NL8900724A - NEW POLYPEPTIDES WITH GROWTH FACTOR ACTIVITY AND NUCLEIC ACID SEQUENCES CODING FOR THE POLYPEPTIDES. - Google Patents

Description

? - V.0. 2062

Novel polypeptides with growth factor activity and nucleic acid sequences encoding the polypeptides.

This invention relates to novel growth factor activity polypeptide compositions, including chimeric polypeptides and methods of their preparation using recombinant DNA techniques.

It appears that a significant number of polypeptides secreted by mammalian cells have growth factor activity. The order of these polypeptides is substantially the same for many mammals. The great interest in these compounds has been stimulated by their association with oncogenicity. It is also interested in how the production of the growth factors is regulated and how these in turn regulate cellular activity.

It has already been noted that infection of mammalian cells by viruses results in the proliferation of the growth of infected cells. Of particular interest for the purpose of this invention are members of the CFPKK virus family, such as variola, vaccinia and viruses associated with certain diseases, such as Shope fibromavirus, Yabatumor virus and Molluscum contagiosum virus (MCV).

It would be important to be able to determine whether viruses that cause cellular proliferation with growth factors would produce associated polypeptides that act either as the growth factor or as the growth factor receptor. These compounds could then be used for the investigation of viral actions, in assays for the presence of the virus, in nutrient media as mitogens and in the development of therapeutic agents for the control of virus infection. It is also important to develop compounds that can form agonists or antagonists of growth factors to use them in growing cell cultures in vitro to detect mitotic processes and in therapy.

30 Relevant Literature

Venkatesan et al, J. Virol. (1982) 44: 637-646 describes the DNA sequencing of the structured gene encoding vaccinia virus proteins unaware that each of these proteins had growth factor activity.

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Cooper et al., Ibid (1981) 37: 284-294, reports the translation of mRNAs encoded within the reverse terminal repeat of the vaccinia virus rancine. Usury diseases for members of the poxveN virus family have been reported for Shope fibromavirus (Shope, J. Exp.

5 Med. (1932) 56: 793-822; Yaba tumor virus (Niven et al; J. Path. Bacteriol. (1961) 81: 1-14) and Molluscum contagiosum virus (MCV) (Postlethwaite, Arch. Environ. Health (1970) 21: 432-452). Descriptions of epidermal growth factor (EGF) can be found in Scott et al., Science (1983) 221: 236-240 and Gray et al., Nature (1983) 10 303: 722-725. The presence of three disulfide bridges in EGF and transforming growth factor (TGF) is reported by Savage et al., J. Biol. Chem. (1973) 248: 7669-7692. See also Doolittle et al., Nature (1984) 307: 558-560 and Autral patent application No. 85006288. It has been suggested that the receptor binding region of the EGF molecule lies in the loop between the third and fourth cysteine residues (Komoriya et al.) Proc, Natl Acad Sci USA (1983) 81: 1351-1355).

New descriptions of vaccinia virus growth factor (VGF) can be found in Brown et al., Nature (1985) 313: 491-492; Reisner, Nature (1985) 313: 801-803 and Blomquist et al., Proc. Natl. Acad.

Sci. USA (1984) 81: 7263-7367. Natural Shope fibroma virus growth factor (SFGF) is described by W.C. Chang et al, in Molecular and Cellular Biol. (1987) 7: 535-540. Shope, J. Exp. Med. (1932) 56: 793-802 reports that infection of adult rabbits by Shope fibromavirus caused rapid proliferation of fibroblasts leading to a benign fibroma. Infection of newborn or adult rabbits with immunosuppression caused rapid proliferation of fibroblasts leading to. an invasive atypical fibrosarcoma. A.C. Allison, J. Natl. Cancer 30 Inst. (1966) 36: 869-876; Smith et al., J. Natl. Cancer Inst. (1973) 50: 1529-1539; A.C. Allison, J. Natl. Cancer Inst. (1966) 35: 859-868.

Natural myxoma virus growth factor (MGF) is described by Upton et al., J. Virol. (1987) 61: 1271-1275. Infection of 35 adult rabbits by myxomavirus causes a rapidly proliferating invasive myxosarcomatous tumor. D.S. Strayer and Sell., D. Natl. Cancer Inst. (1983) 71: 105-116. Found a DNA sequence that is 8900724? # 3 .έτη is capable of encoding a conserved growth factor (Natural Molluscutn contagiosum virus growth factor (MC'GF)). CD.

Porter and L.C. Archard, J. Gen. Virol. (1987) 68: 673-682. Human infection by Molluscutn contagiosum virus induced 5 benign skin tumors characterized by rapidly proliferating cells.

Brown et al., Sex. Transm. Dos. (1981) 8: 227-234. Yaba tumor virus causes subcutaneous histocytoma in monkeys and humans, Bearcroft et al., Nature (London) (1958) 182: 195-196 and is predicted to have a related growth factor due to its association with Shope fibromavirus, Myxomavirus and Molluscum contagiosum virus.

Mouse is known to be EGF (mEGF) (Scott et al, 1983, supra,

Gray et al, 1983, supra) and human EGF (hEGF) (Gregory et al., Int.

J. Pept. Protein Res. (1977) 9: 107-118) are homologous to human, mouse and rat TGF (Marquardt et al., Proc. Natl. Acad. Sci. USA 15 (1983) 8o: 4684-4688) as well as residues 45-85 of a 140 residues, depleted polypeptide, vaccinia growth factor (VGF) encoded by the vaccinia virus genome (Venkatesan et al., 1982, supra).

The expression of foreign peptides using a baculovirus vector in insect cells is described by Maeda 20 et al, Nature (1985) 315: 592-594, and Carbonell et al., J. of Virology (1985) 56: 153-160.

The descriptions of these quotations are incorporated herein by reference.

Summary of the invention

New polypeptide compositions analogous to fragments of viral proteins which are active as mitogens and useful in nutrient media, as reactants for the detection of growth factor receptors or the presence of growth factors and as competing compounds for the transformation growth factor and the metabolites are provided. epidermal growth factor. The compositions may e.g. used therapeutically to promote epithelialization and healing of burns and wounds. The new compositions can be prepared synthetically. The subject peptides can be generated as oligopeptides or fused proteins in a wide variety of hosts using recombinant techniques.

89 00 724.} 4

Brief description of the drawings

Figure 1 is an amino acid sequence comparison of the vaccinia virus protein with known growth factors, wherein the initial amino acid of the VGF is aspartic acid (D) at position 20 of the -24 methianine corresponding to aa of the described peptides; while Figure 2 represents the proposed structure of mature VGF, with asparagine (N) replacements. Potential glycosylation sites in MGF and SFGF are indicated by asterisks.

10

Description of specific embodiments

New compositions which may act as agonists or antagonists of epidermal growth factor (EGF) or transform growth factor (TGF) are envisaged, including various applications in cell culture, diagnostics in vivo therapy and in combination with other peptides in the formation of hybrid polypeptides as immunogens become available for the production of antibodies. Polynucleotide sequences can be isolated and prepared which can then be introduced into an expression vector for expressing the present compositions.

The compositions appear to be analogous to fragments of viral proteins, especially smallpox virus proteins. As illustrated in Figure 1, VGF contains uncharged and hydrophobic residues near the N-terminal between residues 5 and 15 and near the C-terminal between residues 100 and 124. By analogy, these residues can be considered integral membrane glycoproteins with an N- terminal hydrophobic? signal sequence, which is removed proteolytically during or immediately after translation, and a C-terminal transmembraneous sequence, which serves to anchor the mature protein in the membrane. Cleavage of the viral polypeptide at arg-43 and arg-90 of the mature peptide would lead to the release of a soluble polypeptide. The 140-residue VV polypeptide is believed to first form a membrane-associated protein with approximately 121 residues upon removal of the Ig amino acid sequence. After the intracellular processing, a soluble growth factor peptide of about 77 residues is obtained 8900724; jï 5

Cell (1985) 42: 383-393.

A comparison of the Myxoma growth factor (MGF) and Shope fibroma growth factor (SFGF) with the other members of the growth factor family (see figure 1) reveals several noteworthy differences. MGF and SFGF, synthesized as precursor molecules, have a signal sequence at the N-terminal, but unlike the other growth factor members, they do not have a transmembrane domain at the C-terminal, indicating that they are secreted molecules. Sequence analysis shows significant application of the amino-10 acid asparagine in both MGF and SFGF. At six sites, both MGF and SFGF conserved asparagines where the other members do not have asparagine. From these sites, asparagines replace two highly conserved glycine residues at sites 8 and 31, with the first cysteic acid located at site 2. Both of these 15 glycine residues occur in combination with highly conserved thyrosine residues at positions 9 and 32. Due to the highly conserved nature of the amino acids in this plane of the molecule, it is believed that the cysteine loops present in the plane are involved in receptor binding and the function of growth factors in this family.

The amino acids glycine and asparagine are predicted to have the highest beta reversal potential (Chou and Fassman, Ann. Rev.

Biochem. (1978) 47: 251-276). Thus, it has been found that conservation of this reversal potential is extremely important for growth factor activity. In the growth factors other than Myxoma and Shope, glycine is used almost exclusively in key turning areas of the molecule. In MGF and SFGF, these glycines are almost completely converted into asparagines (see figure 2). This indicates an important biological function in connection with the replacement of the highly conserved glycine 30 with the asparagine residue. There is a possibility that replacement of glycine in the conserved reversal regions of the growth factor structure with asparagine may lead to increased growth factor functionality either by increasing binding to the EGF receptor or to a related receptor, causing the direct effect of the growth factor in the cells involved is enhanced or by increasing the stability of the molecule.

89 00724.

6

Two of the novel occurrences of asparagines in MGF and SFGF yield potential N-linked glycosylation sites. Furthermore, these sites are present in the two cystic loops that allow variable numbers of amino acids. Thus, the new occurrences of asparagines can deliver multiple growth factor benefits beyond increased functionality. One of these could be the stabilization of the protein in vivo by protection from degradation of immunogenic sites by glycosylation.

The subject polypeptides are characterized in that they can contain at least one loop or circle, usually three intermediates or circles, due to cysteine bridging, the physiologically active part of the molecule providing a growth factor-related activity about 12 to 65 amino acids, usually covers 15-60 amino acids. Of the three loops, two of the 15 loops have 12 to 15 amino acids (14-17 annular amino acids), excluding the cysteine bridge, more specifically, one has 12 or 13 amino acids (14 or 15 annular amino acids) and the other 15 amino acids (17 annular amino acids), wherein the N-terminal proximal loop usually has 12-13 amino acids, usually 12 amino acids 20, and the middle loop has 15 amino acids.

The third loop or the C-proximal loop has 8 amino acids (10 annular amino acids) and includes flanking amino acids of the following formula:

25 pp3- Ua25-C-aa27) m - C-aa29-aa30-G-Y

I I

PP4- (aa40-aa39-aa38) n-c- R-aa35- G-aa33 in which: the one-letter symbols for the amino acids have a usual

meaning, where C cysteine, G glycine, N asparagine, Y

thyrosine and R is arginine; Aa neutral amino acid which may be aliphatic, especially with 3-4 carbon atoms, or aromatic, especially with 9 carbon atoms, and which contains 0-1 hydroxyl group, e.g. alanine, serine, threonine, tyrosine or phenylalanine; 27 aa a neutral or basic amino acid, in particular 83 00724.

