension parts. Three precursor variants were made having Tyr-Thr, Tyr-Ser and Tyr-Thr-Tyr-Thr extension parts, respectively.

 Example 5. Hybrid proteins that contain HIV-RNase H and N - terminal extension parts.

 The technique for purification of chimeric proteins from recombinant E. coli is based on peptide domains that form a chelate complex with a metal that contain alternating histidines and which have an affinity for immobilized metal ions. The vectors were designed to synthesize fusion proteins using HIV-RNase H as the central part of the protein. As indicated below, hybrid proteins containing alternating histidines were designed for purification using affinity chromatography on immobilized metal ions (IMAC), as well as alternating prolines or alternating alanins for DPP IV cleavage to isolate the peptide (mcp) forming chelated complex with metal.

 In accordance with the present invention, preferred DPP IV-cleavable N-terminal extension parts are listed below.

The HIVRH / mcp N 1 fusion protein includes the N-terminal extension of the Seg ID 15 sequence linked to HIV RNase H
Met ^-11 -Pro ^-10 -Ala ^-9 -His ^-8 - Pro ^-7 -His ^-6 -Pro ^-5 -His ^-4 -Pro ^-3 - His ^-2 -Ala ^-1 - [HIV-RNase H] .

The HIVRH / mcp N 2 fusion protein includes the N-terminal extension of the Seg ID 16 sequence linked to HIV RNase H
Met ^-11 -Ala ^-10 -Pro ^-9 -His ^-8 - Ala ^-7 -His ^-6 -Ala ^-5 -His ^-4 -Ala ^-3 - His ^-2 -Ala ^-1 - [HIV-RNase H] .

The HIVRH / mcp N 3 fusion protein includes the N-terminal extension of the Seg ID 17 sequence linked to HIV RNase H
Met ^-11 -Gly ^-10 -Pro ^-9 -His ^-8 - Pro ^-7 -His ^-6 Pro ^-5 -His ^-4 -Pro ^-3 - His ^-2 -Ala ^-1 - [HIV-RNase H].

 These fusion proteins were cloned and expressed in E. coli and purified using DEAE chromatography and reverse phase HPLC. To characterize the hybrid proteins used N-terminal sequencing. The use of hybrid proteins with alternating histidines for purification of recombinant proteins using IMAC and subsequent removal of the N-terminal extension parts by DPP IV confirms the value of the present invention.

 Construction of chimeric genes containing the HIV RNase H gene.

 All recombinant DNAs were obtained using standard techniques. Oligonucleotides corresponding to the sequence of the chelating complex metal peptide and the cleavage sequence were designed, purified, annealed and doped with the gene encoding HIV-RKase-H to form a chimeric gene.

 To obtain expression vectors encoding alternating histidines / DPP IV-recognizable cleavable residues / HIV-RNase-H, a chimeric gene was introduced into the final expression vector. Expression vectors containing the engineered gene were used to transform E. coli using standard techniques. As a result of the expression of these genes in E. coli, hybrid proteins encoded by chimeric genes are obtained, these hybrid proteins contain HIV-RNase-H amino acids and N-terminal extension parts with alternating histidines (a peptide forming a chelate complex with a metal) and alternating proline or alanine .

 Obtaining the original E. coli extract and isolation of hybrid proteins for sequencing.

Approximately 3 g of E. coli cell paste was suspended in 30 ml of 0.25 M potassium phosphate, pH 7.2, containing 1 mM dithiothreitol (DTT), EDTA, phenylmethyl sulfonyl phosphate (PMSF), and benzamidine HCl, 10 mg / l aprotinin, Leuciptin and Bestain. This suspension was passed three times through a French press to destroy cells. Cell lysates were centrifuged for 1 h at 12,000 rpm. Then the supernatant was removed and ammonium sulfate was added to 70% saturation. After stirring for 1 hour, the suspension was centrifuged for 1 hour at 12,000 rpm. The supernatant was discarded and the pellet was again dissolved in 2.25 ml of 50 mM Tris pH 7.5 containing 1 mM DTT, PMSF and benzamidine. Then, the resulting solution was dialyzed overnight in 20 mM Tris, 50 mM NaCl, 1 mM DTT, 10% glycerol, and 0.1 mM EDTA, pH 7.5 (Buffer A) at 4 ^° C. The dialysate was collected, diluted in one volume Buffer A and introduced into a 10 ml column with washed DEAE-cellulose balanced in Buffer A. This process was carried out in batches and after each batch, the column was washed with 50 ml of Buffer A. The resulting solutions were collected, combined and concentrated by precipitation with 70% ammonium sulfate and then resuspended in 2 ml of Buffer A and dialyzed as described above. Concentrated PH was used for characterization using N-terminal sequencing.

 Purification of fusion proteins using IMAC.

 The possibility of using a chelate complex forming metal peptide to purify recombinant proteins from crude extracts was illustrated using the following chimeric compounds expressed in recombinant E. coli with HIV-PHase H, taken as a protein model. Each of the fusion proteins: HIV-RNase-H / mcp N 1; HIV RNase H / mcp N 2 and HIV RNase H / mcp N 3 were purified.

IMAC columns were prepared as follows. Pharmacia's chelating sepharose rapid flow was thoroughly washed with water (Milli-Q) on a tempered glass filter. Then the gel was resuspended in water to form a suspension. This suspension was carefully poured into a glass column (Pharmacia) to a volume of 6 ml (1 x 7 cm). After gel deposition, the column was washed with 5 volumes of 50 mM EDTA (ethylenediaminetetraacetic acid) (pH 8.0). After that, the column was washed with 5 volumes of 0.2 NaOH and 5 volumes of Milli-Q water. Then the column was loaded with 5 volumes of 50 mm NiSO ₄ (or ZnCl ₂ or CuSO ₄ ). Finally, the column was washed with 5 volumes of equilibration buffer. A balancing buffer was prepared from 20 mM Tris (pH 8.0) containing 500 mM NaCl, 1 mM PMSF, 1 mM benzamidine, 10 mg / L leupeptin and 10 mg / L aprotinin.

 The column was balanced with at least 5 volumes of equilibration buffer. 5-10 ml of the original extract of recombinant E. coli was introduced into the column by gravity. After all the starting material was introduced into the column, this column was washed with 10 volumes (relative to the column volume) of a equilibration buffer containing 1.0 NaCl (instead of 500 mM NaCl) pH 8.0.

