SK60893A3 - Fusion polypeptides - Google Patents

Abstract

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

Description

BACKGROUND OF THE INVENTION

Methods of technology of the desired genetic information modified, modified molecular biology, in particular recombinant DNA, allow the production of relatively large amounts of biologically active polypeptides. In addition, a large number of abv peptides can be produced encoded in polypeptides. Polypeptide modification is often used to improve its activity or to facilitate its production and / or preparation. Therefore, many efforts have been made to determine which modifications enhance or otherwise alter the biological polypeptides of interest. In addition, many polypeptides are required to be purified.

Polypeptides produced by natural processes are often initially considered to be large in size, and are then a series of proteolytic proteins. cleavage to final products. Therefore, there are several proteases that recognize and cleave specific amino acids or amino acid chains. These proteases are involved in the conversion of the precursor protein to the final polypeptide product.

One of these proteases is two or five. and IV (DPP IV) (EC 3.4.10.5). DPP IV described in iiops u-Ha vu, V.K. and G.G. G 1 e n n e r, 11 sure. C h e m i e 3: 1.9 7 - 2 0! (1 9 O 6) and has been demonstrated in many mammalian tissues. Currently, it is commercially available from knzyme Systems Products (Dublin, Calif.). DPP IV recognizes a specific chain of amino acids at the N-termini of the proteins. In particular, DPP IV cleaves the dipeptide from the upstream method, and k i i. The engineers are working on modifications facilitating their production and on N-amino acid amino acids.

N-terminus if the second amino acid from the N-terminus is proline (Pro), hydroxyproline (Hyp), alanine (Ala), series (Ser) and threonine (Thr) is any amino acid, with the third residue from the N-terminus not being proline or hydroxyproline. DPP IV is more effective when the second amino acid from the N-terminus is proline or alanine and is usually most effective when proline is present at that point. DPP activity

The PRO cleavage activity of the precursor moieties naturally

IV for sequentially occurring peptides is extensively documented.

Modern technologies allow the production of biologically active proteins at a high level. Important polypeptides can be synthesized using peptide synthesizers or in host cells using recombinant DNA technology. Biologically active proteins are often used as medicines. There are many examples in which active proteins are used as medicaments, prophylactic agents, or to enhance or suppress certain properties. Because DPP IV and other proteases degrade proteins, these drugs are prone to degrade. Thus, the problem of using biologically active polypeptides as drugs is to reduce their content in tissues, so they need to be administered more frequently. The rapid development of recombinant DNA methodology that enabled the production of polypeptides, proteins and analogs in large quantities over a very short period of time has created the need for a highly efficient and reliable isolation of these proteins in which all proteins are cells together with proteins of complex mixtures in host the media. C i s t e n i. e production of different polypeptides produced by the host cells can be very expensive and otherwise cause denaturation of the cells themselves. An overview of pinon purification methods is the state of the art. The product is included in the overview

137, granted 1.11.1988 11 o p po v i and other th o o o t o o o.

In order to circumvent the limitations of the technique, recombinant DNA technology may be desirable for peptides in lormc artificial proteins containing proteins that can be used. g a n d a 1 e s s u u s p a t e n t e no. d 7 8 2, which is e. We have 1 methods. .3 target for detergents. For example, U.S. Pat. No. 6,782,137, describes the synthesis of an artificial peptide comprising an antigenic linker peptide. An artificial protein containing an infected antibody, a peptide, and thus may be described as purification of the artificial, may pass through a column which binds to U.S. Pat. 6 569 796

1) A thiophene comprising the N-terminus having affinity for immobilized chewed metals. Artificial proteins bind to incubated metals on the column. The problem with these methods is that the linker peptide is often in demand and its removal can be difficult.

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

This invention gives artificial

N-terminal fissile DPP IV.

with DPP IV leads to protein conversion. If artificial screening is used which has an extension so that exposure of the artificial protein to the desired protein is transformed into a biologically active protein by DPP IV in vivo.

When used in scrubbing towers using a sprayer. In the process, it is possible to purify the artificially labeled N-termini as I.I.gand and then treat them with DPP IV. which removes the N-terminal extension and releases the desired protein.

production of the desired proteins, and k later converted to the desired proteins

Protein drugs are

T e n it v y! Artificial proteins, such as those which are derived from DPP IV.

over time, medicines will ensure the continued presence of the active ingredient in the body with the aid of the patient ' s own administration.

z m c n i a n a

DPP IV, what a patient a

Pure desired proteins are possible by the invention by producing and purifying the artificial proteins by processing the artificial proteins in vitro to produce the desired protein.

isolate by this screen and then using DPP IV to

SUMMARY OF THE INVENTION

The present invention comprising an elongated C-terminus to an C-terminus of an artificial fusion protein, a protein portion covalently linked to a base protein portion, wherein the elongated protein portion has the formula:

AXY (X'-Y) _n where A is optional and when present it is methionine, n is 0-20,

X is selected from group containing all of them naturally the occurring residues ain i noky s e 1 í n, X 'is selected from groups o b s a b u u c e j all of them naturally the occurring from y škino aninokyse1 í n except for p r o 1. í n u and hydroxyproline, Y is selected from group containing p r o 1 n n, hydroxypro1 and,

of artificial purification a n a n n, s r n n a t n o n n.

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

U.S. Pat. A 569 79A, granted to 1I.2.19B6 Smith and to o 1. , relates to the third and third. n o v a 1 á t o u t u c t o r h. t ý c h t o tupo t: h. p r o t e n o v,

At the end a c h i? i

The invention discloses a method of isolating linked having biologically active polepeptides to an N-horse linker which is a member of the ovo ions. Connected p e p t. j. d has and t is here to the immobilized metal ions. Impurities by passing the mixture containing the combined immobilized metal ions. metal ions and wash out the metal ion and thus can be removed. protein. a column S p o j t; The protein is only impurities. Upon release, a purified content is obtained which associates with the said protein-associated protein. U.S. Pat. No. 7,821,137, issued Nov. 1, 1988, to H ' s et al., Describes the synthesis of a fusion protein having a highly antigenic N-terminal portion. and the desired polypeptide at the C-terminus. According to IIoppa et al., The pooled proteins are purified from the untreated supernatant by passing the untreated supernatant through a column containing immobilized antibodies to the coupled protein. Immobilized on the column while undesirably distinguishing the antigenic portion of the antibody retaining the protein components of the supernatant are

no. No. 0 220 950, for selective chemical elution. The pooled protein is then eluted and collected.

U.S. Pat. No. 7,334,399, issued March 29, 1988 to Felix et al., relates to analogues of growth hormone releasing factors. A number of analogs having terminal groups Tyr-Ala and liis-Ala are described. However, these molecules are not linked by proteins, but rather by nuclei of proteins. The terminal peptides of Felix et al. Are part of the nucleus of the bGRF analog molecule. European Patent Application, published May 6, 1987, relates to the removal of N-horse residues i. The invention relates to a method and compounds useful for removing N-terminal residues from desired polypeptides. The desired polypeptide exists in the form of a fusion protein having the desired peptide member at the N-terminus of the fusion member of formula X-Pro. After exposure of the coupled protein to specific buffer conditions, diketopiperazine X-Pro portions of the coupled protein are formed and released. This produces the desired polypeptide from the fusion protein. The combined protein poodle EPO 220 958 is not included in this invention because, according to the present invention, if the N-smoke extension is just dipepfide. t. j. when A is absent, n is 0 and

X is a d e s e s a s r a n or t r a n.

occurring amino acid, Y is alanine, ie whenever it is extended (extended)

- () dipeptide, X-Ala, X-Ser or X-Thr. The application 220 958 describes the chemical, not the X-Ala dipeptides, the X-Ser cleavage, of the disclosed enzymatic hydrolysis of the X-Pro dipeptide.

and X-Thr are not susceptible to this type in the application 22 0 950, which shortens (separates) the X-Pro extension from the protein core.

The Australian patent application, document number AU-Al2709 / 88, discloses coupled peptides useful for affinity metal chromatography (IMAC). The disclosed affinity peptides contained at least two adjacent liistidine residues. The purification of IMAC by the compositions described requires special synthetic chemistry for the preparation of nitric acid resins (NTA).

Tallon M.A., and others Biochem. 26: 7767-777A (1987) relates to the synthesis of extended analogues of the tridecapeptide alpha factor Saccharomyces cerevisiae. Synthesized analogs are augmented by alpha factors, which represent chains of the natural pro-factor of the encoded MF - α 1 and -1 structural gene.

G. Kreil et al., Eur. J. Biochem., 111, 99-58 (1980) disclose the sequential cleavage at the N-terminal portion of the melitin precursor (Proinitin) to the peptide of class IV. Promelitin is a major component of bee venom. In the amino acid chain at the N-terminus of the precursor, every other residue is either proline or alanine. Promelithin, unlike the fusion proteins of the invention, is a naturally occurring protein.

M o 1 1 y y C. and others. , Eu r. J.Bi o c h e m. , 16 0, 31-35 (1986) describe the isolation of 1) IV 'from Xenopus laevis skin secretions.

R o z b e r o a s a t i v t a D P P IV.

M e n 11 e i n R. 1.234: No. 2, 25-556 (January 1988) summarizes the role of prolonged residues in the aging and degradation of peptide hormones and neuropeptides. Proteases in mammals are known to be well known in the art. such as D P P 1 V, n i. They may be involved in the synthesis of peptide regulators, but may play an important role in the synthesis of peptides. C h a n i s i n i s that the giant hunters and lower game hunters are procuratorial proteins

relying on DPP IV to convert precursors into mature forms, processing of protein regulators in mammals uses DPP IV usually as a degradation protease.

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

Kubiak T.M. and others: Drug Metaholism and Disposition, 17, no. Ú, 393-397 (1989), describes the metabolic degradation of the in vitro bovine growth hormone releasing analogue (bGRF) in bovine and porcine plasma and its correlation with plasma DPP IV activity. The bGRF analogs tested had an Ala residue at the 2- position of the N-terminus. Metabolic degradation (bGRF) in plasma is reported to be induced by the presence of plasma DPP IV.

Hong W. et al., Biochemistry, 28: 8A7U-8A79 (1989) report expression for enzymatically active DPP IV in cells. Chinese hamster ovaries after transfection.

G. TIBS 15: 23-26 A d d e d i d i s a nd a nd s for end products. The precursors described by Kreil are naturally occurring proteins. The coupled proteins of the invention are artificial proteins.

Boman et al., J Biol. Chem. 26: 58-5 2-5860 (1989) showed that dipeptidylpeptidosis isolated from ceoropia pupae (with similar specificity to DPR IV) was able to remove natural chains at the N-terminus of Ala-Pro-Glu-Pro from the N-terminus of synthetic copies of natural cecropium precursors A and B. The preprocecropin described by Bohman is a naturally occurring protein.

Dalboge Ií. and others Bi o / t.echno 1 ogv, 5: 161-164 (Feb

1987) describe the conversion in vi. tro precursors of human growth hormone (hGH) produced by E. coli to authentic h GII. The terminal elongation of the precursor is removed by dipeptide peptide I.

Dalboge 11 et al. FF13S, 266 (1,2): (March 1989) describe cloning and expression of the precursor IL-1 beta and its conversion to IL-1 beta by removing the precursor extension at the N-terminus.

(January 1990) summarizes the progressive DPP in the conversion of precursors

Krei 1 cleavage d i pepti dy 1pept i give o o I.

Dalboge II. and others, FFB3. (1,2): 1-3 (June 1990) describe in vivo processing in the N-terminal int: t. It has been reported that removal of the N-terminal methionine from the expanded human growth hormone was dependent on the amino acid adjacent to the methionine.

Hopp T. P. et al., Bio / Tech. 6: 1206-1210 (Oct.1988) describe the attachment of an eight amino acid peptide to the N-terminus of a recombinant lymphokine to create an antigenic N-terminus that could be used for immunoassay purification. This publication corresponds to U.S. Pat. 6,702,137 described above.

Smith M.C. et al., J. Biol. Chem. Voi.263, 15: 7211-7215 (1988) describe experimental results supporting the hypothesis that selective metal chelating peptides at the NH2-terminus of a small peptide can be utilized to purify this protein by means of δ new affinities, such as bulk chromatography. . This reference provides specific data relating to one of the above examples 569 796. The nominal use of a peptide coupled to a luteinizing hormone hormone or proinsulin allows the chimeric peptide to be purified by 1M AC, whereas controlled molecules lacking the His-Trp member are not possible. in a similar way.

Hochuli E. et al., J. Chromat. 611: 177-186 (1987) have described an nitriacetic acid absorbent useful for metal ion affinity chromatography in. U.S. Pat. 6 of a His-Trp metal-releasing absorber when it saturates the Ni ²⁺ proteins containing new peptides from the height.

1, j u n g q u i t C. and other descriptions are used

A1 a - H i s - G 1 v - II i s - A r g - P r e s described for the w e n i n i e s s s new useful s e s

Eur. J. B ioc 11 e m. 186: 563-569 (1989) Peptide ebe 1 a fu j ÚE eb of the two metals, r and w FVO eight times it ICIP 11 J ites of iusko 1 O nouobsa hu Jucon would find the van 1 iso Z and E / ⁺ ions. According to Ljungquist, the use of this zinc column metal-ironing peptide provides unexpectedly good purification of the fusion protein.

As used herein, the term 'artificial fusion protein' is understood to include other fusion proteins. Polypeptides and polypeptides refer to interchangeable proteins and polypeptides that do not occur normally in nature and which contain a core protein moiety and an elongated moiety.

As used herein, the core protein-core portion of a protein and a portion of a polypeptide refer to a portion of a fused polypeptide that is located at the C-terminus of the molecule and which without the extended portion would be the desired polypeptide and / or biologically active protein, including naturally occurring biologically active proteins. po lype.pt i do va ich ana 1 ó govamutanto v.

as used herein, the term N-terminal extension of the first up to about 45 amino acids starting at

N-termini that are not part of the core protein.