7 is 3-6 carbon atoms, and in the neutral state contains 0-1 carboxamide group, e.g. arginine, alanine, valine, leucine, isoleucine asparagine or glutamine; 29 aa is a neutral or basic amino acid, either aliphatic or aromatic, an example of the aromatic acid being histidine, and as aliphatic acid containing 2-6, usually 3-6 carbon atoms, with 0-1 hydroxyl group, such as serine, threonine, leucine, valine, isoleucine or arginine; 30 aa is a neutral amino acid, either aliphatic or aromatic, an example of the aromatic acid being histidine and the aliphatic acid containing 3-6 carbon atoms and 0-1 hydroxyl group, especially serine, isoleucine and valine; aa refers to a neutral aliphatic amino acid of 2-6, usually 3-6 carbon atoms with 0-1 hydroxyl group, more particularly serine, threonine, valine, leucine or isoleucine, or an acidic amino acid, for example glutamic acid and aspartic acid;

OC

aa is a neutral or acidic amino acid of 3-6 carbon atoms, exemplifying the neutral acid alanine, valine, leucine, isoleucine and serine, and exemplifying the acidic acid aspartic and glutamic acids; aa an aliphatic neutral substituted amino acid in which the substituent is carboxamide containing 4-5 carbon atoms, or an acidic amino acid, e.g. aspartic, glutamine, aspartic acid or glutamic acid; Aa is an aromatic amino acid or a neutral aliphatic amino acid of 3-4 carbon atoms which usually contains a hydroxyl substituent, preferably is aromatic such as phenylalanine, histidine, thyrosine, serine or threonine; aa40 a neutral substituted or unsubstituted aliphatic amino acid of 3-6 carbon atoms, or a basic amino acid, e.g. alanine, valine, isoleucine, glutamine or arginine ts? m and n are 0 or 1; PP3 and PP4 can be the same or different and are either hydrogen atoms, indicating the termination of the polypeptide, or they can be polypeptide chains with no more than a total of 1000 amino acids, usually no more than a total of about 500 amino acids, wherein preferably at least 90% of the amino-89 00 724/8 * acids present, most preferably at least about 95 een of the amino acids present, are in one of the two polypeptide chains; in some cases, the chain may contain only one amino acid and no more than one hundred amino acids, and usually no longer than about fifty amino acids, depending on the application of the polypeptide and the role of the extended chain; the polypeptide chains may be associated with the naturally occurring polypeptide chains associated with naturally occurring growth factors and smallpox virus proteins or they may be different from the naturally occurring chains or fragments thereof associated with the polypeptide chain which are particularly is shown in the formula, but they are usually unrelated.

The amino acid definitions are summarized below.

15 Neutral (Ne).

Aliphatic (Al)

unsubstituted G, A, V, L, I, P

substituted

oxy S, T

2G thio C, M

amido N, Q

aromatic (Ar)

unsubstituted F

substituted Y.

Heterocyclic H, W

Loaded (at pH 6.0)

basic (Ba) K, R

acid (Ac) D, E

30

The abbreviations in brackets refer to the particular amino acid groups. Unsubstituted means no hetero substituents other than the ca. rboxy and amino groups present in glycine.

The amino acids are naturally occurring L-amino acids.

The neutral amino acids can also be indicated to contain non-polar or polar (but uncharged) R groups.

The amino acids that fall within these definitions are the following; 69 0 07 λj.

9 *

Non-polar amino acids A, V, L, I, P, Μ, N, F, W.

Polar amino acids G, S, T, C, Y, N, Q

The loop between the cysts at positions 28 and 37 in the above formula contains 0-1 acid amino acid (S). The amino acids that immediately flank the cysts outside the loop, (i.e.

amino acids 27 and 38) can include at least one charged amino acid and at least one amido-substituted aliphatic amino acid.

Of the amino acids in the loop (28-37), 4-6 will usually be 5-10 amino acids neutral aliphatic amino acids while no more than 2, usually 1, is an aromatic amino acid.

Of particular interest are compounds in which the polypeptide has less than 130 amino acids, more particularly less than 55 amino acids and at least 44 amino acids, preferably at least about 15 53 amino acids. Of particular interest are polypeptides comprising amino acids 44-48 and fragments thereof of VGF (aa -aa}, see Figure 1.

Preferably aa is a substituted or unsubstituted aliphatic amino acid of 3-6 carbon atoms with 0-1 hydroxyl group, especially serine or valine; or an aromatic amino acid, especially histidine.

Aa is preferably histidine, serine or an unsubstituted aliphatic amino acid of 5-6 carbon atoms, especially iso-leucine; 33 aa is preferably a substituted or unsubstituted alophatic amino acid with 0-1 hydroxyl group and 3-6 carbon atoms; 35 aa is preferably an aliphatic amino acid with 3-6 carbon atoms; 38aa is preferably glutamine or glutamic acid; 39a aa is preferably aromatic, most preferably histidine or phenylalanine; 40 aa is preferably a neutral or basic amino acid.

Of particular interest is the presence of two loops, where the C-proximal loop is joined to the second loop, where the amino acids of the second loop can be very diverse. The second loop preferably contains about 14-16 amino acids 8900724 .: 10 excluding the cysteine bridge, more particularly about 15 amino acids. Of the amino acids, 6-9, preferably 7-9, more particularly about aliphatic amino acids are unsubstituted or substituted, preferably no more than about 3 5 of the amino acids are substituted, most preferably 1- 2 amino acids are substituted; there are 2-4 aromatic amino acids, especially 3 aromatic amino acids, desirably histidine and thyrosine; there are 2-4 acidic amino acids, preferably 3 acidic amino acids, more particularly aspartic acid; and there are 0-2, 10 usually 1 basic amino acid, especially arginine.

Desirably, the cysteine that forms the present loop closest to the cysteine of the other loop is isolated with 0-2, more particularly 0-1 amino acid, especially arginine. Of particular interest for some applications of the present compounds are compounds of the following formula

pp1-aa1-C-aa3-aa4-aa5-aa6-aa7-aa8-F-C-F

N

H “(aa12a) pG-aa14-C-aa16-Ar1- (aa17a) - (aa17b) p 20 aa18-aa19“ aa20-aa2; i — aa22- (aa22a) - (aa22b) -aa23 ir Γ aa24- ( aa25-C-aa27-C-aa29-aa30-GY-aa33-G-aa35

Q

25 R-C-E-aa39-aa40) -aa41-L-aa43-aa44-PP2 where 25 40.

the symbols aa -aa in the formula have already been described; 1 2 PP and PP are the same or different and are hydrogen atoms which indicate the end portion of the indicated polypeptide or are polypeptides having up to about 1000 amino acids in total, usually up to about 500 amino acids which may contain only 1 amino acid in total, either individually or separately polypeptides having 35-100 amino acids, usually about 1-75 amino acids, more particularly about 5-50 amino acids; these polypeptides find specific applications in the modification of, in particular, 8900724.

11 1 2 described sequence for a predetermined purpose; PP and PP may be the same or different from the natural poxvirus polypeptide, but are usually different; aa may be any amino acid, more particularly an aliphatic 5 amino acid, basic amino acid or acid amino acid, preferably an unsubstituted aliphatic amino acid of 2-6 carbon atoms, including glycine, leucine, valine, lysine, glutamic acid and aspartic acid; 3aa is a neutral amino acid, in particular with 2-4 carbon atoms, more in particular asparagine, glysine and proline; aa can be any amino acid, aliphatic or aromatic, especially with 2-6 carbon atoms, which may be neutral or acid, more particularly with 3-6 carbon atoms, including proline, aspartic acid, histidine, serine, leucine; Aa is an acid or a neutral substituted aliphatic amino acid, especially glutamic acid or aspartic acid or a hydroxy substituted amino acid having 3-4 carbon atoms, especially serine; aa® is an unsubstituted aliphatic amino acid of 2-6, usually 2-3 carbon atoms, or an aromatic amino acid, especially glysine, histidine or thyrosine; aa is an acidic, basic or neutral amino acid, especially with 3-6 carbon atoms, such as glutamic acid, aspartic acid, lysine and threonine;

O

Aa is a neutral unsubstituted aliphatic amino acid of 2-3 carbon atoms, or an amino acid substituted with carboxamide of 4-5 carbon atoms, for example asparagine, glutamine or glysine; 11 aa is a neutral amino acid, either aliphatic or aromatic, more particularly aliphatic, especially with 5-6 carbon atoms, more particularly leucine or phenylalanine; 12 aa is an aromatic amino acid or a carboxamide-substituted aliphatic amino acid of 4-5 carbon atoms, especially histidine or asparagine; 12a 35 aa is a neutral aliphatic amino acid or acid amino acid, more particularly glysine, aspartic or aspartic acid; 89 00724: 12 14 aa is an acidic amino acid or a neutral aliphatic amino acid, unsubstituted or substituted, with 4-5 carbon atoms, having 0-1 hydroxyl group, especially aspartic acid, threonine or valine; 1 f aa is a bond or a neutral or basic aliphatic 5 amino acid, unsubstituted or substituted, with 4-6 carbon atoms, especially isoleucine, arginine or methionine;

Ar refers to an aromatic amino acid containing a carbocyclic or heterocyclic ring and including histidine, phenyialanine or thyrosine; 17a 10aa is a neutral aliphatic amino acid, substituted or unsubstituted, with 3-6, usually 3-5 carbon atoms, with 0-1 hydroxyl group including valine, leucine, isoleucine and threonine; 17 aa or b is a neutral unsubstituted amino acid of 5-6 carbon atoms, or an acidic amino acid, more particularly valine, isoleucine or glutamic acid; 18 aa is a neutral aliphatic amino acid, unsubstituted or substituted, with 2-6, preferably 3-6 carbon atoms with 0-1 hydroxy substituent, especially alanine, serine or glutamine; 19aa may be any amino acid, especially aliphatic acidic or basic amino acids, more particularly having 4-6 carbon atoms, most preferably 5-6 carbon atoms, especially arginine, leucine, valine and glutamic acid; 20 aa is an acidic amino acid or an aliphatic carboxamide-substituted amino acid and includes aspartic acid, aspartic, glutamic acid or glutamine; 21 aa is a neutral unsubstituted aliphatic amino acid or basic amino acid of 3-6 carbon atoms and having 0-1 hydroxyl or carboxamide group and includes isoleucine, leucine, asparagine, serine, threonine, lysine and arginine; 22.

Aa is a bond, or an acidic amino acid that is aspartic acid or glutamic acid, or a neutral aliphatic amino acid, especially valine; 22a aa is a neutral aliphatic amino acid, having 0 or 1 hydroxyl group, especially 1 hydroxyl group, more particularly serine; 99 h 35 aa is a neutral aliphatic amino acid with 4-6 carbon atoms, especially leucine or isoleucine; 23 aa is a bond, or a neutral aliphatic amino acid, 8900724.