 The column was then eluted with increasing concentrations of imidazole in equilibration buffer at pH 8.0. In earlier experiments, in each of them, a large number of elutions were performed to determine the concentration at which the chimeric material eluted. Then the number of these elutions was reduced and as a result, only three imidazole concentrations were used: 35 mM, 100 mM and 300 mM imidazole in equilibration buffer, pH 8.0. Used 10 volume layer of each imidazole buffer. Between elutions, the column was washed with 10 volumes of equilibration buffer. Finally, the column was washed with 5 volumes of a 50 mM EDTA pH 8.0 layer to determine if any other protein was bound to the column. The flow rates for the columns were 1.0 ml / min. 5 mm fractions were collected. Work with columns was carried out at room temperature.

 To determine the protein content in the samples, a commercially available Pierce protein assay kit was used.

 The activity of HIV-RNase-H was determined by the method described in FEBS 270 (1,2): 76 - 80 (September 1990), Becerra S.P. and others (this work is introduced into the present description by reference).

 Conversion of fusion proteins with N-terminal extension segments into mature proteins.

For these purposes, commercially available DPP IV isolated from human placenta (Enzyme Systems, Product Dublin, Ca) with a specific activity of 5200 m units was used. / mg protein. One unit of equivalent is the hydrolysis of 1 mM synthetic substrate (Ala-Pro-7-amino-4-2-trifluoromethylcoumarin) per minute at pH 7.8. Enzyme conversion was carried out by incubation of the fusion protein (about 1-100 mg) at a concentration of 1-10 mg / ml with DPP IV at 25 ^° C for 30 min and with the ratio of the enzyme to the substrate 1: 100 (by weight). The desired polypeptide was isolated from the unsplit fusion protein using IMAC. The authenticity of the obtained product was confirmed using N-terminal sequencing.

 Example 6. In vitro processing of bGRF analog precursors in bovine plasma.

In the table. 1, typical experiments are systematized to illustrate the generation of the central part of the peptide [Leu ²⁷ ] -bGRF (1 - 29) NH ₂ (SeqLD 5) from three analogues with N-terminal extension regions Tyr ^-4- Ala- ³ - Tyr ^-2- Ala ^{- 1} - {[Leu ²⁷ ] -bGRF (1 - 29) NH ₂ } (SeqID 19), Ile ^-2 -Ala ^-1 - {[Leu ²⁷ ] -bGRF (1 - 29) NH ₂ } (SeqID 18) and Tyr ^-2 -Ala ^-1 - {[Leu ²⁷ ] - bGRF (1 - 29) NH ₂ } (SeqID 25) by incubation with bovine plasma in vitro. Only those metabolites that were products of DPP-IV-associated cleavage were detected in the incubation mixture.

Tyr ^-4 -Ala ^-3 -Tyr ^-2 -Ala ^-1 - {[Leu ²⁷ } -bGRF (1 - 29) NH ₂ } (SeqID 19) was subsequently converted to Tyr ^-2 -Ala ^-1 - {[Leu ²⁷ ] -bGRF (1 - 29) NH ₂ } (SeqID 25), [Leu ²⁷ ] - bGRF (1 - 29) NH ₂ (the central part of the peptide, SeqID 5) and, finally, in [Leu ²⁷ ] - bGRF (3 - 29) NH ₂ (SeqID 24).

Tyr ^-2 -Ala ^-1 - {[Leu ²⁷ ] -bGRF (1 - 29) NH ₂ } (SeqID 25) was converted to [Leu ²⁷ ] -bGRF (1 - 29) NH ₂ (central part of the protein, SeqID 5) and then in [Leu ²⁷ ] -bGRF (3 - 29) NH ₂ (SeqID 24). The very central part of the [Leu ₂₇ ] protein - bGRF (1 - 29) NH ₂ (SeqID 5) was converted to [Leu ²⁷ ] - bGRF (3 - 29) NH ₂ (SeqID 24) using DPP-IV plasma.

Even if no other metabolites were observed under the HPLC conditions used in the experiments, the peptide [Leu ²⁷ ] -bGRF (3-29) NH ₂ (SeqID 24) disappeared over time, indicating that there should also be other proteolytic reactions not associated with DPP-IV and having significantly lower rates. It was not only shown that a central bGRF peptide was generated from the DPP-IV-cleavable bGRF precursors, but also that the half-life of the central protein generated from the fusion protein was significantly longer in vitro than the half-life of the central peptide (SeqID 5) incubated with bovine plasma (table. 1). Moreover, the time at which the central peptide is released from the precursor obviously correlates well with the length of the extension of the precursor: the half-life of the sequence (SeqID 5) generated from the precursor with 4 amino acid residues in the extension (SeqID 19) is significantly longer than the half-life of a central peptide derived from proGRF having only two amino acids in its extension portion, as in SeqID18 or 25.

 Example 7. In vivo and in vitro - the bioactivity of the precursors of the bGRF analog.

 As shown in the table. 2, plasma levels of growth hormone (GH) in Holstein gobies increased when these gobies were injected with an intravenous SeqID 18 analogue. At a dose of 0.2 nM per 1 kg of body weight, the levels of induced growth hormone in the plasma were compared with the levels generated after injection i. v. the same dose of the central peptide (SeqID 5). It is important to note that the elongated SeqID 18 peptide had only about 5% of its intrinsic activity compared to the central SeqID 5 protein according to an in vitro assay for pituitary cells for GH release. Therefore, a comparison of the in vivo activity for the two peptides obviously indicates that the central peptide was isolated from the extended in vivo peptide.

 The same in vivo GH release pattern was also observed when treated with bGRF precursors having 4 amino acid residues in the extension portion (SeqID 19) (see table 3). As the results showed, there was no significant difference in the in vivo-induced GH release after treatment with precursors having 4 or 2 amino acids in their extension portion (SeqID 19 and SeqID 18 peptides, respectively), despite a significant difference in the in vitro central half-life peptide generated from these two bGRF precursors in virto, as shown in table. 1. In vivo release of growth hormone was fast and the same for the central peptide SeqID 5, as well as for SeqID 19 and 18, with no difference in GH peaks in time after administration of bGRF analogues.