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

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

As used herein, the terms desired protein and polypeptide of interest refer to exchangeable proteins and polypeptides to be obtained in pure form.

As it applies to

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

As used herein, the elongated portion of the N-terminus IV refers to the elongated portion of the fused and moiety digestible DTP protein which is removed by sequential digestion with bP P IV.

me

In the description of all: all of them. n o k s e s n (e n e n e e e s e e v 11) a k o X a a

S e k v. 1 l) e. 3 X a a

The following description applies: represents an amidated C-terminal arginyl moiety

Sek v. 111 č. or Xaa ²⁹ represents an amidated arginyl residue on the Ck onci.

in S e k v. í D t . o Xaa ²⁹ pred í; ta v u j e and m i d o v n y and rginy1 Rest to C-c o n c i. IN SEQ. 1D no. 14 Xaa ²⁹ is amidated arginyl Rest on C-horses i ,. IN SEQ. ID # 18 Xaa ^{3 1} before s tau j t: and m i d o v n y N ^- 1 s gin characterized Rest at the C-terminus. IN SEQ. ID # 19th X aa ^{3 3} p r e d s t a u j e amidated arginyl Rest at the C-terminus. / IN SEQ. I D č. 20 Xaa ³⁹ p r e d and m i d o v n y arginyl Rest at the C-terminus. IN SEQ. ID # 21 Xaa ⁴⁵ p r e d s t a u j e amidated arginyl Rest at the C-terminus. IN SEQ. ID # 2 4 X aa ^{2 7} imagine it amidated arginy1ový Rest at the C-terminus. IN SEQ. ID # 2 5 Xaa ^{3 1} is amidated and rgiy1 Rest at the C-terminus. IN SEQ. ID # 26 X aa ^{3 3} is and m i d o v n y arginy1ový Rest at the C-terminus. IN SEQ. ID # 27 Xaa ^{3 5} p r e d s t a u j e amidated and rg iy Rest at the C-terminus. IN SEQ. ID # 28 Xaa ³⁷ is amidated arginyl Rest at the C-terminus. IN SEQ. ID # 29 Xaa ³³ preds melts e amidated and rg iy Rest at the C-terminus. IN SEQ. 1D no. 30 Xaa ^{3 5} imagine it and m i d o v n y and rgiy1 Rest to C-c o n c i. IN SEQ. II) č. 31 X aa ^{3 7} before s t a u j e and m i d o v n y arginyl Rest to C-ko n e i. IN SEQ. I D č. 3 2 X aa ^{3 9} p r e d s t a u j f; and m i d o v n y argynyl Rest to C-o n e i. IN SEQ. ID # Xaa ^{4 3} before the condition is e and m i d o v n y argieny 1ový Rest on C-kci. IN SEQ. I D č. 3 4 Xaa ^{4 3} before it is e and d i d o v e and r g i. n y 1 o Rest at the C-terminus. IN SEQ. 1 D no. 3 5 Xaa ^{4 3} p r 'e d s ta v u j e a m i d o v »» n a r g i n y 1 Rest at the C-terminus. IN S e k v. I D č. 36 X aa ³ ' p r u <i s i a v 11 j e and m i d <> i Argi. n y i o o

1

Rest at the C-terminus. IN S e k v. I D č. 37 Xaa ^{3 1} is and m i d o v n e and r g i n y 1 o Rest to C-terminal i. IN Seq. II) č. 30 Xaa ^{3 1} p r e d s t. and ainidovaný arginvlový Rest on C - k o c c. . IN SEQ. I D č. 39 Xaa ^{3 1} p r e d s t a u j e amidated and rg i ny 1 o Rest on C-horses i. IN SEQ. 11) no. 4 0 Xaa ^{3 1} is amidated and rg i ny Rest to C-terminal i. / IN SEQ. ID # 41 Xaa ^{3 3} p r e d s t or u j e Whether or not and r g i n y 1 o Rest to C-c o n c i. IN Seq. II) č. 42 Xaa ^{3 1} before s t. and and m i d o v n y arg eny J ový Rest to C-terminal i. IN SEQ. II) č. 43 Xaa ^{3 3} predstavuj e amidated arginy1ový Rest to C-c o n c i.

The invention relates to useful proteins and peptides. In accordance with the present invention, biologically active polypeptides are first prepared as fused proteins comprising two portions: a first portion a second portion, a n-extension or a carboxyl-representing core portion of the protein, and an N-terminus that is covalently linked end to the amine end of the first portion. The elongated portion of the N-terminus of the fused polypeptide has an amino acid chain that makes it sensitive to cleavage by dipeptidyl peptidase IV (DPP IV). The fusion protein of the invention has the formula:

The elongated portion-core portion of the protein means elongation of the N-terminus of the cleavable moiety represents an arbitrary peptide bond wherein the elongated portion by DPP IV, and the core portion of the protein is the desired peptide that is released from the elongated portion by DPP IV treatment.

The elongated portion of the fusion protein of the invention has an amino acid sequence of the following:

A-X-Y (X'-Y) n where Λ either is absent or represents methionine, n is preceded by the number of '1 i.e. This number is from 0 to 20 such groups, preferably from 0 to 10 seconds per pin,

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

It is selected from the group consisting of hydropyloxyproline, alanine. with serine and when n = 0, then Y is selected with alanine, serine and threonine,

X 'is selected from the group consisting of

t. in one for another,

Except for the group consisting of all those except proline and the amino acids occurring with hydroxypropyne.

According to this formula, when n = 1, there are two Y residues. It is also possible to have up to twenty-one Y residues and twenty-X residues in one design. The individual Y and X 'residues may be any residue from the group from which they are selected. That is, individual Y residues may not be similar to one X 'residue in a multiple-s embodiment, each single X' residue present may be any amino acid except proline and hydroxyproin, regardless of the residue is each other X 'residue. Each individual Y and X 'residue must comply with the rules for that particular group, and all that is necessary is that the different individual residues in the specific positions comply with the rules above.

The pooled proteins in which (A) is present as methionine (Met) represent chains useful for the production of biologically active proteins using recombinant DNA methods in E.

The coli or some coli. The Met chain present in these processes is other means which can use the ones with average expert knowledge. Protein synthesis in E. coli is normally initiated by the translation initiation codon of AUG encoding Met. As a result, the newly synthesized polypeptides have a methionine residue as their N-terminal amino acid. E. coli has an enzymatic activity capable of efficiently removing Met on N &apos; horses, although Met. the residue at the N-terminus is adjacent to an amino acid with a relatively small side chain such as Glv, Ala or Ser as well as lro. The highly spherical removal of Met from the N-terminus is possible with the help of Bretane cyan, splitting Met. However, in order for this procedure to succeed, the Met at the N-terminus must be one M e t i n i n i n i n i n i n i n i n i n i n i n e n e n e n e n e n e n e n e n e n e n e n e n e. Prelo pre s o · · · pro pro pro.

3 containing the internal Met chain must be a second amino acid from the N-terminus of Pro, Ala or Ser if Met is to be removed from the E system by E. coli.

In addition to the coupled peptides, the invention relates to molecules having recombinant DNA strands encoding linked polypeptides, to methods of using recombinantly. j DNA, for methods of using the linked DNA. The method of purifying the polypeptides of interest and for drug delivery methods comprising the use of protein drugs that are converted from prodrugs to biologically active forms by sequential proteolytic cleavage of the N-terminal extension in vivo.

The production of linked polypeptides can be accomplished by standard peptide synthesis or both using methods. The average recombinant DNA is well known to those skilled in the art. Synthesis ti preparation of polypeptides, amino acids. For larger peptides, a preferred method is that wherein less than 50 molecules are recombinantly used in recombinant DNA in host cells.

The coupled polypeptides that are recognized and cleaved by DPI 'IV are like unmodified polypeptides, the nucleus. The present invention describes utility. The first protein medicaments have N-prolongation and more usefully and more advantageously, which have only protein-related properties. They are also called active polypeptides useful as drugs covalently associated with N-terminal extension with cleavable DPI &apos; IV. These prototypes can be converted to biologically active forms by cleavage of DIP IV in the human or animal body to which they have been administered as a protein drug. Thus, the present invention relates to the conjugated polypeptides usefully using the conjugated polypeptides in and to a method of administering biologically active polypepsis. The use of the polypeptides of this invention is the purification of proteins, such as protein medicaments, medicaments wherein the N-terminal extension permits purification thereof, and by means of Part IV. unless this is a component of the computer. d u. which cleavable component of the invention relates to linked polypeptides useful; for the purification processes and for the purification process of the desired polypeptides. This serves the usefulness of the invention and is not intended to be limiting.

For both uses,. the core protein portion of the pooled protein releases from the elongated portion by 1 DPP.

In the case of coupled proteins used in purification methods, it is undesirable that the nuclear proteins be substrates such as those in any way that the invention is cleaved by DPP IV so that DPP IV is not preferred for nuclear protein over nuclear; p r o t e n u, v i v o (i.e. v p 1 a z m e

Z n and m t; is capable of splitting the elongation of the elongated portion. Often, it is highly desirable that, in the case where the core protein is a substrate for DPP IV, it has been administered as a protein drug. In such cases, the protein drug can prolong the viability because the DPP IV portion found in (and kidney and liver tissue) will be used to process the N-terminal extension, and thus protein degradation will be delayed. That is, the N-terminal extension may act as a substrate for DPP IV and a competitive inhibitor, delaying the temporal effect of DPP IV on the core protein and thereby temporarily protecting it.

As used herein, a protein drug means a fused N-terminal portion of a cleavable DPP bound to a core protein that is an IV-containing protein and a covalently biologically active polypeptide useful as the drug. administered as with other e f nions

Protein drugs.According to individual preparations of compounds. The individual form of a protein drug may be or may be preferred in the present invention. In all cases, protein drugs are processed by naturally occurring DPP IV, normally found in the body.

An advantage of administering a protein drug in drug formulations is that it has activity and / or ensures the prolonged presence of the biologically active protein. Protein drugs can only actively continue to act as an emodine. molecules. Protein drugs may have existed. in an inactive state until such time has elapsed that a sufficient portion of the extended period of time is inadequate. and the molecule does not happen. P r o t e i no v e

5 medications can be effective if releasing drugs with different N-konea elongations are dependent on their length and in their amino acid chain.

delayed effect. In addition, they degrade at different rates, specific residues present

It is possible to create a different combination of different pro-drug forms having different N-terminal extensions that can ensure a sustained and stable level of active drug in the patient over a long period of time. Protein drugs can therefore act as delayed drug release systems.

As mentioned above, DPI 'IV cleaves from the N-terminus of a polypeptide when certain residues occupy certain positions. As used herein, position one refers to the 11a position of the amino acid residue at the N-terminus. As it relates immediately to the N-terminus. Thus, the position by position is used here to position two, to the position of the amino acid residue adjacent to position one and which is second from as used herein, position three refers to the amino acid residue immediately adjacent to two and which is third from the N-terminus. . Cleavage, which removes the dipeptide from the N-terminus, occurs between position two and position three if and monos with 1 i to three. it is not proline (Pro), hydroxyproline (Hvp), alanine (Ala), serine (Ser) and threonine (Thr).

DPP IV cleaves residues at the N-terminus at different rates, according to am i. n o k y s i i n is in half o n h. In the case of DPP IV, it cleaves most efficiently if the position occupied by Pro is further effective if the position When position one is occupied by th, h, and tidine, DPP-V works at approximately the same speed when position two is occupied by Pro or Ala. DPP IV is then most effective when position two is occupied by Ser. At least, it is effective when position two is occupied by which of the four most two is two is set by Alain e 1 and 1 a n e n a 1 e b o

T hr.

Using e. It is also possible to extend the velocity. Thus, it is possible to administer this specific protein drug, and it will be possible to design differently. various preparations which contain or combine them in the form of prophylactic agents. P r o l e n o v i i. e k v, m and j c. selecte <1 1 e n i. and

6

The N-termini will be processed at a rate that will depend on their amino acid chain. It is possible to formulate a combination of protein drugs to contain a series of protein drugs that will be processed into active peptides over a period of time.

Amino acid chain length and modification are controlling factors for DPP cleavage rates all or most of the alternatives at the fastest, while elongations conversed at the slowest. Next is

IV ί

Extensions containing Y = Thr will be understood to mean that X-Pro units, where X is either Glu or Asp, cleave much slower than their analogs, where X is a neutral or basic amino acid residue. Since the extensions may contain different residues (of four) at each extension position, there may be an extremely large number, variations and permutations.

Any biologically active polypeptide can be used as a polypeptide polypeptide. PCT patent

PCT / US90 / 02923, incorporated herein by reference, application no. PCT patent application no. PCT / US91 / O82A8, incorporated herein by reference, and U.S. Pat. No. 07 / 368,231, incorporated herein by reference, and any description of bovine growth factor-releasing analogues in the pharmaceutical compositions of the invention. Any hormone analogue that can be used with the protein drug of the invention described in these applications can be used as the core peptide moiety. the protein of the invention. Any protein containing such core protein moieties associated with the extension moieties can be prepared by anyone skilled in the art using well known methods.