13 unsubstituted or substituted, with 2-6 carbon atoms, with 0-1 hydroxyl substituent, including glysine, serine, threonine and asparagine; aa * ^ is an aliphatic amino acid, especially thio-substituted aliphatic amino acid; more in particular methionine, proline or an aromatic amino acid, in particular tyrosine; 41 aa is a neutral unsubstituted or substituted aliphatic or acidic amino acid of 4-6 carbon atoms, especially valine, aspartic or aspartic acid; 43 aa is a neutral aliphatic acid or basic amino acid, which may be substituted or unsubstituted, of 4-6 carbon atoms and includes valine, leucine, isoleucine, arginine, lysine and aspartic acid; aa can be any amino acid, especially other than a basic amino acid, and can be acidic, neutral or aromatic, and when other than aromatic contain 3-6 carbon atoms, especially aspartic acid, alanine, leucine, tryptophan or threonine; while p is 0 or 1,

It is of particular importance when PP has the following order 20: PP1'-aa “24-aa“ 23-aa ”22-aa“ 21-aa_20aa-19-aa_i8 -aa “17-aa“ 3 ^ -aa “15 -aa ~ -L4-aa ”13-aa“ 12-aa “· 1 · 1 aa“ 10-aa ”9-aa“ ®-aa ”7-aa ~ 6-aa“ 5-aa ~ 4-aa- 3-aa_2-aa “1 where 11 PP in combination with the following amino acid symbols in Ί the above order is the equivalent of PP. The above 1 11 order falls within the definition of PP; PP can be hydrogen or

30 are an amino acid sequence. One or more of the amino acids, symbolized - X.

sated by aa (X is any number) can be a bond that can reduce the number of amino acids in the N-terminal chain. Thus, all or part of the amino acid sequence may be as indicated. When part of the order is present, the order preferably includes contiguous amino acids, i.e. amino acids in their numerical order without deletions. Usually there is at least one amino acid, usually at least 3, more particularly 8900724i * 14 at least 6, where the residual upstream amino acids may be absent 11 -1-fi, so that PP may be attached to aa, aa and the like; -1aa can be any amino acid, especially aliphatic, with 5-6 carbon atoms, preferably neutral or basic, and more particularly includes lysine, arginine, proline, asparagine or serine; aa can be any amino acid, whether aliphatic or aromatic, especially aliphatic, more particularly with about 10 3-6 carbon atoms; 3 aa may be an aliphatic or aromatic acid, especially aliphatic, either polar or non-polar, with 2-6, usually 2-5 carbon atoms, generally with 0-1 hydroxyl substituent; -4 aa can be any aliphatic amino acid, neutral or basic, generally with 3-6 carbon atoms, usually with 5-6 carbon atoms, especially asparagine, proline or lysine; -5 aa may be any aliphatic amino acid, especially neutral, with generally 3-6 carbon atoms, especially valine or eusoleucine;

_C

Aa is a neutral or acidic amino acid, which when aliphatic contains about 3-6, usually about 4-6 carbon atoms, especially Isoleucine, valine or aspartic acid; -7 aa-8 aa "9 aa-12 aa-14 aa" 16 -17 -19 aa, aa, aa, aa, aa, aa, aa, aa, aa, -20 -21 -23 aa, aa and aa aliphatic amino acids with 3-6 carbon atoms, either polar or non-polar, especially polar with 0-1 hydroxyl substituent or aromatic, aa, and aa ^ are non-polar aliphatic amino acids or acidic amino acids with 2- 6, usually 2-3 carbon atoms; -13 -22 -24 aa, aa and aa are polar aliphatic amino acids or acidic amino acids of 4-5 carbon atoms, in particular with a carboxamido substituent; -11 -18 aa and aa are aliphatic amino acids, either polar or non-polar, or acidic amino acids.

_i

Of particular interest is an N-terminal fragment of aa O Λ _ Λ n 4 mf? 35 to aa H or aa to aa, more particularly aa to aa.

PP may suitably be an unrelated amino acid sequence serving as a fusion protein, in particular to provide an 8900724 immunogenic peptide for the production of antibodies for the compound.

The main aspects of the present compositions are the order 4a-aa -aa, especially the order from the cyst 5 at site 28 to the cyst at site 37 and the welds passing through the cyst at sites 2 and 10 and the cyst at sites 15 and .6 are formed. Desirably, in the present compositions, the loop is formed by the cysts at sites 28 and 37 in conjunction with the loop formed by the cysts at sites 1C and 26.

Chimeric polypeptide sequences can be prepared by combining fragments of different polypeptides in an order substantially the same as the naturally occurring polypeptide chains associated with naturally occurring growth factors and smallpox virus proteins. The resulting chimeric polypeptides then contain about 40-65 amino acids, usually about 45-60 amino acids, especially 49 to 53 amino acids. In either case, the skeletal structure of the cysts is retained, capturing the cyst bridges between with the dimensions previously described. Thus, a fragment from any growth factor with significant homology to VGF (see, e.g., Figure 1) or another growth factor with the same skeletal structure as the present compositions and a similar physiological activity can be used. This is independent of the mammalian source, such as a primate, e.g. a human, a rodent, e.g. rat and mouse, a bovine, a bird, a pig, etc. Up to three junctions may be required depending on the source of the fragments and the length of the desired polypeptide. It is desirable that the intersections be made at some point between Γ 4 44 4 Γ ΠΡ ΛΛ / | g ΡΛ aa ”° and aa; aa and aa, aa ^ 0 and aa ^ and aa ^ and aa. The order between about aa and about aa is referred to as the N-terminal 14 order; that between about aa and about aa is referred to as the central fragment; and that between about aa and the end of the peptide (generally aa ^ to aa ^) is referred to as the C-terminal domain. The exact point of the junction will depend on the position of the restriction sites, etc. Also, an extremely N-terminal sequence comprising approximately aa to aa, usually aa to aa, can also be attached to the chimeric polypeptide 8900724. .

• ί 16 are merged. The fragments are generally about 20 to 50, usually 15 to 45 amino acids in length, since aa has the meaning of the 20th amino acid of the compound but only in the specific order defined <? * - (see Figure 5) ).

Several suitable restriction sites in the synthetic genes used to construct the chimeric polypeptide have been designed. If possible, the restriction sites do not alter the amino acid sequence of the growth factor gene. In some cases, however, the inclusion of the new restriction site produces an altered amino acid sequence. Of particular interest is when the coding sequence of VGF is modified to introduce a Kpn I 13 restriction site by altering the amino acid sequence at aa 15 to aa of GDC and GTC and for entering a Sph I site 15 at aa23 to aa23 by altering the order GMYCRC in GYACVC.

These changes provide suitable sites for linking fragments from the VGF gene and fragments from other growth factors.

The gene fragment aa2 ^ to aa ^ may e.g. suitably to a gene fragment of alpha TGF encoding amino acids corresponding to residues aa to aa are added to form a TGF / VGF hybrid polypeptide. Thus, the order of the chimeric peptide includes aa ”D to aa J (N-terminal) and aa H to aa 3 (central fragments) derived from an alpha TGF, and aa2 ^ to aa ^ (C-terminal) derived from VGF, modified as above.

The preparation of plasmids capable of expressing chimeric proteins with the amino acid sequences derived from fragments of more than one growth factor or smallpox virus polypeptides / with sequence changes as necessary to introduce an appropriate restriction site are described in detail in the experimental part . Fusion proteins can also be prepared when a growth factor or a chimeric polypeptide is expressed joined to the N-terminal amino acids of a signal sequence or the N-terminal amino acids of a highly expressed bacterial, bacteriophage, or eukaryotic gene. Also, an enzymatic or chemical cleavage site can be provided following the leader sequence to provide for cleavage of the mature polypeptide from the leader sequence. The preparation of plasmids 8900724.

Capable of expressing these fusion polypeptides and methods for cleaving and isolating the desired polypeptide are described in detail in the experimental part.

5 Preparation of growth factors

The present compositions can be prepared in various ways depending on the size of the composition. Particularly below about 80, more especially below about 60 amino acids, the composition can be prepared by synthesis in the usual manner. See e.g. Merrifield, Solid-Phase Peptide Sythesis, "The Peptides Analysis, Synthesis Biology",

Special Methods in Peptide Synthesis, Part A, Vol. 2, Gross and Mein-hofer publishers, Academic Press, NY, 1980, biz. 1-284. See also U.S. Patent No. 4127526.

Optionally, one can use the hybrid DNA technology using DNA sequences encoding the desired polypeptide or its precursor. One can synthesize DNA sequences encoding the growth factor of interest using conventional techniques, such as overlapping single strands that can be joined together to establish the desired coding sequence. The termini can be designed to provide restriction sites or one or both termini can be terminated for ligation at complementary end N of an expression vector. For the expression of the sequence, a methionine is initially supplied. Expression vectors are widely available and are clearly described in the literature.

Instead of synthesizing the gene of interest, the growth factor can be isolated by various techniques.

When the growth factor e.g. is a viral growth factor, mRNA can be isolated from the virus encoding the polypeptide including the growth factor, the mRNA is complementarily transcribed ,, (resulting single strand (ss) DNA used as a template to prepare the double strand (ds DNA and the ds DNA gene are isolated Another technique is to isolate a stretch of the viral DNA and to probe it, appropriately degenerate, comprising a region of the most conserved sequences in a growth factor gene. 8300724.

18 encoding a factor in the viral probe may be considerably shorter than the entire sequence, but should be at least 10, preferably at least 14, most preferably at least 20 nucleotides long. Longer oligonucleotides can also be used up to the full length of the growth factor gene. Both DNA and RNA probes can be used.

When used, the probes are typically labeled in a detectable manner (e.g., with p or biotinylated nucleotides) and incubated with single-stranded DNA or RNA from the organism in which a gene is detected. Hybridization is detected by the label after single-stranded and double-stranded (hybridized) DNA (or DNA / RNA) have been separated, typically using nitrocellulose paper. Hybridization techniques suitable for use with oligonucleotides are well known to those skilled in the art.

Although probes are normally used with a detectable label to allow for easy identification, unlabelled oligonucleotides are also useful, both as precursors to labeled probes and for use in methods that provide direct detection of double-stranded DNA (or DNA / RNA). Thus, the term "oligonucleotide" refers to both labeled and unlabelled forms.

Once the desired DNA sequence has been obtained, it can be manipulated in various ways to obtain expression; e.g. chimeric polypeptide sequences can be prepared by combining gene fragments from at least two polypeptides with sequences that are at least more than 30% homologous to naturally occurring growth factors that bind to the EGF receptor (natural ligands). It is particularly desirable that the three-dimensional structure, in particular the loop structure, of the polypeptide be retained, in particular that part or parts of the structure that may be responsible for binding to the EGF receptor and the biological activity of the the naturally occurring growth factors that bind to the EGF receptor.

Depending on the source of the fragments and the length of the desired polypeptide, suitable restriction sites in the synthetic genes used to construct the chimeric polypeptides described above can be designed.

89 00724. " 19

If possible, the restriction site (or sites) leaves the amino acid sequence of the polypeptide unchanged. However, in some instances, the inclusion of a new restriction site (s) may yield an altered amino acid sequence without altering the activity of the protein. When the gene is to be expressed in a host that recognizes the wild-type transcription and translation regulatory regions of the growth factor, the entire gene with its wild-type 5 and 3 'regulatory regions may be in an appropriate expression vector implemented. Several expression vectors exist using mammalian virus replication systems, such as Simian Virus 40, adenovirus, bovine papillomavirus, vaccinia virus, insect baculovirus, etc. These replication systems have been developed to "". to provide the choice of transfectants as well as to provide suitable restriction sites into which the gene can be introduced.

If the gene is to be expressed in a host that does not recognize the naturally occurring wild-type transcription and translation regulatory regions, further manipulation will be needed. Advantageously, a plurality of 3'-trans-20 scripture regulatory regions are known which can be introduced downstream of the stop codons. The non-coding 5 * region upstream of the gene of interest can be removed by endonuclease restriction, Bal31 restriction and the like. Optionally, when a suitable restriction site is present near the 5 'end site of the gene of interest, the gene of interest can be cut and an adapter used to couple the gene of interest to the promoter region. the adapter provides the lost nucleotides of the gene of interest.