 As an interpretation of these results, it can be assumed that the indicated rapid release of GH from precursors in vivo is most likely due to, generally, high DPP IV levels in tissues and organs, which are approximately 100 times higher than plasma DPP-IV concentrations. This assumption also explains the difference in the processing speed of in vivo precursors compared to the proteolysis observed in in vivo experiments, which are systematized in Table. 1. It is also possible that the half-life of the central peptide generated from its precursors was long in vivo, but insufficient to detect altered (prolonged) release of growth hormone. In vivo release of GH is known to be modeled by the stimulatory effects of bGRF and the inhibitory effects of somatostatin (somatotropin release inhibiting factor, SRIF). For the model of calves fed on meat, Moseley et al. (J. Endocr. 17, 253–259, 1988), animals were injected with GRF intravenously two hours before feeding, since the pituitary gland is more susceptible to GRF stimulation before feeding than after feeding. Feeding factors, such as intestinal / pancreatic SRIF, may interfere with the ability of the pituitary gland to release GH. In other words, if there is any amount of SRIF, then GH will not be released from the pituitary gland even in the presence of GRF. Typically, in a model of a bull calf fed to meat that has not been given a stimulant, the concentration of GH in serum drops to its initial levels within 3-6 hours after feeding (the so-called minimum period), while another impulse of exogenous episodic GH occurs after 5 - 8 hours after feeding. When injected 2 hours before feeding, the GH response occurs very quickly, after 5 to 20 minutes, and the GH level remains elevated for 120 to 240 minutes, and then returns to its original level. So far, in the case of test GRF precursors, only the first peak of exogenous GH was elevated, and the second did not show any effect of the administered dose. It is possible that the half-life of the central GRF generated from the precursors in these experiments was not long enough for the central peptide to be present in the bloodstream in concentrations sufficient to affect the second release of exogenous GH, which usually takes place 4-6 hours after first.

 Summarizing the above, it can be argued that the results provide evidence of the basic concept of the use of drug precursors disclosed in this application, since (I) bGRF precursors having DPP-IV-cleaved N-terminal extension sites were successfully processed to form a central peptide in bovine plasma in vitro using DPP-IV-mediated proteolysis; (Ii) the in vitro half-life of the central peptide generated from its precursor was quite long and depended on the length of the N-terminal extension in the precursor; (III) the fact that a bGRF precursor with very low natural activity is as effective as the central peptide in in vivo GH release indicates that, in all likelihood, the central protein is generated in vivo, as expected.

Example 8. Obtaining Gly ^-4 -Pro ^-3- Ile ^-2 - Pro ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, salts of trifluoroacetic acid.

Synthesis of Seq ID 26 Peptide of a GRF Analogue of the Formula Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln -Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in the published PCT patent application / US90 / 029-23, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.16 (4); Thr 1.07 (1); Ser 1.81 (2); Glu 2.07 (2); Pro 0.98 (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. Obtaining Tyr ^-6 -Ala ^-5 -Gly ^-4 - Pro ^-3 -Ile ^-2 -Pro ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

The synthesis of the peptide SeqID 27 GRF analogue containing SeqID 6 in the form of an extension part and having the formula 3H-Tyr-Ala-Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ile-Phe-Thr-Asn-Ser-Tyr- Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt), was performed stepwise, similar to procedure A described in published patent application PCT / US90 / 02923, which is incorporated into this description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.04 (4); Thr 1.03 (1); Ser 1.74 (2); Glu 2.05 (2); Pro 1.99 (2); Gly 2.00 (2); Ala 4.01 (4); Val 1.28 (1); Ile 2.84 (3); Leu 5.09 (5); Tyr 2.94 (3); Phe 0.97 (1); Lys 2.07 (2); Arg 3.00 (3).

Example 10. Obtaining Lys ^-8 -Pro ^-7- Tyr ^-6 - Ala ^-5 -Gly ^-4 -Pro ^-3- Ile ^-2 -Pro ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salts.

The synthesis of the peptide SeqID 28 GRF-analogue containing SeqID 7 in the form of an extension part and having the formula Lys-Pro-Tyr-Ala-Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn- Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt ) was carried out stepwise, similar to procedure A described in published patent application PCT / US90 / 02923, which is incorporated into this description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.04 (4); Thr 0.95 (1); Ser 1.78 (2); Gly 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. Obtaining Gly ^-4 -Pro ^-3- Tyr ^-2 - Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

Synthesis of Seq ID 29 peptide of a GRF analogue having the formula N 6 Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly -Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to procedure A described in published application for PCT / US90 / 029-23, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.01 (4); Thr 0.96 (1); Ser 1.80 (2); Gln 2.02 (2); Pro 0.97 (1); Gly 1.98 (2); Ala 3.91 (4); Val 0.99 (1); Ile 1.89 (2); Leu 5.08 (5); Tyr 3.05 (3); Phe 0.98 (1); Lys 2.03 (2); Arg 3.06 (3).

Example 12. Obtaining Tyr ^-6 -Aka ^-5 -Gly ^-4 - Pro ^-3 -Tyr ² -Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

The synthesis of the peptide SeqID 30 GRF-analogue containing SeqID 8 in the form of an extension part and having the formula # 7 Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser- Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOOH salt) was performed stepwise, similar to procedure A described in published patent application PCT / US90 / 02-923, which is incorporated into this description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.07 (4); Thr 0.96 (1); Ser 1.79 (2); Glu 2.02 (2); Pro 0.99 (1); Gly 1.95 (2); Ala 4.80 (5); Val 0.96 (1); Ile 1.87 (2); Leu 5.09 (5); Tyr 4.11 (4); Phe 0.97 (1); Lys 2.06 (2); Arg 3.08 (3).

Example 13. Obtaining Lys ^-8 -Pro ^-7- Tyr ^-6 - Ala ^-5- Gly ^-4 -Pro ^-3- Tyr ^-2 -Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salts.

The synthesis of the peptide SeqID 31 GRF-analogue containing SeqID 9 in the form of an extension part and has the formula N 8 Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr- Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF salt ₃ COOH) was carried out stepwise, similar to Procedure A described in published patent application PCT / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.06 (4); Thr 0.95 (1); Ser 1.78 (2); Glu 2.01 (2); Pro 1.95 (2); Gly 1.96 (2); Ala 4.81 (5); Val 0.95 (1); Ile 1.87 (2); Leu 5.09 (5); Tyr 4.12 (4); Phe 0.96 (1); Lys 3.08 (3); Arg 3.10 (3).