Other hormones, proteins and proteins

S p e c i f c c e u s u u. o n e n i a t o h t o e z z m y, z. A ss a n d e n d e z. a b r n u j ú:

peptide (V 1 P), a human non-human, protein and blood protein. of the eye. The gastric inhibitory peptide (G11), gastric releasing peptide (GkP), the human peptide H1, the human peptide YY, the phagmenin 7-37 gi of the peptide-like peptide i, the giucagon-like peptide on the id - 2, substance β, neuropeptide Y? human pancreatic polypeptide, insulin-like growth lacquer t.o r-i (i G - - I). Highly effective hormone (b GII)

7 hormone (bGH), pork growth hormone (pGil), prolactin (PRL), human, bovine or pork growth hormone releasing growth factor (GRF), i riter 1 eukin n-1 beta (IT-β); EGF, iGF-2, glucagon, cor-releasing factor. icotropin n (CRF), dynorphin, somatosin 4, endotelin, transforming growth factor alpha (TGF alpha), growth transforming factor β (TGF β), interleukin-4, interleukin-6 , nerve growth factor (NGF), tumor necrosis factor (TNF), insulin, fibroblast growth factor (FGF), in ter f e. and their synthetic or biosynthetic and n-thiol-2 (II-2) or analogs. These polypeptides can also be used to form the core portion of the fusion proteins of the invention. These polypeptides are intended to serve as examples only and are not intended to limit the scope of the invention.

Smaller coupled proteins can be synthesized e.g. using the present invention, solid phase methodologies using peptide synthesizers (Applied Biosystems, Foster City, California) as described in detail in RLT / US90 / 02923 and 07/368 231 are possible.

For larger molecules, the use of recombinant ONA in host cells is preferred. Several methods are available to those skilled in the art wishing to use the recombinant DNA method to prepare fusion proteins. Typically, genes encoding polypeptides are inserted into expression vectors, which are then used to transform or transfect suitable host cells. The inserted gene is then expressed in a host cell and the desired polypeptide is synthesized. To produce the fusion proteins of the invention in the same way, an additional DNA strand is inserted into the gene. The DNA encoding the residues of the N-to-o-c extension is specifically linked to the 5 'end of the gene encoding the polypeptide of interest. This additional genetic material must be placed downs tream eyes

m u s x would ku g i ? n u. p r e d i ž Hli <1 i. d.

activator of the expression vector activate. melting chains in white to white and f in height required by the formula i.d.

so that the fight under control is placed in a suitable reader so that the expressed N-smoke protein covalently bound to

8

In order to obtain the fusion proteins of the invention using recombinant DNA methods, it is therefore necessary to design a gonucleotide. Ids that encode the amino acid chains of the desired N-terminal extension and upstream protein portion insertion beyond these genes must be capable of from the 5 'end of the gene to produce the genomic nucleotides and methods used to encode the nuclear gene. Methods of production of chimary production

Preferably, the desired biologically prepared compounds are well known to those of ordinary skill in the art.

In addition to the usefulness of the coupled proteins as protein medicaments, the present invention relates to the purification and processing of biologically active recombinant polypeptides active recombinant polypeptides in soluble form or by exclusion from the invention can be recognized by purification agents. exclusion medium or extraction solution and then processed to separate the extension portion from the core portion of the protein to prepare the desired protein. Therefore, the proteins of interest most preferably of the invention are biologically active polypeptides that alone substrates for cleavage of DPP IV.

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

Methods and materials for preparing chimeric genes and recombinant vectors, transforming or transfecting host cells, replicating vectors in host cells, and expressing biologically active foreign polypeptides and proteins are described in the art of Mani pu 1 ati, 01 d and Primrose. , 2nd odition, 19131 and Sambrook et al., M. Clecing, 2nd ed., Cold Spring Laboratory Laboratory Press, NY (1989), both of which are incorporated herein by reference.

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

The present invention is applicable. to purify any prokaryotic or eukaryotic o. The protein may be expressed as a product of recombinant DNA technology in transformed or transfected host cells. These recombinant protein products include hormones, receptors, enzymes, storage proteins, blood proteins, and mutant proteins produced by protein engineering methods, or synthetic proteins. The desired proteins of interest may comprise HIV RNase H, t.PA, II. -1, receptor II, -1, CD4, human nerve growth factor, sCD4-P40, human RSV chimeric glycoprotein (see US Patent Application No. 07/543 780, incorporated herein by reference), EGF, IGF -1, IGF-'2, glucagon, corticotropin releasing factor (CRF), dynorphin, endothelin, growth transforming factor alpha (TGF alpha), Pseudomonus endotoxin 40 (PE40), growth transforming factor β (TGF fJ), insulin and their analogs

Examples of immunoaffinity.

cleaning agents from and on

Other detergent compositions which expanded peptides removable by DPP IV are contemplated by the scope of the invention.

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

Immobilized metal affinity chromatography (IMAG) for protein fractionation was first described by Poratb et al., Nature 258: 598-599 (1975). Poratb has described the perforation of the resin with acid. n od i o e f o out (IDA) and heating of metal ions to the derival and chewed 1DA resin.

proteins can be immobilized to ions. This will require that you (1 DA ')

IMAG and use with account. i e ž v

It is desirable that the column containing the bonding conventionally to the matrix of the simulated metalized acid used is followed by treating the ionic ion (s) via the functional groups to provide electrons, the amino acid residues being react with metallic amino acids with

IDA. Proteins bind to capable metal amino acid residues

Polystyrenes of cysteine, histidines and tryptophan. Proteins by ions through one or more electron donor side chains.

Smith and others disclose in the 11S incorporated herein by reference and U.S. Pat. A 569 79A, which is a reference that certain amino acid residues may be bound to metal ions, however. Bound protein elutes by lowering the pH or using competing anti-ligands such as imidazole when the histidine side chains are involved. Di- or tripeptides containing histidine were used by the alignment method

61) 5 p u s s specific they give unexpectedly excellent

US Patent Resin in US Pat

IMAC to demonstrate that IMAC is selective purification with that of Smith and others disclose the use of recombinant DNA methods to produce a fusion protein comprising a metal chelating peptide covalently linked to a desired metal chelating peptide is an elongated portion that is a lever for the desired polypeptide. This control lever can be used to purify the protein.

The use of IMAC technology with metal-chelating peptides having alternating residues II and s is described in U.S. Pat. 07/506 605, incorporated herein by reference. U.S. Pat. 07/506 metal chelating peptides which results in IMAC purification of the coupled protein when the metal chelating peptide contains three to six alternating His residues. According to the teachings of U.S. Pat. 07/506 605 and no. A 569 79A can be utilized by commonly used IDA for purification of fusion proteins. having cationic metals with at least three alternatives h to class 3 new residues that raft and chains recognizable by DPP IV. The structure of the associated compounds and use in the IMAC system is the subject of U.S. Pat. And 569 79A. The structure and use of the metal-containing peptide moiety with alternating His residues are mainly found in U.S. Patent Application Ser. 07 / p06 5 D 5. In this case, it is connected to the system. 1 n y '21

DPP IV Between and Blackening His Residues The present invention yields a fusion protein that can be purified by 1 MAC technology and subsequently processed by DPP IV to the desired polypeptide.

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

AXZ (X'-Y) _n where A is either not or represents methionine n, n represents the number of successively linked X'-Y groups, this number being from 0 to 20 such groups, preferably 0 to 10 groups,

X is selected from the group consisting of all naturally occurring amino acids. na m i

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

X 'is selected from the group consisting of all naturally occurring amino acid residues except proline and hydroxyproline, with at least two to three residues labeled X' and optionally X being histidines (His). Preferably, Y is Pro and n is at least 3. The effect of forming and Y is Pro, IVP, Ala, Ser or Thr. with an example of a fusion protein comprising

DPP IV, the N-terminal extension is progressively cleaved by the biologically active polypeptide, when, however, the biologically active polypeptide is not itself a substrate of DPI ¹ IV.

One example of a fusion protein has an elongated portion of the formula

His-Y (His-Y) n wherein n = 3 to 11 preferably Pro. Other extended part. having the formula:

XY (HIS-Y) _n wherein n = 3 to 0, X is any naturally occurring amino acid and Y is Pro, Hyp, Ala, Ser or 1hr, preferably P ro.

Because Met on the N-horse is a consequence of protein synthesis in

E. coli and is known to be processed by the E-system. t: o 1 i, po k i a j j; s u s e d i n i s o i i n a

P o, U 1 y, A1a and I. e bo

Ser, the following extension represents the strings useful for

IMAC purification and cleavage of biologically active peptides or proteins expressed intramuscularly in E. coli by methods of DNA

M e t -X-Y (IIs-Y) n wherein n = 3 to 8, Y is Pro, Cly, Ser or Ala and Y is Pro, Hyp, Ala, Ser or Thr, preferably Pro.

a k e to 1 a d e, given

In another, the desired peptide or host protein upon transformation or transfection, vectors can be designed to secrete the protein or polypeptide using an elongated portion that facilitates transport, such as

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

Another purification system for protein purification that uses pooled proteins and is well capable of being treated with DI'P purification technology. U.S. Pat. 4 7 (i2 others, included in description)

IV is an immunoaffinity 137 issued on Nov. 1, 1988 to Hoppa and hereby incorporated by reference, describes the synthesis of a fusion protein having a highly antigenic N-terminal portion and a polypeptide of interest in the portion. C-terminus. According to Hopp and other coupled proteins, they are purified from the crude supernatant by passing the antibody through the column containing immobilized to recognize the antigenic portion. protein. Immobilized antibodies retain the protein in the column while undesirable supernatant components elute. Then it is possible to change the conditions in the column so that the complex a n t i. gé n - dissociated.

According to the invention, the highly antigenic N-terminal portion of the fusion protein is an elongated portion that contains residues recognizable by DPP IV. After pooling the fusion protein described in the Hopp patent, the protein is exposed to DPP IV to remove the elongated portion. Those skilled in the art may appreciate. s o t i. and with Hoppa with an extended poodle section, he photographed the invention on the N-horse.

The embodiments and examples described herein are intended to illustrate the invention and not to be capable of processing such that the invention is removed. Also contemplated equivalents of proteins having an N-terminal extension by at least one of the other means of the elongated portion are achieved by a combination of means. The contemplated equivalents also include fused proteins having chemically modified amino acid residues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Example 1

Synthetic protein drugs that: protein drugs having core proteins, DPP IV.

are connected such as su b s t a r v

The coupled polypeptides administered as protein may be synthesized by any of the cleavable DPP IV covalently linked to the N-terminus of the biologically active polypeptide. The structure of these protein drugs may be expressed by the formula:

the elongated portion - the core portion of the protein wherein the elongated portion 'means the N-terminal extension cleavable by DPP IV, represents a covalent peptide bond, and the core portion of the protein means the desired release from the elongated portion by processing having the N-terminal extension. In this example, the nuclear core is one of the most important parts of this example. .

DPP IV synthetic protein porn. Opposite. The n-linked protein is DPP IV by subsequent drug removal can be prepared by peptide synthesis methods well known in the art.

In one embodiment of the invention, the core protein moiety is epidermal growth factor (EGF) and the elongated moiety is Gly - P r - P h e - Λ a:

G 1 y ^Λ - ¾ ro · ^λ - P he ² - A 1 and ¹ - (EGE)

In another embodiment, the core portion is glucagon and the elongated portion is A1 and -Pro -Phe-Ala:

A 1 a '* - For ⁵ - P he ² - A 1 a' - (giukag é 'n)

In another embodiment, the core protein portion is

- ²⁶ (Ala ¹⁵ Leu ²⁷ ) -bGRI: (1-29) NH 2 (SEQ ID 3) and is Tyr-Ala:

Tyr ² -A and ¹ - {[A1 and ¹⁵ Leu ^{2 7} J -bG RF (1-29) NH 2}.

extended Part

Example 2

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

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

the elongated portion-core portion of the protein wherein the elongated portion represents the N-terminal extension cleavable by DPP IV, represents a covalent peptide bond, and the core portion of the protein represents the desired peptide that is released from the elongated portion by DPP IV treatment. In this example, the core protein of the fusion protein is the potency of the DPP IV substrate produced by removal of the elongated portion.

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

In one embodiment, the core protein portion is an 1 µg of bGRF, (Val ² , Ser ⁸ · ²⁸ , Leu ²⁷ ) bGRF (1-33) 0H (SEQ ID 1), and the elongated portion is G 1γ- r r oP h eA 1 a:

G 1 y ^or - P ro ³ -Phe ² - A 1 and ¹ - {| V a 1 ² S er ⁸ - ^{8 8} Leu ^{2 7} ] -bGRF (1. -33) OH}

In another embodiment, the core protein moiety is a corticosteroid release factor (CRF) and the elongated moiety is G ly-Pro-Phe-A 1 and:

G 1 y ^or -Pro1-Phe ² -A 1 and ¹ - (CRF)

In another embodiment, the core protein portion. is dynorphine and the elongated portion is P h t-r r o ľ h t; - And i a:

Phe ⁴ -Pr o ^J -Phe ² - A 1 a ^! - (clays n or 1; í n)

In another embodiment, the core protein moiety is ominated with t i n-2 µl and the elongated portion is G i -P r o - Phe - P r o:

G 1 y '* - P ro ¹ - P h ^z - P ro' - (somatosta L 11 - 2 8)

In another embodiment, the core portion. protein is an elongated part is A 1 a - P r o - P h t: - A 1 a:

A 1 and ⁴ - Pr o ³ - Ph e ² - A1. and ^1- (endothelin)

In another embodiment, the core protein moiety is a bGRF analog [11 e ² Ser ⁸ · ^{2 8} A 1 and ¹⁵ Leu ^{27 7} -bGRF (J. -40) OH (sec. ID 2) and the elongated portion is Phe-Ala:

Phe ² -Ala> - {[I1e ² Ser ⁸ · ^{2 8} A 1 and ¹³ Leu ²⁷ ] -bGRF (1-40) OH}

In another embodiment, the core protein portion is [11 e ² A 1 and ¹⁵ Leu ²⁷ ] -bGRF (1-29) NH 2 (SEQ ID 4) and the elongated portion is Tyr-Ser:

Tyr ² -Ser '{[11 e ² Λ1 and ¹⁵ Leu ²⁷ ] -bGRF (1-29) NH 2}.

endothelin a

Example 3

Prolonged presence of bGRF analogs Leu ^{2 7} - bGRF (1 - 29) NH 2

BGRF analogue, Leu ²⁷ -bGRF (1-29) NH 2, whose chain is shown as Seq. ID 5, may be administered as a medicament comprising the core protein shown as Seq.

advanced protein drugs.