Several strategies can be employed to provide an expression cassette which injects a transcription regulatory region and a translation initiation region in the 5J-3J transcription direction that also inject regulatory sequences permitting the induction of regulation; the gene of interest under the transcription and translation control of the initiation region 35; and a transcription and translation termination region, betfat: *

In selecting appropriate regulatory sequences, the following factors that affect 8900724.20 expression will be taken into account. For transcriptional regulation, the amount and stability of messenger RNA are important factors influencing expression of gene products. The amount of mRNA is determined by the copy number of the particular gene, the relative efficiency of its promoter, and the factors that regulate the promoter, such as enhancers or repressors. The initiation is believed to take place in the region just upstream of the beginning of the coding sequence.

The promoter in prokaryotic cells includes nucleotide sequences which can adversely affect transcription efficiency.

The sequences include the regulatory regions at about -35 and -10 nucleotides from the start of the DNA chain. Efficient promoters include those in which the nucleotide sequence of the -35 and -10 regulatory regions is substantially equal to the corresponding sequences of those regions in the bacterial promoters from highly efficient genes. In general, these regions have a length of resp. about 5 and 6 nucleotides, each sequence varying by about one nucleotide in length and / or in sequence. A preferred order for the -35 corresponding regulatory order is 20 from the trp promoter, namely the TGACA, and for the -10 corresponding regulatory order from the lac promoter, namely TATAAT.

Not only the nucleotide sequences, but also the spacing of the corresponding sequences of the -35 and -10 regulatory regions, with respect to each other, is important to obtain optimal transcription of the gene of interest. Generally, the corresponding sequences of the -35 and -10 regulatory regions are separated by about 16-18 nucleotides, preferably by about 17 nucleotides. Each sequence can vary by about one nucleotide.

Illustration 1 transcription regulatory regions or promoters include, for bacteria, the beta-gal promoter, lambda left and right promoters, trp and lac promoters, trp-lac fusion promoter, etc .; synthetic promoters with sequences substantially similar to these sequences can also be used. A preferred -35 promoter for bacteria is a fusion promoter comprising the -35 regulatory region from the trp promoter and the -10 regulatory region from the lac promoter. Most preferably, the promoter is one in which the -35 trp corresponding sequence is approximately 17 8800724.

21 ΐ nucleotides are placed upstream from the -10 lac corresponding sequence, yeast glycolytic enzyme promoters, such as ADH-I and -II promoters, GPK promoter and PGI. promoter, trp promoter, etc., for mammalian cells SV40 early and late promoters, adenovirus main 5 late promoters, promoter or enhancer sequences and the like.

The transcription regulatory region may also include regulatory sequences with which to modulate the expression time of the gene of interest, e.g. by the presence or absence of nutrients or expression products, in the growth medium, the temperature, etc. Thus, the expression of the gene of interest can be regulated by the temperature of the host cell growth medium, using a regulatory sequence containing the bacteriophage. lambda P1 promoter, the bacteriophage lambda operator and the gene CI857 encoding the temperature sensitive Cj repressor in the expression vector. This permits regulation of the promoter by interaction between the repressor and the operator at low temperatures, e.g. about 30 ° C possible. An increase in temperature to about 42 ° C would inactivate the repressor and allow expression of the gene of interest.

As an example of modulation using growth medium nutrients, regulation of the lac or the trp-lac hybrid promoter can be accomplished using the gene for the lac1 repressor, which binds to the lac promoter region downstream of the -10 regulatory area. The lac1 repressor gene may be present on a 25 episom, preferably the Lac1 enhanced mutant, or

can be included in the expression cassette itself. The presence of a significant concentration of the repressor molecule in the growth medium impairs the promoter function in the absence of inducers. Thus, the addition of I.PTG or lactose to the host cell growth medium enhances promoter function. When the bacterial strain is Lac +, lactose can be used as the inducer instead of I.PTG. In both eukaryotic and procaryotic systems, termination regions may also contain sequences or structures that increase the stability of the nRNA species and allow for higher expression. Thus, the transcription regulatory region may also include regulatory sequences that terminate transcription and yield sequences or structures that degrade the mRNA.

8900724 ...

»22 and thus increase the stability of the mRNA species and allow higher expression. Several examples of procaryotic sequences are known, e.g. the Trp terminator, the gene 32 (T4) terminator or synthetic terminators that are equal in sequence to gene 32.

In eukaryotic systems, transcription termination, RNA cleavage, and polyadenylation regions provide proper maturation for the mRNA transcripts and are necessary for efficient expression. The native 3 'untranslated region may be sufficient, but the polyadenylation signal can be obtained from e.g. SV40, especially including a junction site that provides a more efficient expression. Optionally, the 3 'untranslated region derived from a gene highly expressed in a particular cell type (eg, Ig in myeloma cell and) can be fused to the gene of interest. Such 3 'sequences could also be paired with 5' regulatory sequences from the same highly expressed gene and even enclose coding regions in the reading frame to generate fusion proteins as described below.

Expressed in translation regulation, given the presence of mRNA, expression can be regulated by the initiation rate (ribosome binding to the mRNA), the elongation rate (translocation of the ribosome across the mRNA), the rate of post-translation modifications and the stability of influence the gene product. The extension speed is likely to be affected by codon consumption; the use of codons for rare tRNAs can reduce the translation speed. It is therefore preferred to use codons that repeatedly occur in genes normally expressed by the host cell to increase the translation rate.

3Q In prokaryotes, downstream of the -35 and -10 regulatory regions is a corresponding nucleotide sequence, commonly AGGA / called the Shine-Dalgarno sequence, which is believed to be involved in ribosomal binding. Optimal ribosomal binding and translation initiation can be achieved by using a ribosome binding site functional in the host cell from a highly expressed gene. There are also indications for the presence of nucleotide sequences surrounding the Shine-Dalgarno sequence and sequences within the encoding. B9 0072-4.

23 region that can affect ribosome binding, possibly through the formation of structural motifs through which the ribosome recognizes the initiation site. Thus, altering nucleotide sequences of the coding region is useful for achieving optimal binding and translation initiation. The order of the first about 7 to 30 codons after the initiating ATG codon may also affect binding and expression. Preferably, the major sequence and the Shine-Dalgarno sequence are derived from the same gene or when derived from different genes, the codon 10 of the major sequence can be modified by codon degeneration to the nucleotide sequence of the natural gene following the major sequence as described in co-pending U.S. Patent Application No. 264,098 filed October 28, 1988, the disclosure of which is incorporated herein by reference.

The location of the AGGA sequence relative to the initiating ATG codon may affect expression. Generally, the Shine-Dalgarno sequence is located about 5 to 9 nucleotides from the initiating codon, although unexpectedly higher expression levels can be reached with expression cassettes, in which the Shine-Dalgarno sequence is approximately 10-13 nucleotides, preferably 11-12 nucleotides. from the initiating codon. The stability of the mRNA is controlled by the sensitivity of the mRNA to ribonuclease enzymes.

In general, exonuclease breakdown is hampered by the presence of structural motifs at the ends of the mRNA; palin-25 drome structures, altered nucleotides or specific nucleotides. Endonuclease degradation is believed to occur at specific recognition sites within the mRNA and that stable mRNA would miss these sites. There is also some evidence that mRNA4s that are subject to high translation levels are also protected from degradation by the presence of the ribosomes on the mNRA.

The stability of the expression product can be achieved by providing a synthesis of a fusion protein in which the desired polypeptide is expressed in conjunction with a second polypeptide or fragment thereof. Preferably, the stability of the expression product is achieved by providing a synthesis of a fusion protein in which the polypeptide of interest is expressed associated with a major sequence.

6900724 24

A DNA sequence encoding an N-terminal amino acid sequence from e.g. a highly expressed bacterial or bacteriophage gene, such as the bacteriophage lambda N protein nine, cro gene or beta-galactocydase or a eukaryotic gene is combined with the gene of interest upstream of and in the reading frame. The major sequence usually comprises about 8 to about 50, preferably about 5 to about 45, most preferably 18 to 25 N-terminal amino acids.

The expression of the polypeptide of interest in the form of a fused protein having a major sequence from another gene has several advantages except that stability is provided.

The presence of the N-terminal amino acids provides e.g. a means of applying general purification techniques to purify any of a plurality of polypeptides. For example, the N-terminal amino acids of the N protein are particularly antigenic, and thus specific antibodies raised against the N-terminal amino acids of N-protein can be used as a means of purifying fusion proteins containing the N-terminal site of contain the N protein. Furthermore, the N-terminal site of the N protein has a high positive charge, which facilitates the purification of the desired protein by ion exchange chromatography, etc.

The main sequence can also be a hydrophobic amino acid sequence, which can also function as a signal sequence for secretion. A DNA sequence encoding the signal sequence is joined upstream and in the reading frame with the gene of interest. Typically, the signal sequence includes a cleavage site recognized by a signal sequence peptidase. Thus, by directly placing the polypeptide of interest behind the signal sequence cleavage site, it is possible to specifically cleave the polypeptide of interest from the signal sequence and secrete it as a mature polypeptide. Examples of hydrophobic amino acid sequences include the bacterial alkaline phosphatase signal sequence; the OMP-A-B-C-D-E or F signal sequences; the LPP signal -35 sequence, beta-lactamase signal sequence and toxin signal sequences and mutants thereof. For eukaryotic cells, the signal sequences may be the signal sequence of msisap TGF-beta, P97 gene (human melanoma antigen), K. lactis killer toxin sequence, alpha pair type factor and other killer toxin signal sequences, or the normal 88 00724. "25 signal sequence associated with the gene of interest along with mutants of the signal sequences.

Other main sequences that are useful include hydrophilic sequences, e.g. the N-terminal 41 amino acid residues of amphiregu-5 line, which provide a modification of the function of the polypeptide of interest. In addition, a cytotoxic agent such as toxin A chain fragment, ricin A chain, snake venom growth-inhibiting peptide or a targeting molecule such as a hormone or antibody can be covalently coupled to the main sequence with in most cases minimal effect on biological activity. of the gene product of interest. As described for the other major sequences, a DNA sequence encoding master sequence upstream of and in a reading frame is combined with the gene of interest.

When the major sequence is not a signal sequence or does not contain a suitable natural cleavage site, additional amino acids can be introduced to provide an enzymatic or chemical cleavage site for cleavage of the main peptide, after purification of the fusion protein to allow subsequent purification of the mature 20 polypeptide. possible. The introduction of acid-labile aspartyl-proline linkages between the two segments of the fusion protein facilitates e.g. its separation at low pH. This method is not suitable if the desired polypeptide is acid-labile.

As another example, the fusion protein can be cleaved with e.g.

Cyanogenic "bromide, which is specific for the carboxy side of methionine residues. By placing a methionine between the major sequence and the desired polypeptide, one can release the desired polypeptide. This method is not suitable when the desired polypeptide contains methionine residues.