Example 14. Obtaining Phe ^-10 -Ala ^-9 -Lys ^-8 - Pro ^-7 -Tyr ^-6 -Ala ^-5 -Gly ^-4 Pro ^-3 - Tyr ^-2 -Ala ^-1 {[Leu ²⁷ ] bGRF (1 -29) NH ₂ }, trifluoroacetic acid salt.

The synthesis of the peptide Seq ID 32 GRF analogue containing Seq ID 10 in the form of an extension part having the formula N 9 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-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Asn-Arg-NH ₂ (as CF ₂ COOH salt) was carried out stepwise similar to Procedure A described in published patent application PCT / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.16 (4); Thr 1.01 (1); Ser 1.89 (2); Glu 2.08 (2); Pro 1.91 (2); Gly 1.93 (2); Ala 5.72 (6); Val 0.97 (1); Ile 1.90 (2); Leu 5.09 (5); Tyr 4.09 (4); Phe 1.99 (2); Lys 3.08 (3); Arg 3.04 (3).

Example 15. Obtaining Gly ^-12 -Pro ^-11 -Phe ^-10 - Ala ^-9- Lys ^-8 -Pro ^-7 -Tyr ^-6 -Ala ^-5 - Gly ^-4 -Pro ^-3 -Tyr ^-2 -Ala ^{- 1} {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

The synthesis of the peptide Seq ID 33 GRF-analogue containing Seq ID 11 in the form of an extension part having the formula N 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-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu- Asn-Arg-NH ₂ (as the CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published patent application PCT / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses: Asp 4.08 (4); Thr 0.96 (1); Ser 1.79 (2); Gln 2.07 (2); Pro 2.88 (3); Gly 2.94 (3); Ala 5.74 (6); Val 0.96 (1); Ile 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. Obtaining Val ^-14 -Pro ^-13 -Gly ^-12 - Pro ^-11 -Phe ^-10 -Ala ^-9 -Lys ^-8 -Pro ^-7 - Tyr ^-6 -Ala ^-5 -Gly ^-4 -Pro ^{- 3-} Tyr ^-2 - Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt
The synthesis of the peptide SeqID 34 GRF analogue containing SeqID 12 in the form of an extension part and having the formula N 11 Val-Pro-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-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile- Leu-Asn-Arg-NH ₂ (as the CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published patent application PCT / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.04 (4); Thr 0.96 (1); Ser 1.81 (2); Glu 2.04 (2); Pro 3.80 (4); Gly 2.99 (3); Ala 5.92 (6); Val 1.98 (2); Ile 1.89 (2); Leu 5.10 (5); Tyr 4.09 (4); Phe 1.99 (2); Lys 3.06 (3); Arg 3.08 (3).

Example 17. Obtaining Arg ^-16 -Pro ^-15 -Val ^-14 -Pro ^-13 -Gly ^-12 - Pro ^-11 -Phe ^-10 -Ala ^-9 -Lys ^-8 -Pro ^-7 - Tyr ^-6 -Ala ^{- 5} -Gly ^-4 -Pro ^-3- Tyr ^-2 - Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

The synthesis of the peptide Seq ID 35 GRF-analogue containing Seq ID 13 in the form of an extension part and having the formula N 12 Arg-Pro-Val-Pro-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro- Tyr-Ala-Tyr- Ala-Asp-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala- Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in the published PCT patent application (PCT / US90 / 02923), which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.10 (4); Thr 0.98 (1); Ser 1.84 (2); Glu 2.03 (2); Pro 4.82 (5); Gly 2.97 (3); Ala 5.91 (6); Val 1.99 (2); Ile 1.88 (2); Leu 5.09 (5); Tyr 4.10 (4); Phe 1.98 (2); Lys 3.03 (3); Arg 4.09 (4).

Example 18. Obtaining Val ^-2 -Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

Synthesis of Seq ID 36 peptide of a GRF analogue having the formula N 14 Val-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu -Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published PCT Patent Application / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 3.98 (4); Thr 0.89 (1); Ser 1.76 (2); Gln 1.05 (1); Ala 3.87 (4); Val 1.85 (2); Ile 1.77 (2); Leu 5.17 (5); Tyr 2.04 (2); Phe 0.97 (1); Lys 2.07 (2); Arg 3.06 (3).

Example 19. Obtaining Tyr ^-2 -Thr ^-1 {[[Ala ¹⁵ Leu ²⁷ ] bGRF (1-29) NH ₂ }, salts of trifluoroacetic acid.

Synthesis of the SeqID 37 peptide of the GRF analog having the formula N 15 Tyr-Thr-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser- Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published patent application PCT / US90 / 02923 , which is introduced into the present description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.06 (4); Thr 1.86 (2); Ser 1.77 (2); Gln 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. Obtaining Tyr ^-2 -Thr ^-1 {[[Ile ² Ala ¹⁵ Leu ²⁷ ] bGRF (1-29) NH ₂ }, salts of trifluoroacetic acid.

Synthesis of the SeqID 38 peptide of a GRF analogue having the formula N 16 Tyr-Thr-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu- Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (trifluoroacetic acid salt) was carried out stepwise, similar to procedure A described in published patent application PCT / US90 / 02923, which is introduced into the present description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.06 (4); Thr 1.87 (2); Ser 1.75 (2); Gln 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. Tyr ^-2 -Thr ^-1 {[Thr ² Ala ¹⁵ Leu ²⁷ ] bGRF (1-29) NH ₂ }. Preparation of trifluoroacetic acid salt.

Synthesis of the SegID 39 Peptide of the GRF Analogue of the Formula N17 Tyr-Thr-Tyr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala -Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published patent application PCT / US90 / 02923, which is introduced into the present description by reference. Amino acid analysis (theoretical values are shown in brackets): Asp 4.05 (4); Thr 2.68 (3); Ser 1.77 (2); Gln 2.07 (2); Ala 2.90 (3); Val 1.08 (1); Ile 1.89 (2); Leu 5.20 (5); Tyr 2.87 (3); Phe 0.93 (1); Lys 2.01 (2); Arg 3.07 (3).

Example 22. Obtaining Tyr ^-2 -Ser ^-1 {[Thr ² Ala ¹⁵ Leu ²⁷ ] bGRF (1-29) NH ₂ }, salts of trifluoroacetic acid.