A variety of well known methods can be prepared using the protein moiety. Expansion based n and Seq. ID 5 are Phe-Ala, Gly-P r-11 e-P r0, Seq 6, Seq. ID 7, Tyr-Ala, Gγ-Pro-Tyr-Al and Seq. ID 8, Seq

5 and various at the N-terminus of protein drugs by Seq. ID 5 and the core portion of protein medicines ID

II)

9, Seq. ID 10, Seq. ID T yr - A1 a - T y r - A 1 a a V a 1 - A i a.

I.

SEQ

II) 12, Seq. ID 13,

Example 4

Thr ² A1 a ^{1 r} > Leu ^{2 7} ] -bGR 1 (I -29) NH 2 bGRF

The bGRF analogue, tlr ² Λ 1 a u ie ^{2 7} ] - bGRF (1-29) NH 2, whose chain is shown as May ID 14, can be administered as a drug containing the core protein shown as SEQ ID 1.4 and the N-terminus. extended protein proteins. Prepared by new ijeknv with widening parts of the radius holi three verz.it

Tyr-Tiira. Tvr-Seľ and Tvr-Thr-Tv r-Th r '2 6

Example 1 (i 5

Linked proteins containing JIV RNase H and N-terminal extension

P rinningthe affinity for the other domains of the peptides of the classes with the vectors are, and are, to be purified or by DPP IV purification. cies, based metals containing alternating immobilized metal ions, designed to direct synthesis of fusion proteins using HIV RNase H as the core protein. As shown below, these fusion proteins are designed to have alternating histidines for purification by affinity chromatography (immobilized metal ions) IMAC alternating alanines for cleavage remove metal chelating peptide (incp).

The preferred N-terminal extensions of DPP IV cleavable according to the present invention are outlined as follows:

The linked HIVRH / incp protein # 1 contains Seq. ID 15 linked to H1 in RNase H:

Met ¹¹ -For ¹⁰ -A 1 and ⁹ -H i. ⁸ -Pro ⁷ -P ⁶ -H is ⁵ -H is ⁴ -Pro ³ -H is ² -A 1 and 1- (111 V RNase H).

The linked HIVRH / mcp protein # 2 contains, as an N-terminal extension, SEQ. ID 16 linked to IIIV KNase H:

Me11 ¹ -A 1 and 1 ° -For ⁹ -H is ⁸ -A 1 and ⁷ -H is ⁶ -A 1 and ⁵ -H is ⁴ -AJ and ³ -H is ² -A 1 al - (HI V RNase H).

HIVRH / mcp pooled protein no. 3 contains the N-terminal extension of Seq. 11) 17 linked to H i V RNase clay:

M et ^{1 1} -G 1 y ' ⁰ - P ro ⁹ - H is ⁸ - P ro ⁷ - P ro ^& - H is ⁵ - H is ⁴ - P ro ³ - H is ² - A1 a' - (HIV RNase H).

These fusion proteins are treated and expressed with E. coli and purified by DEAE cassa tore 1 e and RP HPLC. It is used to characterize coupled proteins

N-terminus. Application of coupled histidine alternating proteins to the purification of recombinant proteins by IMAC, followed by removal of the N-terminal extension of DPP IV after claiming utility of the invention.

Construction of Chimeric Genes Containing the RNase H 11IV gene

All methods and methods.

recombinant DNA 0 1 igonucleotides, are prepared corresponding to standard chelating metals / six chains, are prepared, purified, cooled to the gene encoding 11IV RNase to form a chimeric and splice gene .

In order for RNases H, which are cleavable to the expression chimeric, expression vectors encoding Hl V residues are prepared; j a 1 t t r r u u c h i s j. and the DPR IV, the chimeric gene is inserted into the final vector. Expression vectors containing the genes of interest are transformed into E. coli by standard methods. The gene genes in Ľ. coli results in the production of linked proteins encoded by chimeric genes. These linked proteins comprise the amino acids IIIIV of RNase II and the N-terminal extension, containing alternating histidines (metal chelating peptide) and alternating proline and alanine.

Preparation of crude extracts of E. coli proteins for sequencing and isolation associated

About 3 g of E. coli cell paste is suspended in 30 ml of 0.25 mol of potassium phosphate, pH 7.2, containing 1 mmol of di-tritrile (DTT), EDTA, abenzamidine hydrochloride, phenylmethylene. 1 are 1 phonofluoride (PMSF) 10 mg / l aprotinin, leupeptin and bestatin. This suspension was passed three times on a French press to disrupt cells, and the cell lysate was centrifuged at 12,000 rpm for 1 hour. S u p e r on ta n t. was removed from the foil and added, solid ammonium sulfate was 70% saturated. After stirring for 1 h, the suspension was centrifuged at 12,000 rpm for 1 hour. Supernatant. removed and the cake redissolved in 2.2? ml of 50 mmol Tris, pH 7.5, 1 mmol DTT, PNS f and benzamine.

The MAC column is prepared as follows: a. and El for the Ea.st Elfiw from Pharmac and thoroughly wash with 1 ml of water with water. n t. r o v a n o m s i e n o m 1. i I t r 1. Separate water to form a suspension. The suspension is poured carefully into a glass column (Pharmacia) to a volume (> ml (1 x 7 cm). After the gel has settled, the column is washed with 5 volumes of 30 mmol EDTA (ethylenediamine and notetraacetic acid), pH The column is then washed with 5 volumes of 0.2 N NaOH and 5 volumes of Milly-Q water, then the column is saturated with 5 volumes of 0 mmol of both N and SO 4 (or ZriCl 2 or C 11 SO 4). volumes of equilibrium buffer

The buffer solution was prepared from 20 mmol Tri s, pH 8.0, containing 500 mmol NaCl, 1 mmol 1'MSF, 1 mmol benzamidine, 10 mg / l leupeptin and 10 mg / l and pyrrolidine.

The column was equilibrated with at least 5 volumes of equilibrium solution. 5 to E. The organic extracts are loaded onto the column by means of their own raw extracts. After ingress of the column, the column is washed with 10 free volumes of buffer containing 1.0 mol NaCl instead of

500 mmol NaCl, pH 8.0.

eluting by increasing the entire material into

The imidazole column was then adjusted to pH 0.0 with concentrations. In the initial buffer solution in the experiments, a plurality of washes were performed in each experiment to determine the concentration at which the chimera eluted. Later, this elution is simplified and three concentrations of imidazole: 35 mmol.

of imidazole in 11 ml of equilibrium solution, with the use of 100 ml of mole l and 300 m of mole l of pH 8.0. Ten free volumes of each solution are used. The honey is mixed with an equilibrium solution of 11 ml.

the column is washed with 10 dampening buffer

Finally, the column is washed with 5 volumes of 50 mmol EDTA, pH 8.0 to determine if any protein is still bound in the column. The column flow rates are 1.0 ml / min. 5 ml fractions are collected. The columns are operated at room temperature. Commercial protein Pieree assay kits are used to determine the protein content of the samples.

Becerra S.P. and

HIV R N -j z y j. n í. : ľ EEC

H shall be determined by the method described in 27 D (1,2) 76-80 (s epf.1990) for the description given by those. about links.

Conversion of coupled proteins extended (matui'c) proteins n and N -termines of r c 1 '2 9

Commercially available DPP IV, purified from human placenta (Enzyme Systems Products, Dublin, CA), is used, with a specific activity of 5200 rnli per mg protein. One U is equivalent to the hydrolysis of 1 micromole of a synthetic substrate, A 1 α-Pro-7-amino-9-2H-fluoromethyl coumarin per minute at pH 7.8. Enzymatic conversion occurs by incubating the coupled protein (about 1 to 100 mg) at a concentration of 1 to 10 µg / ml with DPP

IV at 25 ° C substrate 1 for 30 minutes and mass

100. The uncleaved fusion protein is IMAC. Reliability is confirmed by chain analysis at the N-terminus.

from

Example 6 Drugs with Analogs of bGPF

In vitro protein bovine plasma processing

The table summarizes typical experiments showing the formation of the core peptide (Leu ²⁷ ) -bGPF (1-29) NH 2 (Seq.ID 5) from its three N-terminal extended analogs:

Tyr ⁴ -A1 and ³ -Tyr ² -A 1 and ^1- (Leu ²⁷ ) bGRF (1-29) NH 2 (SEQ ID 19) e ² -A 1 and ^1- (Leu ²⁷ ) bGPF (1-29) ) NH2 (Seq ID 18)

Tyr ² -A1 and ^1- (Leu ²⁷ ) bGPF (1-29) NH 2 (SEQ ID 25) when incubated in bovine plasma in vitro. The only metabolites found in the incubation mixtures were the cleavage products associated with DPP IV.

Ty r ⁴ -A 1 and ³ -Tvr ² -A 1 and ^1- (Leu ^{2 7} ) bG PF (1-29) NH 2 (Seq. ID. 19) gradually changed to Ty r ² -A 1 and ¹ - (L eu ²⁷ ) bGPF (J.-29) NH 2 (Seq ID 25), (Leu ²⁷ ) -bGR F (1-29) NH 2 (core peptide Seq II) 5), and end to (Leu ²⁷ ) - (i) GPF (3-29) NH 2 (SEQ ID 2 u).

Four ² - 1 A and ¹ - (l ² of ⁷⁾ bGPF (1-29) NLI 2 ⁽² Leu ⁷⁾ -bGPF (1-29) NLI 2 (nuclear PepV (Leu ²⁷⁾ - bGPF (3-29 NH2 (SeqI) (Leu ²⁷ ) - bGPF (I-29) NH2 (Seq ID of plasma DPP IV on bGRF (3-29) NH2 (Seq ID 29)).

Although under HPLC conditions, it is not known.

(Seq. II) 2 5) e changed 11a i. d S (i k in. ID 5) and then on 29). On my own nuclear peptide 5) k 0 n in e r effect equiv. I D 29). use the same in experiments, T P t i. D bGKF (3 -29) Nli 2 (Seq. t.) can e must. also occur

to others. which are not caused by DPP IV, of course at significantly slower speeds. The core bGRF peptide was not only released from the disclosed DPP IV drugs, but also the half-life of the core protein from the fusion protein was significantly prolonged in vitro compared to the half-life of the core protein (SEQ ID 5) directly incubated in bovine plasma (Table 1). ). In addition, the time at which the protein released from the protein drug was released can be well correlated with the length of the protein drug extension: half-life Seq.

The ID generated from the protein drug in both the heavy and amino acid residues was longer than that derived from proGRF, which has only two amino acids in extension as Seq. ID 18 or 25.

drugs with bGRF analogues in vivo and

How the hormone is injected

Example 7

Bioactivity of protein i n i t r o.

shown in Table 2, plasma growth levels increased when the Bo 1 stein-castrate was an intravenous analogue of Seq. ID 10. At a dose of 0.2 nmol / kg body weight, the induced plasma growth hormone levels were comparable to those induced by intravenous injection of the nuclear peptide (SEQ ID 5). It should be noted that the elongated peptide of Seq. ID% of intrinsic peptide action (Sequence both tested in vitro for release had only about ID 5) when

G H from cells comparable to the in vivo activity that the core peptide was released by the pituitary gland. Therefore, these two peptides appear to be from the extended peptide in vivo.

The same picture of GH release in vj in after the I ^- The wall also protein drugs and analogs bGRF to be. amino acid elongation (SEQ ID 19) as shown in Table 3. There was no significant difference between the in vivo GII release induced by protein administration when administered with four residues

1.

or 2 amino acids in elongation (in the case of the elongation)

ID 19 and Seq. (18), despite the significant difference in nuclear protein of the two generated by the two bGRF drugs, as shown in Table 1.

as shown

1

Growth hormone release in vivo was rapid for both the core peptide (Seq. I.b.) and Seq. II) 19 and 18, without any time difference of the maximum GH after irritation of the bGRF analogs.

Our interpretation of these GH release results is as follows: Rapid protein drugs in vivo are caused by generally high levels of tissue and organ DPP IV, which are about 100 times greater than the plasma DPP IV concentration. This explains the difference in in vivo protein drug processing rates compared to the in vivo cleavage summarized in Table 1. It is also possible that the half-life of a nuclear peptide resulting from a precursor protein drug has been prolonged in vivo but not sufficiently to manifest altered (prolonged) release of growth hormone. In vivo release of G11 is known to be. influenced by the stimulatory effect s o m and t. (See also SRIF) · In the example of Moseley and others: J. Endocr. 17

253-259 (1988), animals were injected intravenously with G1F for two hours prior to feeding. the reasons that the pancake is more sensitive to the irritation of GRF before feeding than after feeding. Feeding factors such as secretion of bovine / pancreatic SRIF may impair the ability of the pod to release GH. In other words, no G11 will be released from the pod in the presence of GRF bGRF and the inhibitory effect of the release of somato tropin (bulls) fed by bran during the predominance of SRIF. In the fed bran concentration, the undisturbed example of plasma GH castrates normally decreases at hours after feeding (the so-called glacial period) with another particular unpredictable GH pulse between 5 and 8 hours after feeding. In response to GRF injection 2 hours before feeding, GH response is rapid within 5-20 minutes and GH levels remain elevated for 120 to 2A0 minutes before returning to baseline. In the case of GRF protein drugs previously tested, only the first exogenous GH peak was increased, the second showed no treatment effect. It is possible that the half-life of nuclear GRF released from. of the protein drug in our attempts before 1 d <> s 1, and to allow the patient to be able to do so. is present in the cycle at concentrations sufficient to

2 affecting another particular pulse of exogenous GH, usually A up to 6 hours after the first.