When the major sequence includes signal sequence, genes of interest with secretory major sequences may be expressed with or without the major sequence provided that the sequence can be maintained or spliced. Also, to obtain a large amount of the desired polypeptide as a mature, cleaved and refolded peptide released into the medium, it is preferred to use a promoter, such as the tac promoter, which can operate at a lower temperature, e.g. at on- 8900724.

a 26 about 30 ° C. As a result, unexpectedly higher secretion levels are obtained at the lower temperatures. An exceptionally high level of expression can prevent complete translation modification of the protein from occurring, resulting in aggregation and accumulation of non-cleaved precursor (ie structural protein and main secretary product). Likewise, growth at elevated temperatures, e.g. 42 ° C, the tendency to indicate aggregation and accumulation of non-split precursor. The preparation of fused proteins, including cleavage methods and refolding of the polypeptide of interest, are further described in pending US patent application serial no. 264,098. supra.

After each manipulation of the DNA in the development of the cassette, the plasmid will be donated and isolated and, if necessary, the determined cassette component analyzed in order to ensure that the correct sequence has been obtained. Depending on the nature of the manipulation, the desired sequence can be excised from the plasmid and introduced into another vector, or the plasmid can be cut and the expression cassette component manipulated as appropriate. In some cases, a shuttle vector may be used where the vector can be replicated in different hosts, requiring different replication systems. This may or may not require additional markers that are functional in the two hosts. When such markers are needed they can be included in the vector. The plasmid containing the cassette, two replication systems and the marker (s) can be transferred from one host to another as desired. Any useful marker can be used for selection. The resistance to neomecine or tetracycline is desirably important.

Although a marker for selection is highly desirable due to its suitability, other procedures for examining transformed cells have also been described. See e.g. G. Reipin, et al., Current Genetics, 1982, 189-193. Transformed cells can also be examined by the specific products they make, e.g. the synthesis of the desired product can be determined by immunological or enzymatic methods.

The expression cassette may be stored within an episoma maintenance replication system in a suitable cellular host. 8900724.

27 or can be supplied without a replication system, which can be integrated into the host genome. The DNA can be introduced into the host by known techniques such as transformation, use of calcium phosphate precipitated DNA, transfection by contacting the cells with the virus, microinjection of the DNA into cells and the like. Once the gene of interest has been introduced into the appropriate host, the host can be grown to express the gene of interest. A variety of host cells can be used. Examples of prokaryotic host cells include gram-negative organisms such as E-coli, e.g. JM109, JM101 and JM107; HB1Q1, DH1 or DH5.

Gram-positive organisms such as B. subtilis, which has no periplasmic space and can directly release polypeptide into the growth medium, are particularly suitable. Host eukaryotic cells may include yeast cells, insect cells and warm blood cells, e.g. COS cells, GHO cells, monkey kidney cells and sideworm cells (sf9j.

The structure containing the gene of interest and flanking regions that regulate expression can be introduced into the expression host by any suitable means, e.g.

Transformation, with e.g. calcium phosphate precipitated DNA, transfection, transduction, conjugation, microinjection, etc. The host can then be grown to a high density in a suitable nutrient medium. When the promoter is inducible, allowable conditions will be used, e.g., temperature change, depletion or excess of a metabolic product or nutrient, and the like.

When the regulatory order e.g. the bacteriophage ^ P ^ promoter, the bacteriophage lambda 0f operator and CI857 temperature sensitive repressor / the host cells will be grown at the allowable temperature, generally about 30 ° C, suppressing temperature transfer of the promoter and the host cells unimpeded by the requirements of the synthesis of the foreign gene product may grow, which may also be toxic to the host organism. When the host cells have reached an optimal density, the temperature may rise to an impermissible temperature, e.g. about 42 ° C, at which time the Cl repressor is rendered inactive allowing transcription from the P 1 promoter. Maximal secretion can be achieved by using the lac promoter or a trp-lac promoter and induction with a meta 89 0072 4.

• provide 28 bolic inducers such as lactose for a lac + host taro and lac11 on a vector. Examples of host cells that would be useful with such a system include DH1, DH5 or HB101. When the product is to be maintained in the host cell, the cells are collected, lysed and the product isolated and purified by extraction, precipitation, chromatography, electrophoresis, etc.

When the product is separated, the nutrient medium can be collected and the product in conventional ways, e.g. affinity chromatography are isolated. In order to produce an active protein, it may be necessary to refold the protein. If the protein is expressed as a fusion protein of the major sequence, the major sequence can be treated by treatment with e.g. formic acid or cyanogen bromide are removed.

Preferably, the major order is removed after refolding of the protein.

The recombinant products may or may not be glycosylated with the wild type or other glycosylation. Generally, the glycosylation will differ by no more than about 50%, usually by no more than about 20%, from the wild-type glycosylation.

The amount of glycosylation depends in part on the order of the particular peptide, as well as the organism in which it is produced. Thus, expression of the product in E. coli cells will result in an unglycosylated product, and expression of the product in insect cells will generally result in less glycosylation than when expressing the product in mammalian cells.

Application of growth factors

The present compositions can be used for various purposes in vitro and in vivo as agonists or antagonists for growth factors such as epidermal growth factor (EGF) and transforming growth factor in the alpha alpha TGF. One discussion of growth factors, alone or in combination with other compositions, especially polypeptide compositions, can be used to regulate the growth of cells and for other activities, e.g. found in Handbook of Experimental Pharmacology, Tissue Growth Factors, publisher Baseraga, Vol. 57, Springer-Verlag, Berlin, 1981, chapter 3, in particular pp. 98-109; and Carpenter, Ann. Rev.

8900724.

29

Biochem. (1979) 48: 193-216.

Human EGF appears to be identical to human urogastrone and exerts multiple effects on prenatal and neonatal tissue growth. As an effect, one can mention, among other things, early eye opening, wound-5 healing, incisor breakthrough and accelerated maturation of the lung. EGF receptors are found in many adult tissues. EGF appears to stimulate phosphorylation of its own receptor. EGF also appears to be associated with an increased bone resorption.

Transforming growth factor, in particular alpha-TGF, has many activities analogous to those of EGF. TGF binds to the EGF receptor leading to phosphorylation of the receptor, enhancement of thyrosine-specific kinase activity and stimulation of cell growth. See Cohen, in: Biological Response Mediators and Modulators (ed. J.T. August), Academy, New York, 1983, p.

7-12., Tam et al., Nature (1984) 309: 376-378; Ibbotson et al., Science (1983) 221: 1292-1294.

Myxoma and Shope fibromaviruses induce a significant enhancement of the proliferation potential of cells within the affected area. While vaccinia induces a minor rampant at the site of infection, Shope fibromavirus induces a strong rampant that leads to tumor formation, which is benign in adult rabbits, but rampant and invasive in immunosuppressive adults or newborns. Myxomavirus induces a rapidly spreading myxosarcoma tumor. Although these proliferation phenomena could be attributed to other viral factors, the viral growth factors appear to be strongly implicated in the induced cellular proliferation caused by infection.

The present compounds find particular use as drugs as agonostics for EGF and for wound healing, such as? Epithalization of wounds, such as burns, eye wounds, surgical incisions, etc. The active component can be used in a conventional carrier, e.g. Silvadene are used in an amount sufficient to promote epithalization and / or wound healing, generally in amounts ranging from about 0.01 to 10.0 mg / ml, usually about 0.075 to 05.0 mg / ml ml of EFG equivalents preferably 0.5 to 5 mg / ml. The composition is spread over the wound to completely cover the wound with the composition 8900724.

30 ¥ dress. One can perform four treatments a day or every other day or less depending on the nature of the wound, its response to treatment, the concentration of the active component, and the like.

The present compositions are useful as reagents for diagnostic analyzes or for the preparation of reagents, such as polyclonal or monoclonal antibodies. They can be used as reagents for the detection of analogous growth factors or for the detection of antibodies for the growth factors in physiological fluids, such as blood. Depending on the particular protocol and the purpose of the reagent, the polypeptide may or may not be labeled. Many markers have been used that directly or indirectly provide a detectable signal. These markings include radionuclides, enzymes, fluorescent agents, particles, chemistry.

Nescents, enzyme substrates or cofactors, enzyme inhibitors, magnetic particles, etc. See e.g. U.S. Patent Nos. 3,654,090, 3,817,837, 3,935,074, 3,996,345, 4,277,437, 4,374,925, and 4,366,241.

Several methods exist for coupling the tags to the polypeptides, using the N-terminal amino group for functionalization to form a pyrazolone, while protecting other free amino groups, whereby the pyrazolone can then be contacted with various reactants, e.g. amino groups, to link to the detectable signal-forming unit. By protecting the arginine amino acids associated with the third loop or proximal thereof, other arginines can be functionalized by known methods for conjugation to amino groups or thio groups. Optionally, the polypeptide can be contacted with an active agent, e.g. an activated carboxylic acid and statistically substituted, whereby biologically active material can be separated from biologically inactivated material as a result of the statistical substitution. Finally, depending on the synthesis method, the polypeptide can be modified to provide the desired functionality, as part of the synthetic procedure.

The present compositions can also be used to monitor EGF receptors. The present compositions are also useful for monitoring the cellular response to EGF and / or TGF by ensuring competition between these 88 00 724.

31 naturally occurring materials and a composition of the present invention. In this way, changes in the receptor conformation can be monitored.

The present compositions are also useful for various therapeutic purposes such as growth stimulation or bone formation control. These compounds can be administered intraperitoneally, subcutaneously, intravenously, intraarterially or by application to the site of interest in suitable physiological carriers. Also, the present compositions may be introduced into liposomes, which may or may not involve the use of antibodies for the site direction. Different carriers include phosphate buffered brine, brine, water, etc. The concentration of the additive will vary widely depending on its final use and activity. Other additives may also be included in the composition, such as E6F, TGF, other growth factors, bacteriocides, e.g. antibiotics, bacteriostats, buffers etc.

For the preparation of antibodies, the subject polypeptides in which PP hydrogen or short oligopeptide chains (less than 5 amino acids) are linked to antigenic polypeptides or proteins can be injected into a mammalian host. The antigenic protein contains at least about 60 amino acids and is usually no greater than 100 kilodaltons (kDal). Numerous techniques exist for coupling to polypeptides, either at a specific site or randomly, using bifunctional reagents, e.g. p-25 maleimidobenzoic acid, glutaraldehyde, p, pJ-benzidine, etc. Common antigenic proteins include bovine serum albumin, keyhole limpet hemocyanin, tetanus toxoid, etc. The subject polypeptides are coupled in sufficient number to the antigenic protein. Usually after the initial injection there will be two or more recall injections. For antisera, blood is removed from the immunized host. and isolated the immunoglobulin fraction. For monoclonal antibodies, the spleen is isolated and splenocytes are fused with an appropriate fusion partner by conventional methods. The resulting hybridomas are then screened for antibodies that bind to the epitopic sites of the subject polypeptide. These antibodies are 8900724.

32 are useful for various purposes, such as diagnostic reagents, therapy, etc. The antibodies, when used as reagents, may or may not be labeled as described for the polypeptides.

The following examples are given for illustrative and not limiting purposes only.

EXPERIMENTAL

Content overview

Example I Preparation of synthetic genes of interest 10 A. TGF Synthetic oligonucleotides B. VGF Synthetic oligonucleotides C. EGF Synthetic oligonucleotides

Example II Description of cloning and expression plasmids 15 A. Plasmid pLEBam B. Plasmid pBMll C. Plasmid pBMllM4 D. Plasmid pBMl1M5

E. Plasmid pBMll / NDP

F. Plasmid pBMll / PAD

G. Palsmide pBMll / PAK

H. Plasmid pTCPt I. Plasmid plac / crogal J. Plasmid ptac / crogal

25 K. Plasmid pRSV

L. Plasmid pAc610 M. Plasmid pToxl and piox2 N. TakPak 30 Example III Combination of growth factor genes for expression in prokaryotic cells

A. Preparation of plasmid pLEBam / TVV

B. Preparation of plasmid pLEBam / TVV

VGF and fragments thereof 35

Example IV Expression of the polypeptides of interest as a fusion protein with the N protein

A. Modified synthetic TGF

8300 724.