Synthesis of the SegID 40 Peptide of the GRF Analogue of the Formula N18 Tyr-Ser-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Leu -Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was performed stepwise, similar to Procedure A described in published PCT patent application / US90 / 02923, which is introduced into the present description by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.11 (4); Thr 1.82 (2); Ser 2.64 (3); Glu 2.05 (2); Ala 2.90 (3); Val 1.04 (1); Ile 1.87 (2); Leu 5.16 (5); Tyr 2.92 (3); Phe 0.94 (1); Lys 2.01 (2); Arg 3.04 (3).

Example 23. Obtaining Tyr ^-4 -Thr ^-3 Tyr ^-2 - Thr ^-1 {[Thr ² Ala ¹⁵ Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

Synthesis of Seg ID 41 peptide of a GRF analogue having the formula N19 Tyr-Thr-Tyr-Thr-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala- Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in published application for PCT / US90 / 02923, which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 4.06 (4); Thr 3.66 (4); Ser 1.85 (2); Glu 2.05 (2); Ala 2.93 (3); Val 1.09 (1); Ile 1.91 (2); Ser 5.15 (5); Tyr 3.91 (4); Phe 0.95 (1); Lys 2.00 (2); Arg 3.04 (3).

Example 24. Obtaining Tyr ^-2 -Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, trifluoroacetic acid salt.

Synthesis of the peptide Seg ID 42 GRF analogue having the formula Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser -Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was performed stepwise, similar to Procedure A described in published PCT patent application / US90 / 02923, which is introduced into the present description by reference. Amino acid analysis (theoretical values are shown in parentheses): 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 2.18 (2); Arg 3.03 (3).

The following compounds were prepared according to Method A described in the form of their trifluoroacetate salts and confirmed by amino acid and mass spectral analyzes.
N-α - (Tyr-Thr) - [Thr ² Ala ¹⁵ , Leu ²⁷ ] bGRF (1-29) NH ₂ , trifluoroacetate.

H-Tyr-Thr-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu- Leu-Gln-Asp-Ile-Leu-Asp-Arg-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis (theoretical values are given in parentheses): Asp 4.06 (4); Thr 2.69 (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); Tyr 2.87 (3); Phe 0.93 (1); Lys 2.01 (2); Arg 3.07 (3).

N-α - (Tyr-Thr-Tyr-Thr) - [Thr ² Ala ¹⁵ , Leu ²⁷ ] bGRF (1-29) NH ₂ , trifluoroacetate.

H-Tyr-Thr-Tyr-Thr-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg- Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (CF ₃ COOH, salt).

 Amino acid analysis results; theoretical values are given in parentheses: Asp 4.06 (4); Thr 3.66 (4); Ser 1.85 (2); Glu 2.05 (2); Ala 2.93 (3); Val 1.09 (1); Ile 1.91 (2); Leu 5.15 (5); Tyr 3.91 (4); Phe 0.95 (1); Lys 2.00 (2); Arg 3.04 (3).

N-α - (Tyr-Thr) - [Ile ² Ala ¹⁵ , Leu ²⁷ ] bGRF (1-29) NH ₂ , trifluoroacetate.

H-Tyr-Thr-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu- Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: 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).

N-α - (Tyr-Ser) - [Thr ² Ala ¹⁵ , Leu ²⁷ ] bGRF (1-29) NH ₂ , trifluoroacetate.

H-Tyr-Ser-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu- Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 4.11 (4); Thr 1.82 (2); Ser 2.64 (3); Glu 2.05 (2); Ala 2.90 (3); Val 1.04 (1); Ile 1.87 (2); Leu 5.16 (5); Tyr 2.92 (3); Phe 0.94 (1); Lys 2.01 (2); Arg 3.04 (3).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Hse ²⁸ ] bGRF (1-28) NH ₂ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Hse-NH ₂ (CF ₃ COOH).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.05 (2); Thr 0.99 (1); Ser 2.46 (3); Hse 0.25 (1); Glu 2.06 (2); Ala 3.09 (3); Val 2.02 (2); Ile 1.86 (2); Leu 5.01 (5); Tyr 1.96 (2); Phe 0.96 (1); Lys 2.00 (2); Arg 2.01 (2).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets 3213.5 (3213.8).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ Hse ³⁰ ] bGRF (1-30) NH ₂ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.03 (2); Thr 0.95 (1); Ser 3.24 (4); Hse 0.61 (1); Glu 2.06 (2); Ala 3.28 (3); Val 1.98 (2); Ile 1.84 (2); Leu 4.97 (5); Tyr 1.89 (2); Phe 0.90 (1); Lys 1.98 (2); Arg 2.92 (3).

Mass spectrum; theoretical values (M + H) ⁺ are indicated in brackets 3456.6 (3457.0).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³³ ] bGRF (1-33) NH ₂ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Hse-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.04 (2); Thr 0.93 (1); Ser 3.23 (4); Hse 0.22 (1); Glu 4.19 (4); Gly 0.95 (1); Ala 3.21 (3); Val 1.93 (2); Ile 1.82 (2); Leu 4.96 (5); Tyr 1.91 (2); Phe 0.91 (1); Lys 1.99 (2); Arg 3.03 (3).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets 3770.1 (3770.4).

[Val ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ⁴⁵ ] bGRF (1-45) NH ₂ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Lys-Val-Arg-Leu-Hse-NH ₂ (CF ₃ COOH, salt )

 The results of amino acid analysis, theoretical values are indicated in parentheses: Asp 3.05 (3); Thr 0.93 (1); Ser 3.20 (4); Hse 0.21 (1); Glu 8.41 (8); Gly 2.04 (2); Ala 4.17 (4); Val 2.83 (3); Ile 1.79 (2); Leu 5.96 (6); Tyr 1.88 (2); Phe 0.83 (1); Lys 2.94 (3); Arg 4.93 (5).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets 5179.3 (5179.9).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Hse ²⁸ ] bGRF (1-28) NHC ₂ H ₅ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3241.8 (3241.8).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³³ ] bGRF (1-33) NHC ₂ H ₅ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Gln-Gln-Gly-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3797.9 (3798.4).

[Val ² , Ser ⁸ Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NHC ₂ H ₅ , trifluoroacetate.

H-Tyr-Val-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 1.93 (2); Thr 0.87 (1); Ser 3.20 (4); Hse 0.42 (1); Glu 2.00 (2); Ala 3.08 (3); Val 1.98 (2); Ile 1.85 (2); Leu 4.99 (5); Tyr 1.96 (2); Phe 0.97 (1); Lys 2.19 (2); Arg 3.03 (3).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets 3485.4 (3485.1).

[Thr ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NH ₂ , trifluoroacetate.