Overall, our results support the general concept of protein drugs described herein because: i) GRF protein drugs with N-terminal extension with cleavable DPP IV have been successfully prepared to produce a nuclear peptide in bovine plasma in vitro via DPP IV mediated cleavage; desalted

the half-life of the in vitro nuclear protein from the pooled protein has been significantly prolonged and is a function of the length of the N-horse and protein drug elongation, iii) the fact that bGRF protein drugs with very low intrinsic potency were as effective as the core peptide that the core peptide was formed in vivo as expected.

Example 8

Preparation of Gly ^from '-Prol-Ile ² -Pro ^I - {(i, eu ²⁷ ) bGRF (1-29) NH2} trifluoroacetate salt.

Synthesis of GRF Peptide Analog Seq. 11) 26 having the formula: Gly-Pro-Ile-Pro-Tyr-Ala-Asp-Ala-11le-Phe-Thr-Asn-Ser-Tyr-ArgLys-Val-Leu-Gly-Gln-Leu-Ser- Ala-Arg-Lys-1, eu-Leu-Gln-Asp-IleLeu-Asn-Arg-NH 2 (as the trifluoroacetate salt) is carried out sequentially according to Procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values in

in parentheses: Asp A, 16 mm (6) , Thr 1.07 (J), S e r 1.81 (2), G 1 u 2.07 (2), For 1.98 (2) , G l y 1, 99 (2), Ala 2 99 (3), Val 1 L, 1 3 (1), 2.86 (3), 1, e u 5.0 8 (5), Tyr 1.93 (2), Ph e 0.96 ABOUT) , Lys

2.06 (2), Arg 2.97 (3).

Example 9

Preparation of Ty r - A 1 a - G 1 y ^{4 -} Pro ^J - 1 e ^{2 -} Pro ¹ - {(Leu ^{2 7} ) b GRF (.1-29) NII 2} triploorofosso. .

Synthesis of GR 1 Analog Analog Seq. ID 27, comprising Seq. clay 6 as an elongated part having the formula:

3H - Ty r - A 1 a - G 1 y - ¾ re - 1 1 e - i 'ro - Ty r - A J. a - A sp - Λ ia - 1 ie - P h - T hr - A sn S e r-Ty r - A rg - lys -Val- L e uG i yG] n - le u-Se r - A ia - Arg - ly s - le u - L eu 3 3

G 1 η - A sp -1 1 e - L eu - A sn - A rg - N Η 2 (as three 1 1 · acetic acid) is carried out sequentially according to procedure A. which is described in the published PCT patent included in the description of the following amino acids, theoretical values in brackets: Thr 1.03 (1), Ser 1.74 (2), Glu 2.05 (2), Pro Ala 4.01 (4), Val 1, -28 (1), clay appended to PCT / US 9 0/0 2 92 3, hereby incorporated by reference. Results of analysis A with p 4.04 (4),

1.99 (2). Gly

2.00 (2)

2.84 (3), Leu 5.09 (5), Tyr 2.94 (3), Pbe 0.97 (1), Lys 2.07 (2), Arg 3.00 (3).

Example 10

Preparation of Lys ⁸ -Pro ⁷ -Tyr ⁶ -A 1 and ⁵ -G 1 y ⁶ -Pro ³ -I1 e ² -Pro '- {(Leu ^{2 7} ) bG RF (1- 2) N112} Trifluoroacetate Salt

Synthesis of GRF peptide analog sckv. II) 28, comprising SEQ ID NO 7 as an elongated portion having the formula: Lys-Pro-Tyr-Ala-Gly-Pro-11e-Pro-Lyr-A1 and Asp-A1 and -11e-Phe-ThrAsn -Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-I.eu-Ser-Ala-Arg-Lys-LeuLeu-Gln-Asp-White-Leu-Asn-Arg-NH2 (as tr 111uoroacetate) is carried out sequentially according to procedure a, which is described in published PCT patent application PCT / US90 / 02923,

included to popi s u t. ý m t0 reference. The results and n and i z amino acids, t e 0 r e t i c values 0 0 0 0 0 0 0 0 0 0 0 A with p 4.04 (4), Thr 0.95 (1), Ser 1, 78 (2), Glu 2.04 (2), Pro 2.91 (3), Gly 1.98 (2), Ala 3.91 (about) . Va 1 0.96 (1), ile 2,86 (3), Leu 5.08 (5), Tyr 6 (3) , Pbe 0.97 (s), Ly.s 3.06 (3), Arg 3.08

(3).

Example 11

Preparation of G 1 y ^Λ -For ³ -Tvr ² -A ia ¹ - {(Leu ^{2 7} ) bGRF (1-29) NH 2} tria 1 for the 6-acetic salt

Synthesis of GRF Peptide Analog Seq. II) 29, which has the formula:

6-G 1 yP r o-Ty r- A 1 a - Ty r - Λ 1 a - A sp - Λ 1 a - I 1 e - Pii e - T li r - A sn - S e r-Ty r - And rgLys-Va i-Leu-G iy - G! n-L eu-S e rA J a-Arg-Lys-Leu-cuG 1 n-A more preferably Leu-Asn-Arg-NH2 (as tri-trifluoroacetate) is carried out sequentially according to Procedure A. which is described in published PCT patent application PCT / US9002923, incorporated herein by reference. The results and theories of the seine, theoretic theses are not formed by: A sp 4.0.) (4), fnr 0.9 6 (1), S cr .1, 8 0

6

(2), Glu 2,02 (1), ľr o 0.97 (1), G 1 y 1.98 (2), A la 3.91 wall 0.99 (1), White 1.89 (2), L e u 5.0 8 (5), Ty r 3.05 (3) D, 98 (1), Lys 2.03 (2), Arg 3,0 6 (3).

Example 12

Preparation of Tyr ⁶ -A 1 and ^5- G 1 Y ⁴ -ro ³ -Tyr ² -A 1 and ¹ - {(Leu ²⁷ ) bGRF (1-29) NH 2) trifluoroacetate.

/

Synthesis of GRF Analogues of Peptide Sek v. II) 30, comprising Seq. ID 8 as an elongated portion having the formula:

7-Tyr-Ala-Gly-1-Tyr-A 1.a-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser -Ala-Arg-Lys-Leu-Leu-GlnAsp-11e-Leu-Asn-Arg-NH 2 (as a 1 ^: 1 urea) was carried out sequentially according to Procedure A described in the published PCT patent PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical

values they are in parenthesis : Asp 6.07 (A), T h r 0.96 (D, S e r 1.79 (2), Glu 2.02 (2), LRO 0.99 (D, G 1 y 1 95 (2), A l and 6 , 80 (5), Val 0.96 (D , clay 1.87 (2), L e u 5.09 (5), Ty r 6.11 (A), Phe 0.97 (1) , Lys 2.06 (2), Arg 3,0 8 (3). Example 13 preparation Lys ⁸ - P ro ⁷ - Ty r ⁶ -A la ⁵ Gly * -Pro ³ - Ty r ² -A la ¹ - {(Leu ²⁷ ) bGRF-

(1-29) N H 2} trifluoroacetate.

Synthesis of GRF Peptide Analog Seq. ID 31 containing Seq. ID 9 as an elongated portion having the formula:

8-Lys-Pro-Tyr-A1a-G1y-lro-Tyr-Ala-Tyr-Al-Asp-Al-11e-Phe-Thr-

A s n - S e r - Ty r - A r g-Lys-Va-1-Le u - G 1 γ- G i n - L e u - S e r - A 1 a - A r g - Lys-Leu- Leu-Gn-Asp-11. e - L e u - A with n - A r and - o ΝΙΙ 2 (a 1) o c a e t á t) the carried out gradually according to <1 po s i u}> u A which j < described in audience not j PTC approx k e P CT / US90 / 02923 and broken down to description here reference. E n s 1 e d k y and n a 1 z z a m i n o y s e i í n, theoretical values are in z á t v o r 6 p (6) , Thr 0.95 (1), S e r 1.7 8 (2), Glu 2, 0 i (2) . lro 1.95 (2) , G 1 y 1, 96 ('2), A 1 and 6,81 (5), Va i 0.9 5 (1 ) 1 i e 1.87 (2), i. e u 5.0 9 (5), Ty r 6.12 (6), e 0.96 (i 1, 1 y) WITH 3.08 (3), Arg 3, i 0 (3).

Example 14

Preparation of Phe ¹⁰ -A 1 and ⁹ -Lys ⁸ -Pr o ⁷ -Tyr ⁶ -A 1 and ⁵ -G 1 y ⁴ -For 3-Tyr ² -A 1 and ¹ {(Leu ²⁷ ) bG RF (1- 2 9) NH 2} three 1 uorace tu.

Synthesis of GRF Analog Peptide Anal. 11) 32 comprising SEQ ID NO 10 as an elongated portion having the formula:

9-PheA 1 a-Ly s-Pr o-Tyr-A 1 aG 1v-Pro-Tyr-A1 a-Tyr-A 1 aA with pA 1 a-11 e Phe-Thr-Asn-Se r'- Tyr - A rg - Ly s - V and 1 - L eu - G 1y - G 1n-Leu-Ser-Ala-ArgLys-Leu-Leu-Gln-Asp-Ile-Leu-Asn-Arg-Nil2 ( as trifluoromethyl a) is carried out successively according to procedure A described in U.S. Pat

* published incorporated into PCT paten p 0 f) i u t u m 10 PCT / US90 / 02923 reference. 1 and n is 1 • * amino acids, theoretical values s ú v á á tvorkách: A with p A, 16 (A), Thr 1.01 Ser 1.09 (2) G 1 u 2.0 0 (2), Pro 1.91 (2), Gly 1.93 (2), A la 5.7 2 (6), Val 0.97 (1), 90] (2), Leu 5.09 (5), Tyr A, 09 (A), Phe 1.99 (2), Lys 3.00 (3), Arg 3.0 A

(3).

Example 15

Preparation G 1 y ^{1 2} - Pr o ¹ -Phe ¹⁰ -Ala ⁹ -Lys ⁸ -Pro ⁷ -Tyr ^G -Alo- ^> -GLy ⁴ -Pro ³ Tyr ² -Ala ¹ - {(Leu ²⁷ ) bGKF (1 -29) N J12} tr. f 1 uóra ce ta tu.

Synthesis of GRF Peptide Analog Seq. ID 33 containing Seq. ID 11 as an elongated portion having the formula:

10-Gly-Pro-Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val Leu-Gly-Gln-LeuS e r-A1 a-Arg-Ly s-Le u-Leu-G 1 nA with p-11 e-Le u-Λ sn -A rg-NH 2 (as

three f 1 uórac et.át.) take place e p 0 s t p e p 0 d t a p 0 s t u p u A, which is described v p u b 1. i k 0 in n PCT Patent p r i h 1 a k e PCT / US9O / O2923. included in with this link. Res products amino acids analysis s e 1 i 11, t e 0 r e t i. c k é values are in brackets : A s p A, 00 (A), Thr 0.96 (T), Ser 1., 7 9 (2), G 1 at 2.07 (2), for 2,0 0 (3), Gly 2.9A (3), A1 and 5.7 ó ((>), Val 0.96 (J.). Ile 1, clay 0 (2), Leu 5.13 (5), Tvr A, j 1 (A), l’h e 1.99 (2), Lvs 3.10 (3) , Arg 3.09 (3). Example 16 EXAMPLE ^- and law in the 1 ^{"Z |} - Pr 0 ^{! !} - G 1 y * ² - Pt "11- Phe ¹⁰ -A 1 and ⁹ - L ys ⁸ - pr 0 ⁷ Tyr G _

A 1 and ⁵ - G 1 y ^h - Pr o - Ty r ² - · A 1 and ¹ - {(L eu ^{2 7} ) bG R 1 (1-2 9) N11 2} three 1 u u lation 36 here .

Synthesis of GRF Peptide Analog Seq. ID 36 containing Seq. ID 12 as an elongated portion having the formula:

11-Va 1 -Pro-G 1 y-1'r-1'li eA 1 a - Ly s - Pr o-Ty r-Λ 1 α-G 1'-yr-Ty r-A1 and Tyr-Ala-Asp -Ala-Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-GlyG1 n-L eu-S er-A 1 a-A rg-Lv s-L ou-L cu -G 1 n-A with p-11 e-L eu-A sn-A rg-NH 2 (as triforate) is carried out up to the post-up A described in the injected PCT PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values are in brackets:

A with p 6.06 (about) , Thr 0.96 (D, Ser 1.01 (2), Glu 2.06 (2), for 3.80 (6), Gly 2.99 (3). A 1 a 5.92 (6), wall 1.98 (2), Ile 1, 89 (2), Leu 5.10 (5), Ty r 6.09 (a) Phe 1.99 (2), I.y s 3.06 (3), Arg 3.08 (3).

Example 17

Preparation Arg ^{1 6} - Pro ^{1 5} - Val ^{1 4} - Pro ^{1 3} - G 1 y ^{1 2} - Pro ¹ t-Phe 4 -Ala 4 -Lys ⁸ Pro ⁷ -Tyr ⁶ -A1 and ⁵ -G 1 y ⁴ - For ^3- Tyr ² -A 1 and ¹ - {(Leu ²⁷ ) bGRF (1-29) NH 2} trifluoroacetate.

Synthesis of GRF Peptide Analogs Seq. ID 35, comprising Seq. ID 13 as an elongated portion having the formula:

12-Arg-Pro-Val-Pro-Gly-Pro-Phe-Ala-Lys-Ala-ProTyr-Gly-Ala-ProTyr-Tyr-Ala-Asp-Ala-Ile-Thr-The-Asn-Ser- Tyr-Arg-Lys-ValLeu-Gly-Gln-Leu-Ser-Ala-Arg-by-Leu-Leu-Gln-Asp-Ile-Leu-AsnArg-NI12 (as three fluoroacetate) is carried out sequentially according to Procedure A, which is described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Results of amino acid analysis, theoretical values are in

parenthesis : A with p 6.10 (6), Thr 0.98 (1). S e r 1.86 (2), Glu 2.03 (2), For 6 , 82 (4), ( 11 y 2.97 (3), A1 and 5 , 9 i. (6), Val] 1., 99 (2), ile 1,88 (2), 5 e -09 (6), Ty r 6, 10 (ABOUT .· Pb e 1.98 (2), , by s

3.03 (3), Arg 6.09 (6).