33 B. Modified synthetic TGF-VGF hybrid

Example V Preparation of the polypeptide of interest as a fusion protein with the N protein and a cleavage site

A. Modified synthetic VGF

B. Modified synthetic TGF / VGF hybrids

C. Synthetic EGF

Example VI Preparation of the polypeptide of interest as a fusion protein with the modified alkaline phosphatase signal sequence

A. Preparation of pBM11 / PAD / EGF

B, Preparation of pBM11 / PAD / nVGFa 15

Example VII Expression of the polypeptide of interest as a fusion protein with an alkaline phosphatase signal A. Preparation of pBM11 / PAK / nVGFa (Alkaline phosphatase signal sequence / nVGFa with natural VGF N-

20 final rankings and sequences GTC and GYACRC

B. Preparation of pBM11 / PAK / EGF

C. Preparation of TacPak / EGF (alkaline phosphatase signal sequence / human EGF) Example VIII Expression of a polypeptide of interest as a fusion protein with the alkaline phosphatase signal sequence using an expression cassette comprising a transcription termination region A. Preparation of pTCPt / EGF (/ trp-35j 16bp / "lac-ioj 30 / lacSDjl1bp / ATGj / alkaline phosphatase signal /

human EGF / trans-term-NEO

B. Preparation of pTCPt / nVGFa (^ trp-35jl6bp / lac-wj [l acSD.//croSD.7 11 bp [pJG] / al kal isch phospha-phase signal / n-terminal VGFa in order 35 GTC and GYACRC) / trans-term-NEO) C. Preparation of pTNPt / EGF (£ trp-35ji7bp / ac-10j / nSDj8bp ^ ATGj / alkaline phosphatase signal / human

EGF / trans. term.-NEO

89 0 07 M.

34

Example IX Combination of growth factor genes for expression in eukaryotic cells

A. Preparation of piasmi's PRSV / VGF

B. Preparation of plasmid pAc / VGF

5 C. Preparation of pAc / SFGF

Example X Preparation of polypeptides

A. Solid phase synthesis of BGF and TGF

B. Isolation of the recombinant polypeptides prepared in prokaryotic cells C * Isolation of VGF and SFGF produced by eukaryotic expression of the natural genes

D. Natural EGF

Example XI Activity Assays A. Mitogenic Assay B. Soft Agar Colony Growth Stimulation Assay C. EGF Receptor Binding Inhibition Assay D. Radio Immunoassay 20'E. Wound healing

Example XII Biological activity of recombinant growth factors prepared in prokaryotic cells A. EGF receptor binding B. Mitogen activity C. Wound healing

Example XIII Biological activity of FGV prepared in eukaryotic cells 30 A. VGF prepared in monkey (BSC-D cells B. VGF prepared in CHO cells C. VGF prepared in silkworm

D. Immunological comparison of VGF and TGF

35 Organic depots

The following expression plasmids, all transformed into E. coli HB101, were deposited at 8900724 on the indicated data.

American Type Culture Collection, 123Ü1 Pariclawn Drive, Rockville, MD 20852 whose identification in ATC designations are given below: 5 Identification ATCC designation Deposit date PBMll 67366 PBM14 67367 PBMll / PA / VGF 67417 June 3, 1987 PBM11 / DP / VGFa 67418 June 3, 1987 PBM11 / PA / EGF 67419 3 June 1987 10 PBM11 / M5 67436 PBM11 / C2 67437 PBMl 1 / NDP / EGF 67547 23 October 1987

Methods, JG General cloning techniques were used as described in Maniatis et al, 1982, "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, CSH, New York. All DNA modifying enzymes were obtained from commercial suppliers. They were used according to the manufacturer's instructions. Materials and equipment for DNA purification and separation were used according to the instructions of the suppliers.

Example I

Synthesis of Synthetic Genes of Interest Synthetic growth factor genes were designed using host cell codons optimized for high expression levels. Various suitable restriction sites in the synthetic genes were also designed. If possible, the new restriction sites leave the amino acid sequence of the growth factor gene 20 unchanged, but in some cases the inclusion of the new restriction site produced an altered amino acid sequence. These sites roughly divide the synthetic genes into three parts namely the N-terminal, central and C-terminal domains.

The natural VGF gene product contains an extreme N-terminal domain that has no counterpart in mature TGF. VGF fragments missing this domain are indicated as truncated. The restriction sites were used for the initial build-up of the end genes 88 0072.4.

36 from partial synthetic oligonucleotide fragments extending from one restriction site to another. The oligo-nucleotides were synthesized with an oligonucleotide synthesizer from Applied Biosystems and purified on an acrylamide gel. The oligonucleotides were phosphorylated at the 5 'end using T4 polynucleotide kinase and each oligonucleotide was then attached to its complement.

A. TGF Synthetic oligonucleotides 10 1. Human TGF N-terminal domain: TGF ->

BssHIINcol

MVVSHF N DCPDSHTQ FC

5 'cgcgccatggttgtttctcactttaacgactgcccggactctcatactcagttttgc 3' GGTACCAACAAAGAGTGAAATTGCTGACGGGCCTGAGAGTATGAGTCAAAACG 15

Kpnl F H G T

TTTCATGGTAC 3 'TGF104 AAAGTAC 5' TGF103 20 2. Modified human TGF central domain where the human sequence QEDK has been changed to QEEK, which sequence is found in rat TGF:

Kpnl Sphl ~ CRFLVQEEK PAC 25 5 'CTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATG 3' TGF101 3 'CATGGACGGCAAAAGACCAAGTCCTTCTTTTTGGCC 5' TGF102 3. Human TGF C-terminal domain: 30 ^ h1

VCHSGYVGARCEHAD LL 5 'CGTTTGCCATTCTGGCTACGTTGGCGCACGTTGCGAACACGCTGACCTGCTG 3' GTACGCAAACGGTAAGACCGATGCAACCGCGTGCAACGCTTGTGCGACTGGACGAC

BamHI

A Ter 35 GCTTAAG 3 'TGF205 CGAATTCCTAG 5' TGF206 8900724.

37 B. V6F Synthetic oTigonucleotides 1. VGF Extreme N-terminal domain:

HindiII

EDSGNAIETTSPEI TNATT 5 'AGCTGACTCTGGTAACGCTATCGAAACTACTTCTCCGGAAATCACTAACGCTACTACT 3' V 5 3 'CTGAGACCATTGCGATAGCTTTGATGAAGAGGCCTTTAGTGATTGCGATGATGA 5' V

2. Modified VGF N-terminal domain containing the Asp-Pro cleavage site, the sequence HGT replacing the natural sequence HGF; 10

BamHI

IDPM DIPAIRLCGP EGDGY 5 "GATCGATCCCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTAC 3 'CTAGGGTACCTGTAGGGCCGATAGGCAGACACGCCGGGCCTTCCGCTGCCGATG

Kpnl

ις C L H G T

TGCCTGCATGGTAC 3 'VGF104a ACGGACGTAC 5' VGF103a 3. Modified VGF central domain where the sequence GYAC 20 replaces the natural sequence GMYC:

Kpnl Sphl T.CIHARD ID GYAC 5 'CTGCATCCATGCACGTGACATCGACGGCTACGCATG 3' VGFlOla 3 * CATGGACGTAGGTACGTGCACTGTAGCTGCCGATGC 51 VGF102a 25 4. VGF Terminal domain, 5J

Sphl EcoRl

CRCSHGYTG 51 CCGTTGCTCTCATGGCTACACTGG 3 'VGF1A 30 3' GTACGGCAACGAGAGTACCGATGTGACCTTAA 5 'VGF2A

5. Modified VGF C-terminal domain, S 'terminus, the sequence VCS replacing the natural sequence RCS; 35 Sphl EcoRl VCSHGYTG 5 'CGTTTGCTCTCATGGCTACACTGG 3' VGF1 3 'GTACGCAAACGAGAGTACCGATGTGACCTTAA 5 "VGF2 8900724.

fl 38 6. VGF C-terminal domain 3 'fragment, terminating at YQR instead of PNT, the derived C-terminal from naturally secreted VGF:

EcoRI BamHI

5 IRCQHVVLVDYQR Ter 5 'AATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGTTAAGGATC 3' VGF3 3 'GCAACGGTCGTACAACAAGACCAGCTGATGGTCGCAATTC 5' VGF4 C. EGF Synthetic Oligonucle

Three sets of overlapping synthetic oligonucleotides 1 (A, B), 2 (A, B) and 3 (A, B) encoding human EGF were synthesized on an oligonucleotide synthesizer from Applied Biosystems and purified on an acrylamide gel. The oligonucleotides were phosphorylated at the 5 'end with T4 polynucleotide kinase. Each oligonucleotide was attached to its complement.

15 1.

NcoIEcoRI

M-NSDSECPLSHDGY 5 'CATGAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTAC 3' EGF1A

3 'TTAAGACTGAGACTTACGGGCGACAGAGTACTGCCGATGACGGAC 5' EGF2A

2.

Nsil Sphl

CLHDGVCMYIEALDKYAC

25

5 'TGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATG 3' EGF1B 3 'GTACTGCCGCATACGTACATGTAGCTTCGAGACCTGTTCATGC 5' EGF2B

3.

30 Sphl

NCVVGYIGERCQYRDLK 5 'CAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAA

3 * GTACGTTGACGCAACAACCGATGTAGCCGCTTGCAACGGTCATGGCACTGGACTTT

BamHI

W W E L R * 35 TGGTGGGAACTGCGTTAAG 3 'EGF3 ACCACCCTTGACGCAATTCCTAG 5' EGF4 S9 ¢ 07 2 4 .1 39 *

Example II

Description of cloning and expression plasmids A. Plasmid pLEBam was used to donate synthetic oligonucleotide fragments i.v.m. are suitable BssHII 5 and BamHI restriction sites. A plasmid with Mcol and BamHI restriction sites, such as pBM11 or pBM11 / NDP (as described below) are useful for donating the synthetic nucleotide fragments and other DNA sequences of interest.

B. Plasmid pBM11, described in co-pending U.S. Patent Application No. 264098, filed October 28, 1988, allows a gene of interest downstream of the DNA sequences encoding the 33N terminal amino acids of the bacteriophage N gene at a Donate BamHI restriction site. After induction of XPL promoter-activation of the C1857 temperature sensitive repressor at 42 ° C, the foreign gene product becomes as the C-terminal part of a fusion protein, the N-terminal sequence of which is of the N gene. , expressed.

C. Plasmid pBM11M4, described in co-pending United States Patent Application No. 264098, filed October 28, 1988, is derived from pBM11 and thereby allows one to donate a gene of interest to a BamHI restriction site immediately after the initiating methionine of the N gene. Plasmid pBM1l / M4 also contains an Ncol site in the neomycin gene.

D. Plasmid pBM11M5 described in the aforementioned US patent application is derived from pBM11 in which an Ncol site present in the neomycin resistance gene is removed by site-directed mutagenesis. Therefore, no partial digestion of the vector with Ncol is required to donate a gene of interest in pBM11 / M5.