H-Tyr-Thr-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NH ₂ (CF ₃ COOH, salt).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3458.7 (3459.0).

[Ile ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NH ₂ , trifluoroacetate.

H-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NH ₂ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.04 (2); Thr 0.93 (1); Ser 3.41 (4); Hse 0.56 (1); Glu 2.04 (2); Ala 3.01 (3); Val 1.13 (1); Ile 2.93 (3); Leu 5.11 (5); Tyr 2.00 (2); Phe 0.96 (1); Lys 1.99 (2); Arg 2.93 (3).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3470.4 (3471.1).

[Ile ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NHC ₂ H ₅ , trifluoroacetate.

H-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.01 (2); Thr 0.94 (1); Ser 3.35 (4); Hse 0.47 (1); Glu 2.01 (2); Ala 3.03 (3); Val 1.14 (1); Ile 2.89 (3); Leu 5.12 (5); Tyr 1.99 (2); Phe 0.97 (1); Lys 1.97 (2); Arg 3.00 (3).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3498.7 (3499.2).

[Ile ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NHC ₂ H ₅ , acetate.

H-Tyr-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NHC ₂ H ₅ (CH ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 2.16 (2); Thr 0.93 (1); Ser 3.31 (4); Hse 0.62 (1); Glu 1.98 (2); Ala 3.01 (3); Val 1.04 (1); Ile 2.79 (3); Leu 4.97 (5); Tyr 1.94 (2); Phe 0.96 (1); Lys 2.18 (2); Arg 3.63 (3).

Mass spectrum, theoretical values (M + H) ⁺ are indicated in brackets: 3499.9 (3499.2).

[His ¹ , Ile ² , Ser ⁸ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NHC ₂ H ₅ , trifluoroacetate.

H-His-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 1.94 (2); Thr 0.91 (1); Ser 3.23 (4); Hse 0.91 (1); Glu 1.97 (2); Gly 0.96 (1); Ala 1.93 (2); Val 0.97 (1); Ile 2.80 (3); Leu 4.90 (5); Tyr 0.96 (1); Phe 0.96 (1); His 0.94 (1); Lys 1.93 (2); Arg 3.70 (3).

Mass spectrum, in parentheses are the theoretical values (M + H) ⁺ : 3460.3 (3459.1).

[His ¹ , Ile ² , Ser ⁸ , Ala ¹⁵ , Leu ²⁷ , Ser ²⁸ , Hse ³⁰ ] bGRF (1-30) NHC ₂ H ₅ , trifluoroacetate.

H-His-Ile-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Val-Leu-Ala-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln- Asp-Ile-Leu-Ser-Arg-Hse-NHC ₂ H ₅ (CF ₃ COOH, salt).

 The results of amino acid analysis, in parentheses are the theoretical values: Asp 1.91 (2); Thr 0.88 (1); Ser 3.39 (4); Hse 0.26 (1); Glu 1.90 (2); Ala 2.86 (3); Val 0.99 (1); Ile 2.79 (3); Leu 5.02 (5); Tyr 0.95 (1); Phe 0.95 (1); His 0.92 (1); Lys 1.96 (2); Arg 3.73 (3).

Mass spectrum, theoretical values in parentheses (M + H) ⁺ : 3473.0 (3473.1).

Example 25. Obtaining Tyr ^-4 -Ala ^-3 Tyr ^-2 - Ala ^-1 {[Leu ²⁷ ] bGRF (1-29) NH ₂ }, salts of trifluoroacetic acid.

Synthesis of the SeqID 43 Peptide of the GRF Analogue of the Formula N13 Tyr-Ala-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln -Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-NH ₂ (as CF ₃ COOH salt) was carried out stepwise, similar to Procedure A described in the published patent application PCT (PCT / US90 / 02923), which is incorporated herein by reference. Amino acid analysis (theoretical values are shown in parentheses): Asp 3.97 (4); Thr 0.90 (1); Ser 1.74 (2); Gln 1.98 (2); Gly 1.04 (1); Ala 4.85 (5); Val 0.91 (1); Ile 1.77 (2); Leu 5.13 (5); Tyr 4.14 (4); Phe 0.99 (1); Lys 2.07 (2); Arg 3.05 (3).

 In the table. Figures 4-6 show the results of studies of the response to the administration of an injection of derived peptides of the GRF series to the bovine.

(3) Данные последовательности ID N 2;
(1) Характеристики последовательности:
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(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID N 2:

(4) Данные последовательности ID N 3:
(1) Характеристики последовательности:
(A) Длина: 29
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(XI) Описание последовательности ID N 3:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 29
(XI) Описание последовательности ID N 3:

(5) Данные последовательности ID N 4:
(1) Характеристики последовательности:
(A) Длина: 29
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 29
(XI) Описание последовательности ID N 4:

(6) Данные последовательности ID N 5:
(1) Характеристики последовательности:
(A) Длина: 29
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C- терминально амидированный аргининиловый остаток.Sequence List
(I) General information:
(I) Applicant
(II) Title of the invention: hybrid polypeptides
(III) Number of sequences: 42
(IV) Postal address:
(A) Address:
(B) Street:
(C) City:
(C) State:
(E) Country: USA
(F) Zip / Postal Code: 49001
(V) Machine read form:
(A) Media type: floppy disk (3M 3.5, double-sided DS - 1.0 MV)
(B) Computer: compatible IBM PC
(C) Operating System: PC-DOS / MS - DOS
(D) Software: WordPerfect 5.1
(VI) Current application data:
(A) Application Number:
(B) Date of submission:
(C) Classification:
(Vii) Data from a previous application:
(A) Application Number: US 07/626, 727
(B) Date of submission: 13/12/90
(Vii) Data from a previous application:
(A) Application Number: US 07/614, 170
(B) Date of submission: 14/11/90
(Vii) Data from a previous application:
(A) Application Number: PCT / US90 / 02923
(B) Date of submission: 30/05/90
(Vii) Data from a previous application:
(A) Application Number: US 07/368, 231
(B) Date of submission: 16/06/89
(Vii) Data from a previous application:
(A) Application Number: US 07/506, 605
(B) Date of submission: 09/04/90
(Viii) Information and Attorney / Agent:
(A) Name; surname:
(B) Registration number: 33229
(C) Document / Dossier Number: 4595
(IX) Telecommunication information:
(A) Phone: 616 385 5210
(B) Facsimile: 616 385 6897
(2) Data sequence ID N 1:
(1) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 1:

(3) Data sequence ID N 2;
(1) Characteristics of the sequence:
(A) Length: 40
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 2:

(4) Data sequence ID N 3:
(1) Characteristics of the sequence:
(A) Length: 29
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 3:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 29
(XI) Description of the sequence ID N 3:

(5) Data sequence ID N 4:
(1) Characteristics of the sequence:
(A) Length: 29
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 29
(XI) Description of the sequence ID N 4:

(6) Data sequence ID N 5:
(1) Characteristics of the sequence:
(A) Length: 29
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C- terminally amidated arginine residue.