Example 1 B

P r {[> ra va 1 ² - A ia ¹ - {(ie u ^{2 7} ) b ((RF (1-2 9) NT fz) tri lu l race tá ovej 1 1 i i i i i..

Synthesis of a n n i o g (, R 1 of the peptide Seq. D 3 (> has the formula:

14-Va1-Ala-Tyr-A1a-Asp-A1a-11e-lhe-Thr-As n-Ser-Tyr-Arg-Lys Va1-Leu-Gly-G1n-Leu-Ser-A1 a-Arg- Lys-Leu-Leu-G 1 - -Asp-1β-LeuAsn-Arg-NH 2 (as a three-fluor phase) is carried out sequentially according to procedure A described in published PCT / US9O / O2923 , incorporated herein by reference: Amino Acid Analysis Results, Theoretical Samples: A sp 3.9 B (4), Thr 0.89 (1), Ser 1.76 (2), G 1 µ 2.02 (2), Gly 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

Preparation of Tyr ^{2 -} Th ¹ - {(A 1 and ¹⁵ Leu ^{2 7} ) bGKF (1 - 29) N 11 2} tri-o-theta.

Synthesis of GRF Peptide Analog Seq. II) 37, which has the formula:

15-Tyr-Thr-Tyr-A1a-As pAi-1e-Phe-Thr-As n-Ser-Tyr-Arg-Lys Va 1-Leu-A1 and G 111-Leu-Ser-A 1 and - Arg-Ly s-Leu-Leu-G 1 nA sp - 11 e-LeuAsn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference. Amino acid analysis results, theoretical values are enclosed in brackets: sp sp 4.06 (4), hrhr 1, clay 6 (2), Ser 1.77 (2), G 1 2,0 2.07 (2), Ala 3 , 90 (4), Val 1.08 (1), Clay 1.89 (2), Leu 5.1.1 (5), Tyr 2.94 (3), Phe 0.96 (1), Lys 1.99 (2), Arg 3.04 (3).

Example 20

Preparation of Tyr Thr ^{1 2} - {(I 1 Λ ² E ^{1 r} 1 a ^'2 in Le ⁷⁾ bGRľ (1-29) NH2}, three delta L in d r and s are GMT.

Synthesis of GRF Peptide Analog Seq. clay) 38, which has the formula:

16-Tyr-Thr-Tvr - 1 1 eA with p-Λ I a - 1 1 e-lhe - Thr-A with nS e r-Tyr-Arg-Ly with Va 1 - Leu-A 1 aG I n - Ie u-S e rA 1 a-A rg-Ly s-Le u-1. e uG 1 nA sp-1 ie-L, eu Asn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially as described above. in published

PCT / OS90 / 02,923th For the purposes of amino acid analysis, the theoretical values are based on the sample: A sp 6, 0 7 (4), T 1, 8 7 (2), S er 1.75 (2), G 1, 2.0 7 (2), Ala 2.94 (.3), Val 1.0 d (i), .11 e, 2.87 (3), 1.e.i. 5.12 (5),

PCT p a t e n t o v c; j p o p i s t t m. on procedure A, e i b 1 a

s)

Tyr 2.92 (3), Phe 0.96 (1), Lys 2.00 (2), Arg 3.05 (3).

Example 21

Preparation of Tyr ² - Thr ¹ - {(Thr ² -A 1 and ¹⁵ - Leu · ²⁷ ) bGRF (1 - 29) NH 2} trifluoroacetate.

Synthesis a n a 1 ύ g u P F p e t i d S e k v. 11) 3 9, which has:

17-Tyr-Tlir-Tyr-Thr-Asp-A 1 and -11 e-Plie - Thr-Asn-S er-Tyr-Arg-Ly with Va 1-Leu-A 1 aG 1 n - L. e uS e r-A 1 a-Arg-1, y-Leu-Leu-G 1 n-A sp-1 e-LeuAsn-Arg-NH 2 (as trifluoroacetate) is carried out sequentially according to Procedure A, which is described in published PCT / US90 / O2923, incorporated herein by reference, in amino acid analysis results, in parentheses: Asp 4.05 (4), Thr 2.68 (3), Ser 1.77 (2), Glu 2.07 (2), Ala 2.90 (3), Val 1.08 (1), Clay 1.89 (1), Leu 5.20 (5),

Tyr 2.87 (3), Phe 0.93 (1), Arg 3.07 (3).

PCT patents are hereby incorporated by reference

Example 22

Preparation of Tyr ² -Se r ¹ - {(Thr ² -A 1 and ¹⁵ -Leu ^{2 7} ) bGRF (1-29) NH 2} tr 1-theta.

Synthesis of GRF Peptide Analogs Seq. ID 40, which has the formula:

18-Tyr-Tyr-Tyr-Tlir-Asp-A1 and -11e-Plli-Thr-A with nSe-Tyr-Arg-Ly with Val-Leu-Ala-Gln-Leu-Ser-Ala -Arg-Lys-I.eu-I.eu-Gln-Asp-Ile-LeuAsn-Arg-NH2 (as three fluoroacetate) is carried out sequentially according to procedure A described in published PCT patent application PCT / US90 / 02923 , incorporated herein by reference. Amino acid analysis results, theoretical values are shown in brackets: Asp 4.11 (4), Thr 1.82 (2), Ser 2.64 (3), Glu 2.05 (2), Ala 2.90 (3) , Val 1.04 (1), Clay 1.87 (2), Leu 5.16 (5),

Tyr 2.92 (3), Phe 0.94 (1), Lys 2.01 (2), Arg 3.04 (3).

Example 1 and d_2 3

Preparing Tyr ⁴ -T hr ^J -Tyr ^{2- to} hr ¹ - {(T hr ² -A 1 and ¹ '- L eu ^{2 7} ) h GRP (1-29) NH 2} trif 1. at óra ce ta here.

Synthesis of and GRF peptide logo Seq. í D 41, which has the formula:

19-Ty r-Ty r-Ty r-ΐ ii r - Ty r - T li rA sp - A ia - iie - P h - T h r- A s 11 - S e r-Ty r Arg-Lys-Va 1 - L eu - Λ i, 1 - (I I n - i, eu - S e rA ia - A r g- L ys -Leu-leu - G 1 n - As pI 1 e - L eu - A sn A rg-M1 2 (as i. U. I. T.) Is followed stepwise according to procedure A as described in PCT patent application PCT / IJ 890/02923, incorporated herein by reference. Amino acid analysis results, theoretical values are in brackets: Asp 4.06 (4), Thr 3.66 (4), Ser 1.85 (2), Clu 2.05 (2), Ala 2.93 (3) ), Val 1.09 (1), Clay 1.91 (2), L.eu 5.15 (5), Tyr 3.91 (4), The 0.95 (1), Lys 2.00 (2) Arg 3.04 (3).

Example 24

Preparation of Tyr ² -AJ and ¹ - {(Leu ^{2 7} ) bGRF (1-29) N ^{1 2} } three µl of here.

Synthesis of GRF peptide analogue of SEQ ID NO 42 having the formula:

Tyr-A 1 a-Tyr-A1 a-As p-A1 and -11 e-Phe-Thr-A sn-S er-Tyr-Arg-Lys-Va-Leu-G 1 yG 1 n-Leu-Se r-A ja - Arg-Ly s-Leu-l.eu-G 1 n- A s p-I 1 e - L eu - A n Arg-NI 2 (as the three-molar here) is followed sequentially according to procedure A, which is described in published PCT patent application

PCT / 1JS90 / 02923, incorporated herein by reference for amino acid analysis, theoretical values 4.01 (4), Thr 0.97 (1), Ser 1.88 (2), Glu (1), Ala 3.88 (4) , Val 0.97 (1), Ele 1.86 by reference. Results in parentheses: Asp

2.00 (2), Gly 1.02 (2), at 5.03 (5),

Tyr 3.03 (3), Phe 0.96 (1), Lys 2.08 (2), Arg 3.03 (3).

Example 25

Preparation of Ty r ⁴ -A 1 and ³ -Ty r ² -A 1 and ¹ - {(1 ^{2 7} ) bGRF (1 - 29) N 1 2} trifluoroacetate.

Synthesis of GRF (S e k) 11) 43, which has the following formula:

13-Tyr-A 1 a-Tyr-a 1 a-Tyr-A 1 a-As p-A1 a-11 e-Phe-Thr-Asn-S-r-TyrArg-Ly s-Va 1 - Leu-G 1 yG 1 nl.eu-Se rA la-Arg - by s - L.eu - L e uG 1 n-A with p11e-Leu-Asn-Arg-NH2 (as tr if 1 u tation) is governed by sequentially according to Procedure A described in published PCT patent application PCT / US90 / 02923, incorporated herein by reference

reference. Analysis results aminokv s e 1 í n , t c : 0 t e c how much t y s ú in brackets: Asp 3.97 (4) , Thr 0.90 (D i . S e 1 ’ 1.74 (2), G .1 u 1.98 (2), Gly 1.04 (1), AI and 4, 8 5 (5), V and i 0,9 1 (1), I 1 e 1.77 (2), 1, u 5, i 3 (5), T y r 4, 1 6 (6), 0 , 9 9 (. 1 j, by s 2.0 7 (2),

Arg 3.05 (3).

Table j

In vitro and in vitro plasma activity of GRF and n a 1 o g o v

Peptide chain SekvID NO U č i. n n o s ť A Plasma * * ti / 2 (min) (Leu ²⁷ ) bGRF (1-29) NH 2 5 1:00 2A. 0 (2) Ile ² -Pro '- (Leu ²⁷ ) bGRF (1-29) Nil 2 10 0.04 5 43.2 (1) Tyr ² -A 1 and ^J - (Leu ²⁷ ) bGRF (1-29) NH 2 2 5 12:13 38.5 (1) Tyr ⁴ -A 1 and ³ -Tyr ² - A 1 a ^] - (Leu ²⁷ ) bGRF (L-29) NH2 *** 19 ^} 0052 297.1 (2)

notes:

* peptides were tested in anterior pituitary cells culture as described by Friedman et al., J. Peptide and Protein Res. 37, 14-20 (1991).

** peptides were incubated in 30 micromoles of bovine plasma in vitro at 37 ° C as described by Kubiak et al., Drug.Met.Disp. 17, 393-397 (1989). The values given here refer to the half-life of (Leu ²⁷ ) bGRF (1-29) NH 2 (Seq. II) 5) incubated directly in plasma or to (Leu ²⁷ ) bGRF (1-29) NH of (Seq ID 5) generated from the extended peptides SEQ. II) 18, 25 or 19. Two experiments were performed with two different plasma samples. The trial number is given in brackets.

*** peptide Seq. ID 19 was tested against (Leu ²⁷ ) bGRF (1-29) NH'2 different bovine plasma samples.

(Sequ. ID Half Time Sequ

5) using II 15 in this plasma was 5D, 2 minutes.

.1

Table 2

GH response to

IV different doses of (Leu ²⁷ ) bGRF (1-29) NH 2 (Seq ID 5) and e ² -Pro ¹ - {(I.eu ^{2 7} ) bGRF (1-29) NH 2 (Seq ID 18) for Ho 1 steins of bran-fed castrates *

Treatment Dose nmo 1 / kg P ο č e t cl Height and peak ng / kg 1 n t. e r v a 1 Area *** And ^a B a And ^a B ^a And ^a B ^a The solution ** C) 11 ^b 32.4 ^b 3 2.4 ^p 8 9 ^p 89 ^p 4.3> ’ 4. 3 ^b SekvID18 12:02 8/1 0 ^c · 7 .1. . 2 ^b · ^c 7 6.9 ^b · 23 « 18 « 4. 6 ^b - « 5.0 ^b - ^c Sequence 5 12:20 9/1 0 ^y - 119.8 « 130.9 « 23 « 14 « 6.8 ^d 7.0 ^d SekvID18 12:20 10/10 ^c 10 1.4 c. D 101.4 « 26 « 26 « 6.3 «- ^d 6.3 «· ^d SekvID18 20.0 10/10 ' 1 2 7.8 ^d 138.8 « 2 3 « 23 « 10.1 « 10. l ^e PULL 12:04 9.1 9.4 8 8 0.3 0.3 P value 0.0001 0.007 0.007 12:04 0.0 3 0.00 01 0.0001

notes:

Animals were challenged intravenously at the indicated doses two hours prior to feeding by the procedure described by Moseley et al., J. Endocr. 17, 253-259 (1988).

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

a Animals with a response * * saline * * * range 0-8 hours (units) b, c, d, e values with different indexes in the column are significantly different (P less than 0.05).

Table 3 Response s é r o v o o G H for IV injections r h z n y c h d á v o k (Leu ²⁷ ) bGRF (1-29) NH ₂ (Seq. 10 5) and T yr ⁴ - A 1 and ³ -1 1 e ² ~ P ro ¹ - {(Le u ²⁷ ) -1) GKF (1-29) NH2) (Seq. II) 19) u Ho 1 stein kastrá to v fed c b abrasion m i *

Treatment dose nmo 1 / kg Count and -7Peak height ng / m 1 interval Area *** A D A B A B The solution ** 0 11 ^b 30.9 ¹ ' 30.9 ^b A5 ^b A 5 ^b 3.9 ^b 3.9 ^b Sequence 5 12:02 9/12 ° · ^d 91. 9 ^C 101.3'- Ai b 20 ^c And. 7 ^b · c And. 7 ^b 'c SekvlD19 12:02 10/12 c · ^d 80.5 ^C 92. Ac 30 ^p 22 ^c And. 8 ^c And. A ^b · c Sequence 5 12:20 11/12 ^d 7 9.3 ^c 79.1 ^b - ^c 21 ^p 10 ^c 5. A ^c 5.3 ^C SekvID19 12:20 7/12 ^c 9 7. A ^c J. 2 0.1 c 63 ^p 8 ^C 5. Ac 5. A c. <1 SeqIDl 8 20.0 9/12 ^c · ^d 7 A. 5 ^c 70.8 ^b - ^c 2A ^b 18 c 6.9 ^d 6.8 ^d PULL 0.1 15.9 17.3 1 A 0. A 0.3 0. A phodno ta 0.0001 12:06 12:06 12:28 12:11 0.0000 0.007 EMS 0.1117 30 A 2 362 3 2260 7 63 2 2 81 2A83

notes:

Animals were fed over two hours prior to feeding by the procedure described in Moselle, et al., J.Endocr. 17, 253-259 (1900).