E. Plasmid pBM11 / NDP, described in the aforementioned US patent application, is derived from pBM11 and has DNA sequences encoding an acid labile aspartic proline acid dipeptide which, among the sequences encoding the N gene, encode a gene of interest 8900 72 4 .

4) 40 is inserted. The plasmid contains an Ncol site and a C1al site for donating a gene of interest downstream of the promoter.

F. Plasmid pBM11 / PAD, described in the aforementioned US patent application, is derived from plasmid ρΒΜ11M4 and one can thus donate a gene of interest in a HindIII, Narrow and BamHI downstream of a modified alkaline phosphatasial sequence.

G. Plasmid pBM11 / PAK, described in the aforementioned US patent application, is derived from plasmid pBM11 / M4 and one can thus donate a gene of interest in a HindIII, Narrow or BamHI downstream of alkaline phosphatase signal sequence.

H. Plasmid pTCPt, is designed to possess the tac promoter elements and use the cro SD to express the gene of interest behind the alkaline phosphatase signal sequence. An example of the construction of this plasmid is given below in the construction of pTCPt / EGF.

SO

Structure of pTNPt (/ trp-357l7bp /Ïac-10jvnSDj;8bp/'AIGJ/alkaliV.c phosphatase signal / coupler / trans. Term.-NEO

This plasmid is designed to contain the tac promoter elements <2 // to use the N gene ST to express a particular gene behind the alkaline phosphatase signal sequence.

It has a pBR322 background with the neomycin resistance gene. Plasmid pTNPt was constructed as follows: (a) Preparation of the 2.8kb EcoRI-BamHI fragment from pBM16t / VGFa lacking the HindII site.

Plasmid pBM16t / VGFa was digested with EcoRI and BamHI

and the 2.8 kb fragment was isolated. The 2.8 kb fragment was ligated to an EcoRI-BamHI linker and a correct structure was isolated by restriction analysis, and designated as intermediate I.

The unique HindIII site near the neomycin resistance gene was removed from the intermediate I plasmid by degradation with HindIII, blunt ends with the Klenow fragment S900724.

41 were created, which were reconnected. This resulted in intermediate II plasmid that missed the Hind III site.

The 2.8kb EcoRI-BamHI fragment of pBM16t / VGFa that missed the 5 HindIII site was isolated by p intermediate II

with EcoRI and BamHI. The resulting 2.8 kb fragment was isolated by agarose gel electrophoresis.

(b) Preparation of the 15Qbp BamHI-Bsml fragment from pBM11 / PAK

Plasmid pBM11 / PAK is identical to pBM11 / PAK / EGF with the exception that it contains a linker region with HindIII, Sma and BamHI sites downstream of the alkaline phosphatase signal sequence instead of the EGF gene. pBM11 / PAK was digested with Bsm1 and BamHI and the 150bp fragment containing the N gene $ D, the alkaline phosphatase signal sequence and a linker region was isolated.

(c) Preparation of oligonucleotides TacA + and TacA-

Oligonucleotides TacA + and TacA- were synthesized with an oligonucleotide synthesizer from Applied Biosystems and designed to be. Had EcoRI overhang at the 5 'end in which the trp-35 matched sequence of the lac-tQ matched sequence was separated by 17 nucleotides within which an SstI site was located. The sequence also included the 5 'end of the lac mRNA, the lac repressor binding site 25 and a Bsm1 overhang.

TacA + 5'AATTACTCCCCATCCCCCTGTTGACAATTAATCATCGAGCTC

GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAG 3 '

30 TacA- 5'GTGTGAAATTGTTATCCGCTCACAATTCCACACATTATA

CGAGCTCGATGATTAATTGTCAACAGGGGGATGGGGAGT 3 '(d) Ligation and Isolation of pTNPt

The 2.8kb EcoRI-BamHI fragment, the 150bp Bsml-BamHI fragment 31 and oligonucleotides TacA + and TacA- were bound together with DNA ligase and the DNA was used to transform competent JM109 (1aclq). Correct build was isolated by 8900724.

42 restriction analysis and DNA sequencing.

(EcoRl instead of pbmh)

- I

GOESTACTCCCCATCC

5

Ss tl trp-35 {17bp) Xac-10 CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG) 10

Bsml 5'lac mRNA-> n mRNA->

TGTGG / AATTGTGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCT

TTGGGGTGTGTGATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGC

15

TGCCAATGTGCCAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACACACC

Pvul mSD (8bp) si anal full aoYde ">

ACCAAAGCTAACTGAC {AGGA} GAATCCAG ATGAAACAAÏCTACGATCGCCC

MKQSTIAL

Narrow

HindiII BamHI

TCGCACTTCTCCCACTGCTGTTCACTCCAGTGACAAAAGCTTCCCGGGATCCGTG

ALLPLLFTPVTK

25 (BamHI instead of pBMll) Trans. Term. |

ACTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC

30 I. Plasmid plac / cro- /? bile consists of the operator promoter region of E. coli lactose (lac) operon, as well as the ribosomal binding sites of lac and cro. The plasmid was constructed as described in the aforementioned U.S. Patent Application 264,098. Fusion proteins expressed by this vector consist of the N-terminal position of bacteriophage X cro-protein, the amino acid sequence encoded by the introduced DNA and the C-terminal position of β-galactosidase. The controlling elements of this 8900724.

& 43 vector consist of the operator promoter region of the E.coli lactose (lac) operon, as well as the ribosomal binding sites of lac and cro.

5 j. PIasmide ptac / cro-ft-gal allows to donate a gene of interest downstream of the N-terminal 21 amino acids of the bacterial cro protein. The plasmid was constructed as described in the aforementioned U.S. Patent Application 264,098. the expression vector ptac / cro- / S-gal is equal to plac / cro-yS-gal, 10 with the exception that the promoter of ptac / cro- ^ - gal from the -35 region of the promoter of the trvotophamic uron and the Pribnow box of the lac operon exists. A higher level of expression is possible with this hybrid promoter than with plac / cro-e-gal.

K. Plasmid pRSV allows expression of a gene of interest in eukaryotic cells with the RSV LTR promoter,

Gorman et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6777-6781.

20 L. Plasmid pAc61Q, Smith et al., Molec. Cell. Biol. (1983) 12: 2156-2165, allows expression of a foreign gene in insect cells.

M. Plasmids pTox1 and Ptox2 allow expression of a gene of interest in yeast gene using the X. lactis killer toxin signal sequence / EMBO 6, 229-234 (1987); Biochem »

Biophys. Res. Comm. 144, 613-619 (1987] /. This structure contains the URA3 gene for selection in yeast and the AMP gene for selection in bacteria ·

Transcription is initiated with the CYCI promoter and GAL 30 "upstream activation sequence" (Methods Enzymol., 101, 181-191, 1983) and terminates within the 3 'end of the FLP gene (Mol. Cell. Biol., 5, 2770-2780 (1985) The build contains both the two micrometer origin and the pBR322 origin for replication in yeast and E. coli, respectively. The killer toxin N-terminal region 35 with the signal sequence and the Kex2 cleavage site was introduced opoligo nucleotides and contained the AT rich region immediately before the initiator codon to provide optimal translation initiation and 8900724.

ν '44 length signals. Several restriction sites were constructed to allow insertion of genes of interest downstream of both the signal cleavage site and the Kex2 cleavage site.

5 1. Preparation of the 3kb EcoRI-EspI fragment of pBR322 containing the origin and the AMP resistance gene.

Plasmid pLGSD5 (-ATG), a yeast expression vector (Methods Enzymol. 101: 181-191 (1983) was digested with EcoRI and Espl and the 3.0kb fragment was isolated.

2. Preparation of the 2.1 kb EcoRI-EspI fragment of the 2micro-meter plasmid containing the 3'-end of the FLP gene

Plasmid pLGSD5 (-ATG) was digested with EcoRI and Espl and the 2.1 kb fragment was isolated.

15 3. Ligation and Isolation of Intermediate Plasmid.

The 2.1 kb and 3kb EcoRI-EspI fragments were bound together with DNA ligase and the DNA was used to transform Competent HB101. Correct construction was isolated by restriction analysis.

4. Preparation of the 1.8kb EcoRI-BamHI fragment of pLGSD5 (-ATG) containing the URA3 gene, the GAL UAS and CYC1 promoter.

The plasmid pLGSD5 (-ATG) was digested with EcoRI and BamHI and the 1.8kb fragment was isolated. The BamHI site is contained within a linker 4bp introduced upstream of the CYC1 initiator.

5. Preparation of oligonucleotides pTox1A +, 2A-, 1B +, 2B-.

Oligonucleotides were designed to link the codons for the signal sequence of K. lactis killer toxin to the BamHI site downstream of the CYC1 initiator. To conserve the optimal translation initiation, 13bp of the A-T rich sequence was introduced upstream of the killer toxin initiator codon. This order complies with the corresponding order determined for yeast initiating codons. A Bg11I site was placed immediately upstream of the A-T region to allow mutagenesis of the initiating region and the signal sequence. A HindIII site 10 nucleotides was placed upstream of the signal cleavage site every 8900724.

45 to allow mutagenesis of downstream sequence and ligation of the gene of interest. In pToxl, an Aval, Xhol site was placed directly downstream of the signal cleavage site, while pTox2 a Nael site was placed at the signal cleavage site to donate the blunt end directly from the Gly-Leu to Ala-Glys donation directly over the signal cleavage site. . Resolution of the heteroduplex at the cleavage site would result

You are in the formation of clones that contain .pToxl sequence and those that contain the piox2 sequence. In both builds, the subsequent 1G sequences encode the remainder of 12 killer toxin precursor sequences down to the Kex2 Lys-Arg cleavage site, at which point several cleavage sites (Stul, Sail, AccI, Hindi, and BamHI ^ were placed to clone a gene of interest ease.

With the Stul site, blunt end donation is possible immediately after the Arg codon of the Kex2 cleavage site. The oligonucleotides have a BamHI overhang at the 5 'end and a HindIII junction at the 3' end in the intermediate vector. The resulting ^ s / 4th <¢ / ¾ sequence no longer contains either of the two places.

(BamHI) BglII HindII

MN IFYIFLF LLS 1A + 5 · gatcagatctatatetataaaatgatatattttacatatttttgtttttgctaagc 2A- 3 1 TCTAGATTATTAATATTTTACTTATATAAAATGTATAAAAACAAAAACGATTCGA,

Splitting site signal Kex2splijtingSplSats (pToxl) / Aval, Xhol / Stul SalI, Acci Ba fvqglehthrrgslvkrplstdp

1B + 5TTCGTTCAGGGCCTCGAGCATACTCATAGAAGAGGCTCCTTAGTCAAAAGGCCTTTGTCGACGGATCC 2B- 3AAGTCCGGCCGCTCGTATGAGTATCTTCTCCGAGGAATCAGTTTTCCGGAAACAGCTGCCTAG

A G

30 (pTox2) Nael

These oligonucleotides were synthesized with an Applied Biosystem synthesizer and purified by gel electrophoresis. Then they were phosphorylated with T4 kinase and the complementary pairs were attached, ligated and gel purified.

6. Preparation of EcoRI-HindIII intermediate vector.

8900724.

46

Intermediate vector was digested with EcoRI and HindIII and then treated with calf alkaline phosphatase. This treatment removed a small EcoRI-HIndlII fragment and left a HindIII site upstream of the FLP 3 'sequence.