(7) Данные последовательности ID N 6:
(1) Характеристики последовательности:
(A) Длина: 6
(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID N 6:

(8) Данные последовательности ID N 7:
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(B) Тип: аминокислотная
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(9) Данные последовательности ID N 8:
(1) Характеристики последовательности:
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(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID N 8:

(10) Данные последовательности ID N 9:
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(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID N 9:

(II) Данные последовательности ID N 10:
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(B) Тип: аминокислотная
(D) Топология: линейная
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(12) Данные последовательности ID N 11:
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(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности: ID N 11:

(13) Данные последовательности ID N 12:
(1) Характеристики последовательности:
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(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID N 12:

(14) Данные последовательности ID N 13:
(I) Характеристики последовательности:
(A) Длина: 16
(B) Тип: аминокислотная
(D): Топология: линейная
(XI) Описание последовательности ID N 13:

(15) Данные последовательности ID N 14:
(I) Характеристики последовательности:
(A) Длина: 29
(B) Тип: аминокислотная
(D): Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 29
(XI) Описание последовательности ID N 14:

(16) Данные последовательности ID N 15:
(I) Характеристики последовательности:
(A) Длина: 11
(B) Тип: аминокислотная
(D): Топология: линейная
(XI) Описание последовательности ID 15:

(17) Данные последовательности ID N 16:
(I) Характеристики последовательности:
(A) Длина: 11
(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Описание последовательности ID 16:

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(I) Характеристики последовательности:
(A) Длина: 11
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(D) Топология: линейная
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(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 31
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(I) Характеристики последовательности:
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(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 33
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(21) Данные последовательности ID N 20:
(I) Характеристики последовательности:
(A) Длина: 39
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 39
(XI) Описание последовательностей ID N 20:

(22) Данные последовательности ID N 21:
(I) Характеристики последовательности:
(A) Длина: 45
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 45
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(I) Характеристики последовательности:
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(B) Тип: аминокислотная
(D) Топология: линейная
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(B) Тип: аминокислотная
(D) Топология: линейная
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(I) Характеристики последовательности:
(A) Длина: 27
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 27
(XI) Описание последовательности ID N 24:

(26) Данные последовательности ID N 25:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 31
(XI) Описание последовательности ID N 25:

(27) Данные последовательности ID N 26:
(I) Характеристики последовательности:
(A) Длина: 33
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 33
(XI) Описание последовательности ID N 26:

(28) Данные последовательности ID N 27:
(I) Характеристики последовательности:
(A) Длина: 35
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 35
(XI) Описание последовательности ID N 27:

(29) Данные последовательности ID N 28:
(I) Характеристики последовательности:
(A) Длина: 37
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 37
(XI) Описание последовательности ID N 28:

(30) Данные последовательности ID N 29:
(I) Характеристики последовательности:
(A) Длина: 33
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa 33
(XI) Описание последовательности ID N 29:

(31) Данные последовательности ID N 30:
(I) Характеристики последовательности:
(A) Длина: 35
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа35
(XI) Описание последовательности ID N 30:

(32) Данные последовательности ID N 31:
(I) Характеристики последовательности:
(A) Длина: 37
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа37
(XI) Описание последовательности ID N 31:

(33) Данные последовательности ID N 32:
(A) Длина: 39
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа39
(XI) Описание последовательности ID N 32:

(34) Данные последовательности ID N 33:
(I) Характеристики последовательности:
(A) Длина: 41
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа41
(XI) Описание последовательности ID N 33:

(35) Данные последовательности ID N 34:
Характеристики последовательности:
(A) Длина: 43
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа43
(XI) Описание последовательности ID N 34:

(36) Данные последовательности ID N 35:
(I) Характеристики последовательности:
(A) Длина: 45
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название ключ/: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа45
Описание последовательности ID N 35:

(37) Данные последовательности ID N 36:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название ключ/: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа31 (XI) Описание последовательности ID N 36:

(38) Данные последовательности ID N 37:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа31
(XI) Описание последовательности ID N 37:

(39) Данные последовательности ID N 38:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа31
(XI) Описание последовательности ID N 38:

(40) Данные последовательности ID N 39:
(I) (A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(XI) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа31
(XI) Описание последовательности ID N 39:

(41) Данные последовательности ID N 40:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа31
(XI) Описание последовательности ID N 40:

(42) Данные последовательности ID N 41:
(I) Характеристики последовательности:
(A) Длина: 33
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Хаа33
(XI) Описание последовательности ID N 41:

(43) Данные последовательности ID N 42:
(I) Характеристики последовательности:
(A) Длина: 31
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa31
(XI) Описание последовательности ID N 42:

(44) Данные последовательности ID N 43:
(I) Характеристики последовательности:
(A) Длина: 33
(B) Тип: аминокислотная
(D) Топология: линейная
(IX) Отличительные особенности:
(A) Название/ключ: C-терминально амидированный аргининиловый остаток
(B) Локализация: Xaa33
(XI) Описание последовательности ID N 43:

й(B) Location: Xaa 29
(XI) Description of the sequence ID N 5:

(7) Data sequence ID N 6:
(1) Characteristics of the sequence:
(A) Length: 6
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 6:

(8) Data sequence ID N 7:
(1) Characteristics of the sequence:
(A) Length: 8
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 7:

(9) Data sequence ID N 8:
(1) Characteristics of the sequence:
(A) Length: 6
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 8:

(10) Data sequence ID N 9:
(1) Characteristics of the sequence:
(A) Length: 8
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 9:

(II) Data sequence ID N 10:
(I) Characteristics of the sequence:
(A) Length: 10
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 10:

(12) Data sequence ID N 11:
(1) Characteristics of the sequence:
(A) Length: 12
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence: ID N 11:

(13) Data sequence ID N 12:
(1) Characteristics of the sequence:
(A) Length: 14
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 12:

(14) Data sequence ID N 13:
(I) Characteristics of the sequence:
(A) Length: 16
(B) Type: amino acid
(D): Topology: linear
(XI) Description of the sequence ID N 13:

(15) Data sequence ID N 14:
(I) Characteristics of the sequence:
(A) Length: 29
(B) Type: amino acid
(D): Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 29
(XI) Description of the sequence ID N 14:

(16) Data sequence ID N 15:
(I) Characteristics of the sequence:
(A) Length: 11
(B) Type: amino acid
(D): Topology: linear
(XI) Description of the sequence ID 15:

(17) Data sequence ID N 16:
(I) Characteristics of the sequence:
(A) Length: 11
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID 16:

(18) Data sequence ID N 17:
(I) Characteristics of the sequence:
(A) Length: 11
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID 17:

(19) Data sequence ID N 18:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 31
(XI) Description of the sequence ID 18:

(20) Data sequence ID N 19:
(I) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 33
(XI) Description of sequence ID 19:

(21) Data sequence ID N 20:
(I) Characteristics of the sequence:
(A) Length: 39
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 39
(XI) Description of sequences ID N 20:

(22) Data sequence ID N 21:
(I) Characteristics of the sequence:
(A) Length: 45
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 45
(XI) Description of sequences ID N 21:

(23) Data sequence ID N 22:
(I) Characteristics of the sequence:
(A) Length: 10
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 22:

(24) Data sequence ID N 23:
(I) Characteristics of the sequence:
(A) Length: 16
(B) Type: amino acid
(D) Topology: linear
(XI) Description of the sequence ID N 23:

(25) Data sequence ID N 24:
(I) Characteristics of the sequence:
(A) Length: 27
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 27
(XI) Description of the sequence ID N 24:

(26) Data sequence ID N 25:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 31
(XI) Description of the sequence ID N 25:

(27) Data sequence ID N 26:
(I) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 33
(XI) Description of the sequence ID N 26:

(28) Data sequence ID N 27:
(I) Characteristics of the sequence:
(A) Length: 35
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 35
(XI) Description of the sequence ID N 27:

(29) Data sequence ID N 28:
(I) Characteristics of the sequence:
(A) Length: 37
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 37
(XI) Description of the sequence ID N 28:

(30) Data sequence ID N 29:
(I) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Location: Xaa 33
(XI) Description of the sequence ID N 29:

(31) Data sequence ID N 30:
(I) Characteristics of the sequence:
(A) Length: 35
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa35
(XI) Description of the sequence ID N 30:

(32) Data sequence ID N 31:
(I) Characteristics of the sequence:
(A) Length: 37
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa37
(XI) Description of the sequence ID N 31:

(33) Data sequence ID N 32:
(A) Length: 39
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa39
(XI) Description of the sequence ID N 32:

(34) Data sequence ID N 33:
(I) Characteristics of the sequence:
(A) Length: 41
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa41
(XI) Description of the sequence ID N 33:

(35) Data sequence ID N 34:
Sequence Characteristics:
(A) Length: 43
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa43
(XI) Description of the sequence ID N 34:

(36) Data sequence ID N 35:
(I) Characteristics of the sequence:
(A) Length: 45
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Key name /: C-terminally amidated arginine residue
(B) Localization: Haa45
Description of the sequence ID N 35:

(37) Data sequence ID N 36:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Key name /: C-terminally amidated arginine residue
(B) Localization: Haa31 (XI) Description of the sequence ID N 36:

(38) Data sequence ID N 37:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa31
(XI) Description of the sequence ID N 37:

(39) Data sequence ID N 38:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Xi) Features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa31
(XI) Description of the sequence ID N 38:

(40) Data sequence ID N 39:
(I) (A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Xi) Features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa31
(XI) Description of the sequence ID N 39:

(41) Data sequence ID N 40:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa31
(XI) Description of the sequence ID N 40:

(42) Data sequence ID N 41:
(I) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Haa33
(XI) Description of the sequence ID N 41:

(43) Data sequence ID N 42:
(I) Characteristics of the sequence:
(A) Length: 31
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Xaa31
(XI) Description of the sequence ID N 42:

(44) Data sequence ID N 43:
(I) Characteristics of the sequence:
(A) Length: 33
(B) Type: amino acid
(D) Topology: linear
(Ix) Distinctive features:
(A) Name / key: C-terminally amidated arginine residue
(B) Localization: Xaa33
(XI) Description of the sequence ID N 43:

th

Claims (9)

1. A fusion protein, not found in nature, containing an extension peptide fragment, covalently linked by its C-end to the N-end of the Central part of the protein, General formula
AXY- (X'-Y) _n -protein,
where A is hydrogen or Met, and if A is hydrogen, then the protein is an analog of bGRF,
if A is Met, then Bur-PHkazaH protein;
X is an amino acid residue selected from the group Ala, Arg, Gly, Lys, Pro, Phe, Ser, Tyr, Val;
Y is an amino acid residue selected from the group Ala, Ser, Thr, Pro;
X 'is an amino acid residue selected from the group Ala, Gly, Ile, His, Lys, Phe, Tyr;
n = 0 - 7, and if n = 0, and A is hydrogen, then Y is Ala, Thr, Ser.  2. The protein of claim 1, wherein the central portion of the protein is selected from bGRF analogues.  3. The protein according to claim 1 or 2, in which A is Met, and X is Ala, Gly, Pro, Ser.  4. The protein of claim 1, wherein n = 3 to 5. 5. The protein of claim 4, wherein all of the X ¹ residues are His.  6. The protein of claim 5, wherein the three Y residues are Pro. 7. The protein of claim 1 or 2, wherein the extension peptide fragment is selected from the group
Tyr - Ala, Gly - Pro - (Ile - Pro), Gly - Pro - (Tyr - Ala), Tyr - Ala - (Tyr - Ala), Val - Ala.  8. The protein according to paragraphs. 1 - 7 as an intermediate for purification of the native protein under the action of the enzyme DPPIV. 9. A method for isolating a fusion protein according to claim 1 of the general formula
AXY- (X'-Y) _n -protein,
where A is Met;
n is 4;
X = Ala, Gly, Pro;
Y = Ala, Pro;
X 'is His;
protein - Bur - PHase H,
by column chromatography on Sepharose, including immobilized metal ions.

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