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

a Animal with a response ** Saline saline *** 0-0 hours (units) b, c, d, e values with different indices. they are significantly different in the column (P less than 0.05).

List of strings string information Sek v. II) č. J

C i i r a k f e r i s i. i k a r e a a z c a:

) A) D 1 : 3 3 B) T y i): or m i n o y s e i in D) To po) ú g i a: i i n e i r

(xi) Chain description: Seq. ID e. 1

5 10

Tyr-Val-Asp-Ala-Ile Phe-Ser-Ser-Tyr-Arg-Lys-Val-Leu 15 2 0 2 5

A1a-G 1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G Jn-As p-11e-Leu-Ser-Arg 30

Gln-Gln-Gly-Glu

Sequence Information Sequ. III (i) 2 Characteristics of the chain: i) A) Length: 90

B) Type: amino acid

D) Topology: linear xi) Chain description: Seq. ID # 2 1 5 9

Tyr-Ie-Asp-Ala-Ie-Phe-Thr-Ser-Ser-Tyr-Arg-Lys-Va1-Leu 15 20 25

Ala-Gln-Le-Ser-Ala-Arg-Lys-Leu-l.eu-Gln-Asp-Ile-Leu-Ser-Arg 30 35 90

G1 n-G1n-G1y-G1u-Arg-A with n-G1n-G1u-G1n-G1y-A1 and

Sequence Information Sequ. Chain characteristics: (i) A) Length: 29

B) Type: amino acid

D) Topology: 1 line i x) Features:

A) Name / Key: Arginyl

B) Location: Xaa29 xi) Chain description: Seq. II) č

5

Tvr-A 1a-Asp-A1 and -1 and Phe-Thr-Asn 1 5 21)

A 1 a - 0 1 n - Le: u-S e: r-A 1 a - A g - L y s - I. e u

Sequence Information Sequ. Cha ra kle r i s t th e r a z z a:

residue ninidated at the C terminus

S e r - Ty r - A r g - Ly s - V and I - I. c u 2 5

Le: u-G i n - A s i »- I 1 e - L t: u-A s n -Xa a

II) č. and

(i) (A) (I): '29

D) TyP ^: amino acid

D) T o p o 1 o g i: Linear ix) Features:

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

(B) Location: Xaa29 (xi) Pope s: c. I L) Nr. 6

5 1D Ty Tyr-11 e-Asp-A1 and -Ie-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Va1-Leu

20 25 * A 1 a-G 1 n-Leu-S e r-A 1 a - Arg-Lys-1.eu- Leu-G 1 n-Asp-1 e-Leu-Asn-Xaa

Sequence Information Sequ. ID č.5

Characteristics of the chain: i) A) Length: 2 9

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

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

B) Location: Xaa29 xi) Chain description: Seq. ID # 5

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

20 25

Gly-Gln-Leu-Ser-Ala-Arg-Lys-l.eu I.eu-Gln-Asp-Ile-Leu-Asn-Xaa

I n f o r m a c e e r e r e a n e e e v e. ID # 6

C h a r a k t e r: i. t i c t i s: i) A) Length: 6

B) Type: amino acid D) Topology: linear xi) Chain description: Seq. 1D no. 6

5

Ty r-A 1 and -G 1 y-Pr o - I 1 e-P ro

Sequence Information Sequ. II) No.7 Characteristics: i) A) Length: 8

B) Type: amino acid

D) Topo y: 1 i ne x xi) Chain description: Seq. 11) no. 7

5

Ly s - P r o - Ty r - A 1 a - G 1 y - P o -11 e - P o

Sequence Information Sequ. ID # 8 Characteristics of the chain: i) A) Length: 6

(B) Type: amoocyte 1 ina

D) Topology: linear, xi) Chain description: Seq. II) č. 8

5

Tyr-Ala-Gly-Pro-Tyr-Ala

Sequence Information Sequ. ID # 9 Chain Characteristics: i) A) Length: 0

B) Type: amino acid

D) Topology: Linear xi) Chain description: Seq. ID ó. 9

5

Ly s-Pro-Ty r-A 1 and-G 1 y-Pro-'i'yr-A 1 a

I n o o r m a c o e r e r. and z c i S 'k v. ID # 1D Chain Characteristics:

i) A) Length: 10 B) Type: am i no k y.s e 1 i na D) T o p o 1 ó g i. and : i i e e r 11 a xi) P opi with chain. C a: S (d. v.) D no . 1 0

5 10

Phe-A 1 a - Ly s - Pr o-Tyr-A 1 a - G i y - i'ro-Tyr-a a

About Sek v. II) č. 11 Chain characteristics: i) A) Length: 12

B) Type: amino acid

D) T o p o 1 ó g i: 1 i n e a r n a x i) Description r. a z c a: S e k v. I D č. 11

10

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

Sequence Information Sequ. ID No.12 Chain Characteristics: i) A) Length: 14

B) Type: amino acid

D) Topology: linear x i) Chain description: Seq. 11) no. 12

5 10

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

Sequence Information Sequ. Chain Characteristics:

i) A) Length: 16 B) T y (i): a m i n o k y s e 1 i n a D) T o p o 1 ó g i a: 1 ineárna (xi) Description of the solution c. e » : Seq. I D č . 13

5 10

A r g - P r o - V a 1 - P r o - G 1 y - P r o - P h e - Λ 1 a - L y s - P r o - Ty r - A1 a - G 1 y - P r o 15

Tyr-A 1a

Sequence Information Sequ. ID # 14 Chain Characteristics:

Length: 29

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

A) Name / Key: Arginyl residue amidated at the C terminus D) Location: Xaa29 x i) Description: S e: k v. 11) no. 14

5 10

Tyr-Thr-As p-A1 and -11e-Ph e-Thr-A sn-Ser-Tyr-Arg-Lys-Va1-Leu 15 20 25

A1a-G1n-Leu-S e r-A1a-Arg-Lys-Leu-Leu-G1n-Asp-11e-Leu-As n-Xaa

Sequence Information Sequ. ID No.15 Characteristics of the chain: i) A) Length: 11

D) Type: amino acid

D) Topo 1 ogiči: 1 i river x i) Chain description: S e k v. ID # 1.5

5 10

Met-Pro-A 1 a - Hs-Pro ~ Hs - Pr o-ILi s - Pro-Hi s-A 1 a

Sequence Information Sequ. II) No.16 Characteristics of the chain: i) A) Length: 11

B) Type: amino acid

D) Topology: linear xi) Description: Seq. II) č. i 6

]. 5 10

M e i - A 1 a - P r o - H i s - Λ 1 a - H i .s - A 1 a -11 i s - A i a - il i s - A 1 a

String information. SEQ. 11) no. 17 Charak't. e r i s th e r i n d a c: a) A) Length: 11

B) Tv p: a m i no k y s e i i na

D) Topology: linear (xi) String description: Seq. ID # 17 1 •about . 10 Met - G 1 y - P r - H i - P r - H i - P r - H i - P r - H i - A 1 a Sequence Information Sequ. 11) no. 10

/

Chain Characteristics:

i) Length: 31 ii B) Type: amino acid D) T o g g: linear ix) features: A) Name / Key: Arginyl residue amidated to C horses B) Position: X and a 3.1 xi) String description: Seq. II) č. 10 1 5 10 ΙΙθ- -Pro-Tyr-A1a-Asp-A1 and -11e -Phe-Thr-Λ with n-S e r-Tyr-Arg-Lys - Ι 5 20 2 5 Val - Leu-G1y-G1n-Leu-Scr-A1 and -Arg-Lys-Leu-Leu-G1n-A with p-11 e-Leu '30 Asn -Xaa Information about S t: k v. 11) no. 1 9

Characteristics of the chain

i) Length: 33 B) Type: a m i n o y s e 1 i na ix) D) Topology: 1linear features: A) Name / Key: Arginyl residue amidated at the C terminus (xi) B) P o 1 o h; X a a 3 3 The chain: Seq. II) č. 19 1 5 10 Ty r- -A la - Ty r - A 1 a - Ty r - A 1 a - A with p-A i a - I 1 e - h h e - Thr - A with n - S e r-S e r- 1 5 2 0 2 5 Arg -l.ys-Vii 1 - i, e u - A 1 and - G J n ·· i. e - S e r - A i a - A r g - L y s - l, e u - L e u-G 1 u - A s p

1LE-Leu-Ser-Xaa

Sequence Information Sequ. II) č. 20 Characteristics of the chain: i) A) Length: 39

B) Type: amino acid

D) Topology: linear ix) Features:

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

B) Location: Xaa39 xi) Chain description: Seq. II) č. 20

10

Phe-Ala-Lys-Pro-Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala15 20 25

Ile-Phe-Thr-Asn-Ser-Lyr-Arg-Lys-Vai-Lu-Ala-Gln-Leu-Ser-Ala 30 35

Arg-Lys-Leu-Leu-Asp-G1N-I1e-Leu-Asn-Xaa

String information. SEQ. III č. 21 Characteristics of the chain: i) A) Length: 45

B) Type: amino acid

D) T o p o 1 o g i: 1 i n e a r i x) Features:

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

B) Location: Xaa45 xi) Chain description: Seq. ID č.21

5 10

A rg - P r ^- o - V a 1 - P ro - G 1 y - P ro - P he - .A 1 a -1, ys - P ro - y yr - A 1 a - G 1 y - P ro 15 20 25

Ty r - A1 a - T y r - A L a - A with p - A 1 a - i i - P h t? - T h r '- A s n - S e r - T yr - A g - L y s - V and I

35 40

Leu-G 1 y-G 1 n - Leu - S e r-A i - A r g - f s - Leu - Le u - G 1 n-As p - I 1 e - I.e u4 5

Sequence Information Sequ. II) No'22 C h a r a k t e r a c a r e a a c a: i) A) Length: 10

B) Type: amino acid

D) Topology: linear x i) Description c a: Seq. I D č. 22 /

5 í 0

Phe-A 1 a-Lys -Pro-Ty r-A 1 a-G 1 y-Pro-Tyr-A 1 a

Chain Information Seq. U) no. 23 Characteristics of the chain: (i) A) (I): 16

B) Type: a m i n o y s e 1 i. on the

D) Topology: Linear xi) Chain description: Seq. ID # 23

5 10

Arg-Pro-Va 1-Pro-G 1y-Pro-Phe-A 1 a-Lys-Pr o-Tyr-A 1 a-G 1y-Pro15

Tyr-A la

Sequence Information Sequ. ID # 29 C h a r a k e r j. s i s a n d i n a: i) A) Length: 27

D) Type: amino acid

D) Topology: linear i x) Features:

A) Has v / key: Argir 1 ovv residue amidated at the C terminus

II) Po J oh a: X a a 2 7 xi) Chain description: Seq. ID No, 29

5 1 0

As p-A 1 a - I 1 e - Phe-Th r-A with n-Se r-Ty r-A rg-1.-y with -Val-Leu-G 1 v-G i n15 '2 0 2 5

Le u - S e i '- Λ i a - A r g - i. y s - L e u - L e u - G 1 n - A s ρ - I I e -1, e u - A s n - X a a

Sequence Information Sequ. II.) No.25 Chain characteristics: i) A) Length: 31

D) Type: a m i n o y s e 1 i n a

D) Topology 1: linear i x) Features:

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

B) Location: Xaa3l xi) Chain description: Seq. II) č. 25

5 K)

Tyr-Ala-Tyr-Ala-Asp-Ala-Il and -Plie-Thr-Ser-Ser-Tyr-Arg - Ly s 15 20 25

Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-lle30

Leu-Asn-Xaa

Sequence Information Sequ. ID No.26 Chain Characteristics: i) A) Length: 33

B) Type: amino acid

D) Topology: linear ix) Features:

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

D) Position: X a a 3 3 xi) Chain description »: Seq. II.) No. 26

I 5 10

G 1 y - P r -1 -1 e - - P r - T y r - A 1 a - A with p - A 1 a - 1 1 e - P h e - T r - A s n - S e r - T r 15 20 25

Arg-i, ys - Va 1 - I, e uG 1 y - G 1 n - Le u - S er - A 1 a - A rg -1, ys - L eu -1, eu -G 1 n - As p 30

II e-Leu-Asn-Xaa

Sequence Information Sequ. II) No.27 Characteristics of the chain: (i) A) Length: 3 5

B) T y p: a m i n o y s e i i n a

D) T o p o 1 ó gi: 1 i n e \ n ix) Features:

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

B) Position: Xaa 35 x i) Description of the sequence: Seq. II) č. 27 /

5 10

Ty r - A 1 a - G 1 y - P r o - I 1 e - P r c> - T y r - A 1 a - A s p - A 1 a - I 1 e - P h e - T h r - A s n 15 20 25

Ser-Tyr-Arg-Lys-Val-Len-Gly-Gln-Leu

Leu-G 1 n-A with p-11 e-Leu-A.s n-Xaa

Sequence Information Sequ. ID No.28 Chain Characteristics: i) A) Length: 37

B) Type: amino acid

D) Topology: linear ix) Features:

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

B) Location: Xaa37 xi) Chain description: Seq. II) č. 28

5 10

Lys-Pro-Tyr-Ala-Gly-Pro-Iie-Pro-Tyr-Ala-Asp-Ala-Ile-Phc15

Thr-Asn-Se r-Tyr-Arg-I.y s-Va 1-Leu-G 1 y-G 1 n-Leu-Se r-A 1 a - Arg-Ly s 30 3 5

Leu-Leu-G1n-As p-11e-Leu-Asn-Xaa

Sequence Information Sequ. ID n.29 C h a r a k t e r t s t a k a r e. and from c: a:

i) A) LENGTH: 3 3

B) Tvp: amino acid

D) Topology: linear ix) Features:

A) Name / Key: Arginyl residue amidated at the C terminus B) Position: X a a 3 3 x i) Chain description: Seq. II) č. 29

5 10

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

Arg-Lys-Va1-Leu-G1y-G1n-Leu-S-r-A1a-Arg-Lys-Leu-Leu-Gn30

A with p -11 e - L e u - A with n - X and a

Sequence Information Sequ. ID no.30 C h a r a k t e r a t a k a chain: i) A) Length: 35

B) Type: amino acid

D) T o p o 1 o g i: 1 i n e a n i i) Features:

A) Name / Key: Arginyl residue amidated at the t-terminus

B) Position: Xa and 35 xi) Chain description: Seq. II) č. 30

5 10

20 Tyr-Ala-Gly-Pro-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn15

Ser-Tyr-Arg-Lys-Va1-Leu-G1y-G1 n-Leu-S e r-A1 α-Arg-Lys-Leu30

Leu-G1 n-Asp-Ie-Leu-Asn-Xaa

Sequence Information Sequ. ID # 31 Ch a r a c t e r i s tic t a r c a:

i) A) Length: 37

B) Type: amino acid

D) Topo Legion: Linear i x) Features:

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

B) P o 1 o h a: xi) Description of the chain

SEQ. II) e. 3.1

- 55 10

Ly s - Pro-Tyr-A 1. a - G 1 y-Pr υ-Tyr-A 1 n - Tv r-Λ 1 a - A with p-A 1 a - 11 e-Phe15 20 25

Thr-Asn-Ser-Tyr-Arg ~ Lvs-Va1-Leu-G J y-G 1 n-Leu-Ser-A 1 a-Arg30 35

Lys-Leu-Leu-G 1. n-Asp-11a-Leu-Asn-Xaa /

Sequence Information Sequ. ID No.32 Chain Characteristics: i) A) Length: 39

B) Type: amino acid

D) T o p o 1 o g i: 1 i. n e á r n a ix) Features:

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

B) Location: Xaa39 xi) Chain description: Seq. II) No.32

I 5 10

Phe-ala-lys-pro-tyr-ala-gly-pro-tyr-ala-tyr-ala-asp-ala15 20 2 5

II-e-Phe-Thr-Asn-Ser-Tyr-Arg-I, ys-Va 1 -I.eu-G 1 y-G 1 n-Leu-Ser30 35

A1a-Arg-Lys-Leu-Leu-Gin-Asp-11e-Leu-Asn-Xaa

I n f o r m a c o o chain. SEQ. ID # Characteristics of the chain: i) A) Length: 41

B) Type: amino acid

D) Topology: linear ix) Features:

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

B) Location: Xaaúl (xi) Chain description: Seq. 11) no. 33

5 10

G 1 y - Pro-Phe-A 1 a - Lys-Pro-Tyr-A 1 a - G i-Pro-Tvr-A 1 a - Ty r-A 1 a 15 2 0 2 5

A s p-A 1 a - I 11: - Pli e - Tli r - A s n - S e r - Ty r - Arg - l.y s - Va I - l.e u - G i y-G .1 n -30 35 40

Le u-Ser-A1 and-Arg-Lys-Leu-Le u ~ G1 η-A sp-11e-Leu-Asn-Xa and

Sequence Information Sequ. ID No.34 Chain Characteristics: i) A) Length: 43

B) Type: amino acid

D) T o p o 1 o g i: 1 i n e a r i i) Features:

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

B) l´oloha: Xaa 4 3 x (i) Chain description: Seq. II) č. 3 4

10

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

Tyr-Al a-Asp-A1 a-11 e-Phe-Thr-A with n-Ser-Tyr-Arg-I, y-Va 1 - Leu30 3 5 4 0

G 1y-G 1n-Leu-Ser-A1 and -Arg-Lys-Leu-Leu-Gin-A sp-11 e-Leu-As n-Xaa

Sequence Information Sequ. II) No.35 Characteristics of the chain: i) A) Length: 45

B) Type: amino acid

D) Topology: 1 i n e r n o ix) Features:

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

B) Position: Xa a 4 5 x i) Description of the sequence: Seq. 11) no. 35

5 10

Arg-Pro-Va i - lro-G 1y-Pro-Phe-A 1 and -Lys -Pro-Tyr-A 1a-G1 y-Pro15 20 25

Ty r-A 1 a - Ty r-A 1 a - A with ρ-A 1 a - I I e - P h e - Thr- A s n-S c r-'lv-Arg - l.y s 3 0 35 4 0

V a 1 - Leu-G i-G 1 n-Leu-Sc r-A 1 a - Arg-1-y-1, eu - Leu-G 1 n-Asp-I e 5

Leu-Asn-Xaa

Sequence Information Sequ. II) č. 36 Characteristics: i) A) Length: 31

B) Type: amino acid

D) Topology: 1linear ix) Features:

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

B) Position: X a a 31 x i) Chain description: Seq. II) č. 36

5 10

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-Ile30

Leu-Asn-Xaa

Sequence Information Sequ. II) No.37 Chain characteristics: i) A) Length: 31

B) Type: amino acid

D) Topology: linear ix) Features:

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

B) Location: Xaa 31 xi) Chain description: Seq. II) č. 37

5 10

Tyr-Thr-Tyr-A1a-As p-A1a-11e-Phe-Thr-Asn-Ser-Tyr-Arg-Lys 15 20 30

Va 1-Leu-A 1 α-G 1 n-Leu-Ser-A1 and -Arg-Lys-Leu-Leu-G 1n-Asp-11 e31.

L e u - A with n - X and a

Chain information Seq. 11) no. 3íl C ha r a k t e r 1 s V. i k a r e a z c a: i) A) Part 7: 3 I

B) Type: amino acid

D) Τορυ1ógia: 1linear ix) Features:

A) Name / Key: Arginyl residue arnidated to C horses;

B) Location: Xaa 31 xi) Chain description: Seq. 11) no. 30

5 10

Tyr-Thr-Tyr-11e-A sp-A 1 a -1 Ie - Phe-Thr-A sn-Ser-Ty-Arg-l.y s 15 20 25

V a 1-Leu-A1 a-G 1 n-1 e-S e r-A 1 a-Arg-1 y s -Leu-Leu-G 1 n-A s p-11 e30

Leu-Asn-Xaa

Sequence Information Sequ. ID # 39 Chain characteristics: i) A) Length: 31

B) Type: amino acid

D) Topology: linear ix) Features:

A) Name / Key: Arginyl residue arnidated to C horses

B) Po 1oha: Xaa 31 (xi) Chain description: Seq. ID # 39

5 10

Tyr-Thr-Tyr-Thr-Asp-A1-a1e-Phe-Thr-Asn-Ser-Tyr-Arg-Lys15 20 25

Va1-Leu-A1a-G1n-Leu-S e r-A1a-Arg-Lys -Leu-Leu-G1n-As p-1 1 e30

1. e u - A with n - X and a

Chains Information Sequ. ID No.AO C h a r a k t e r i. s f i k a r z a a z c a:

(i) A) D 1 item : 3 i B) Type: am i no k ys e i i na D) T o p o 1 ó g i a: i i n e a r n a

i. (x) C r t y:

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

B) Location: Xaa31 xi) Chain description: Seq. ID # 60

5 1.0

Tyr-Ser-Tyr-Thr-As p-A1 1 and -11 e -Phe-Thr-As n-Ser-Tyr-Arg-Lys 15 '2 (Jan '25

V a 1 - L e u - A1 a - G 1 n - L e u - S e r - A 1 n - A g -1, y s -1, e u - L e u - G 1 n - A s p -11 e 30

l.eu-Asn-Xaa

Sequence Information Sequ. ID No.61 Characteristics of the chain: i) A) Length: 33

B) Type: amino acid

D) Topology: linear ix) Features:

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

B) Location: Xaa33 xi) Chain description: S e k v. II) č. 61

10

Tyr-Thr-Tyr-Thr-Tyr-Thr-Asp-A1 and -Ie-Phe-Thr-Asn-Ser-Tyr15 20 25

Arg-Lys-Val-Leu-Ala-Gln-Ser-l.eu-Ala-Arg-Lys-Leu-Leu-Gln30

Asp-11 f: -l.eu-Asn-Xaa

Chain information Seq, II) no. 6 2 C h a r e c e: r i s t. i a c a i c a: i) A) Length: 31

B) Type: amino acid

1.)) To p o 1 ó g i a: 1. I n o r n a i x)

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

B) Location: Xaa3i xi) Chain description: Seq. ID # 62

Other orb characteristics * i 1 A) Length:

5 10

Tyr-A 1 a - Tyr-A 1 a -A sp- A 1 <i - I 1 e - P h e -Thr-Ser-S er-Tyr-Arg-Ly s 15 20 25

V a 1-Leu-G ly-G 1 n-Leu-S e r-A 1 a-Arg-L y s-Leu-), e-G 1 n-A sp -11 e 30

Leu-Asn-Xaa of Seq. ID No.4 3 S e c a:

3

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

A) Name / Key: Arginyl residue and C to C C) B) Position: Xa a 3 3 x i) Chain description: S e k v. I D č. 4 3

5 1.0

20 Tyr-Ala-Tyr-Ala-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Ser-Ser-Tyr

Arg-Lys-Va 1-Leu-A1a-G 1n-Leu-Ser-A1a-Arg-Lys-Leu-Leu-G 1n30

Asp-Ile-Leu-Ser-Xaa

P Λ 'ľ. 'N. 0'

Claims (1)

An artificial linked protein comprising an elongated peptide moiety covalently linked from its C-terminus to the N-koric of the core protein moiety, characterized in that its elongated protein moiety has the formula: A-X-Y (X'-Y) n wherein A is either not or is methionine, n is 0 to 20, X is selected from the group consisting of all amino acid residues occurring X 'is selected from the group consisting of occurring amino acid residues with hydroxyproline, Y is selected from the group of serine and nature is all known in nature. n a and hydroxypropyl, alanine, cases where N - 0, then alanine, s n a n a n a n a n a n a n a n o n o n n a n a n a n a n a n a n a n a n a n a n a n a n Y is selected from the group consisting of Artificial linked protein according to claim 1, characterized in that A is present and X is selected from the group consisting of Pro, Gly, Ala or Ser. 3. The artificial fusion protein of claim 1, wherein n is U to 10. 4. The protein-coupled protein of claim 1, wherein said biologically active polypeptide is selected from the group consisting of bGRI ·, EOF, IGF-2, glucagon, corotho-tropin dyno-analog; in, endothelin, growth hormone, and fact, vasoactive intestinal peptide, human B-ka zomorl. in, gastric inhaled peptide, gastric release peptide, human peptide H1, human peptide YY, fragment 7-37 of glucagon-like peptide-du-1, gucucone below the human peptide 1d-2, I P, neuropeptide Y, human pancreatic polypeptide, insulin-like raschol-1, human growth hormone, growth hormone factor, bovine growth hormone, pork hormone, prolactin, human growth hormone releasing factor, bovine growth hormone releasing factor, pork releasing growth factor hormone, a sheep factor that releases growth hormone, interleukin n-1β, interleukin-2. 5. artificial coupled protein pod ľa n o r o k u 1 , indicating the because of said extended peptide portion is selected z 0 groups containing γ-P r-11 e - P r o, , Seq. ID 6, Seq. ID 7 Tyr-A 1a, G 1 y - Pro-Tyr-Ala, Seq . ID 8, Seq. ID 9, Seq. ID 10, Seq . ID 11, Seq. 1D 12, Sekv, ID 13 T T y r - Λ 1 and - T y r - A 1. and, Va1-A 1a, SEQ. ID 15, Seq. ID 16, p I e k v. I D 17, Seq. ID 22, SEQ. ID i 23. 6. U m e 1 ý united by claim 2 , indicating the because of n j e 3 to 5. 7. artificial coupled protein pod ľa year 6 , s n i n i i n i s a because of all of them X 'residues are histidines. 8. Ume J ý with a protein pod ľa year 7 , s a because of X is h i s t i d. 9. artificial coupled protein p o d year 7 , s a because of three Y the residues are proline. 10. Use of artificial combined drug preparation. P o r 11: o n u o n d o n o n o o n n o The use of an artificial fusion protein according to claim 10, characterized in that said medicament comprises further fusion proteins having identical biologically active moieties and a variety of different proteins. The use of an artificial fusion protein according to claim 10, wherein said bioingically active portion of said fusion protein is a bOK1 analogue. 3. S p o o u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u s. 2. The method of claim 1, wherein said protein-linked protein is selectively contacted with a material that immobilizes said protein-linked protein. n é n t: purity s and o d s t i n a, stated coupled ' .... n s a o d d e 1 í. o d / kombinuj e u leads it combined p r o t e n s a u e d e n e requested p r o t e n the recovered. of said material, with DPP IV and 16. The method of claim 13, wherein said material is fixed in a column. The purification method of claim 13, wherein said material is antibodies having an elongated portion. characterized in that they are related to said The method of purifying that said material is an elongated portion comprises histidines. according to claim 13, characterized in that it is a bilaz. and at least 3 following X 'remnants that