5 7. Ligation and isolation of pToxl and piox2

The 1.8kb EcoRI-BamHI fragment was ligated to the oligonucleotide fragment using DNA ligase, and the resulting fragment was gel purified and then ligated to the EcoRI-HindIII intermediate vector, after which the DNA was used for transformation of competent HB101. Correct constructs were identified by restriction analysis and DNA sequencing and a clone with the order of pTox1 and one with the order of pTox2 were isolated. Both clones missed the BamHI at the intersection of the oligonucleotides and the intermediate vector. 15

Example III

Combination of growth factor genes for expression in prokaryotic cells.

A. Preparation of pi asmide pLEBam / TTV 20 The synthetic chimeric growth factor, designated TTV

or (TGF / TGF / VGF) was combined in the donating vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene with the exception of the sequence QEEK modified from the natural human sequence QEDK. The carboxy end site was derived from the amino acid sequence of VGF and ended with the sequence YQR upstream of the natural sequence PNT. PI asmid pLEBam was degraded with BssHII and BamHI. BssHII-BamHI pLEBam was then ligated to oligonucleotides TGF 101, 102, 103 and 104 and VGF 1, 2, 3 and 4 using DNA ligase and the resulting pioms used for the transformation of competent HB101. The transformants were selected for ampicillin and sorted by restriction analysis using EcoRI, Ncol and BamHI. and by nucleotide sequencing using the Maxam-Gilbert 35 protocol. A correct build-up was designated as pLEBam / TTV.

8900724.

47 TGF *

Ncol

CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT MVVS HFND CPDSHTQFCF

5 VGF ->

Kpnl Sphl

TCATGGTAGCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT HGTCRFLVQEEKPACVCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT 10 HGYT GIRCQHVVLVDYQR

BamHI

TAAGGATCC

Ter 15

B. Preparation of pi asmide pLEBam / TVV

The synthetic chimeric growth factor, designated TVV or (TGF / VGF (VGF)) was combined in the cloning vector pLEBam This hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain of the gene. The central and C-terminal domains are derived from the truncated VGF sequence and terminate with the sequence YQR upstream of the natural sequence PNT Also the synthetic gene has the modification GYACVC for GMYCRC.

(A) Preparation of a 4.3kb KpnI-Sphl fragment from pLEBam / TVV PIasmid pLEBam / TVV was digested with kPnl-Sphl and the 4Kb KpnI-Sphl fragment was gel purified. This degradation removes the central TGF domain from the synthetic gene TVV in the cloning piasmid pLEBam.

(b) Ligation and isolation of pLEBam / TVV

Oligonucleotides VGFIOta and 102a were ligated to the 4.3kb KpnI-Sphl fragment of pLEBam / TTV using DNA ligase and the resulting mixture was used for transformation of competent HB101. The transformants were selected for oNpicillin and sorted by nucleotide sequencing using the Sanger dideoxy method. A correct build-up was isolated. 89 00 724.

35 48 taught by pLEBam / TVV TGF -

Ncol

5 CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT MVVSHFNDCPDSHTQFCF

VGF -►

Kpnl Sphl

TCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT

, "Hgtcihardidgyacvcs 10

EcoRI

catggctacactggttcgttgccagcatgttgttctgctcgactaccagcgt hgytgircqh ^ vlvdyqr

15 BamHI

TAAGGATCC Ter

Example IV

Expression of the polypeptide of interest as a fusion protein with the N protein

(A) Modified synthetic TGF

1. Preparation of pBM11 / N / TGF

The modified human TGF was expressed in this system as part of a fusion with the 33N terminal amino acids of the N gene and has the sequence QEEK replacing the human sequence QEDK.

(a) Preparation of a 780bp SphI-Pvul fragment from pBM11 / N / TTV PI as the pBM11 / N / TTV was degraded with Sphl and Pvul after which the 87bp SphI-Pvul fragment was gel purified. This fragment contains a portion of the pBM11 plasmid at the Pvul end and at the Sphl end, the N gene and the N-terminal two-thirds of the human TGF gene.

(b) Preparation of the 5kb BamHLP fill fragment from pBM11 / N / TTV Plasmid pBM11 / N / TTV was digested with BamHI. and Pv.ul and the 5 kb BamHi-Pvul fragment was gel purified.

C. Ligation and Isolation of pBM11 / N / TGF

8900724.

49

Oligonucleotides TGF205 and 206, the 780 bp SphI-Pvul fragment and the 5 kb BamHI-PvuI fragment of pBMI1 / N / TTV were joined together and used for transformation of competent HB101. The transformants were selected for neomycin and sorted by restriction analysis using EcoRI and nucleotide sequencing according to the Sanger dideoxy method.

A correct build-up was designated as pBM11 / N / TGF.

N gene -

'u- ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA MDAQTRRRERRAEKQAQW K

BamHI

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCCGCAT

ADD PLVVVSAKPVRIRM

15 TGF -

GGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTTTCAT VVSHFNDCPDSHTQ FCFH

2ü Kpnl Sphl

GGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCCATTCTG GTCRF LVQEEK PACVCHSG

BamHI

GCTAGGTTGGCGCACGTTGCGAACACGCTGACCTGCTGGCTTAAGGATCC YVGARCEHADIiLA Ter 25 B. Modified synthetic TGF-VGF hybrid

1. Preparation of pBMI1 / N / TTV

In this construction, a synthetically modified TTV 3Q chimeric gene was expressed as the C-terminal part of a fusion protein with the first 33 amino acids of the N gene at the N-terminal. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal two thirds of the gene except for the sequence QEEK, which was altered from the natural human sequence QEDK. The carboxy end site was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

8900724 · 50 (a) Preparation of Ncol (blunt) -BamHI TTV synthetic gene Plasmid pLEBam / TTV was digested with Ncol and blunt ended by filling in the overhangs using the Klenow fragment of DNA polymerase. The DNA was then degraded with BamHI and the 170 bp Ncol (blunt) BamHI TTV fragment was gel purified.

(b) Preparation of BanHI degraded pBM11 Plasmid pBM11 was degraded with BamHI

10 (c) Ligation and Isolation of pBM11 / N / TTV

BamHI degraded pBM11, the Ncol (blunt) -BamHI TTV fragment and BamHI linkers (5'GATCCG3 ") were bound together using DNA ligase and the resulting mixture was used for transformation of competent HB101. The transformants were selected on neomycin and sorted by restriction analysis and nucleotide sequencing using the Sanger dideoxy method. A short build was isolated, designated pBM11 / N / TTV.

on N gene

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA MDAQTRR RERRAEKQAQWK

BamHI

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCCGCAT AANPLLVGVSA KPVRI RM

TG F ^

GGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTTTCAT

VVSHFNDCPDSHTQFCFH

... Kpnl Sphl VGF - * ·

JU GGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCTCATG GT CRFLVQEEKPACVC S H G

EcoRI

GCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGTTAAG YTGIRCQHVVLVDYQR Ter 35

BamHI

GATCC

8900724.

51

Example V

Preparation of the polypeptide of interest as a fusion protein with the N protein and a cleavage site A. Modified synthetic VGF 5 1. Preparation of pBff11 / NDP / VGFA:

The N-terminal sequence of the synthetic VGFA gene is a truncated version of the natural VGF sequence beginning with the DIPAIR sequence. In this plasmid, the VGFA fragment is placed downstream of 32 amino acids of the lambda N-protein 1 µ and the dipeptide aspartic proline. In order to preserve the Kpnl cloning site, the synthetic sequence was changed to encode CLHCGTC instead of the natural VGF sequence CLHGDG and it ends with the sequence IQR upstream of the natural sequence PNT. Also, the VGFA gene encodes the sequence GYACVC which replaces the natural sequence GMYCRC.

a. Preparation of a KpnI-BamHI 80bp C-terminal fragment of the synthetic VGF gene PI asmid pLEBam / TVV was degraded with Kpn1 and BanHI and the 8o bp KpnI-BamHI fragment was gel purified.

This fragment contains the C-terminal two-thirds of the synthetic VGF gene with the Kpnl site located at the 5 'end.

b. Preparation of BamHI degraded dephosphorylated pBam! 1 Plasmid pBM11 / N / TTV was degraded with BamHI and the 5'-phosphates removed by treatment with calf intestinal alkaline phosphatase. The 5.6 kp BamHI plasmid fragment was gel purified.

30 C. Ligation and Isolation of pBM11 / NDP / VGFA

Oligonucleotides VGF 103a, 104a, the 5.6 kb BamHI fragment of pBM11 and the 80 bp KpnI-BamHI fragment of pLEBam / TVV were bound together using DNA ligase and then used to transform competent HB101. The transformants were selected for neomycin and sorted by restriction analysis using C11 al and nucleotide sequencing according to the Sanger-dideoxy technique.

8900724.

5 52

A short build was isolated, designated pBM11 / NDP / VGFA. This construction has the orders GTC and GYACVC instead of the authentic VGF orders GDC and GMYCRC.

N gene - * ·

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA

MDAQTRRRERRAEKQAQWK

* * * * 10 Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC A ANPLLVGVS AKPVRI DP

Ncol VGF

CCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT

MDIPAIRLCGPEGDGYCL

15

Kpnl Sphl

GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT HGTCIHAR DIDG YACVCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT H GYTG IRCQHVVLVDYQR

BamHI

TAAGGATCC

Ter 25 2. Preparation of pBM11 / NDP / VGFa

The N-terminal sequence of VGFa is a truncated version of the natural VGF sequence and starts with the sequence DIPAIR.

30 The VGFa sequence also contains the changed orders GTC and GYACRC

instead of the natural VGF sequences GDC and GMYCRC. In this piasmid, the VGFa gene is located downstream of 32 amino acids of the lanbda N protein and the dipeptide aspartic proline. Treatment of the purified fusion protein with formic acid results in the cleavage at the acid labile aspartic proline peptide bond, allowing the VGFa protein to be separated from the lambda N-proteT.ne 8900724. "53 amino terminal site. The cleavage is such that the VGFa portein remains with the proline residue at the amino terminal site.

a. Preparation of Sphl degraded, dephosphorylated pBM11 / DP / VGFA

Plasmid pBM11 / DP / VGFA (10 microg.) Was degraded with 30 units of Sphl and the 5'-phosphates were removed from calf intestines by treatment with alkaline phosphatase. The 5 kb piasmid fragment was collected after electrophoresis on an agarose gel.

b. Preparation of an EcoRI-Sphl 70bp fragment from pBMI1 / DP / VGFA

Plasmid pBM11 / DP / VGFA (10 microg) was digested with 30 units of EcoRI and then with 30 units of Sphl. The 70 bp fragment was collected after electrophoresis on an agarose gel.

3. Ligation and isolation of pBMU / NDP / VGFa

The 24 bp fragment containing oligonucleotides VGF 1a and 2a, the 5 kb Sphl fragment and the 70 bp EcoRI-Sphl fragment of pBM11 / DP / VGFA were bound together and the mixture was used to transform the competent E.coli HB101 cells. The transformants were sorted by nucleotide sequencing using the Sanger dideoxynucleotide method. A correct clone designated as pBM11 / NDP / VGFa was isolated.

N gene - *

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA

MDAQTRRRERRAEKQAQWK

25 * * * *

Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATCC

ADDLVGVSAKPVRIDP

Ncol VGF -

30 CCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT MDIPAIRLCGPEGDGYCL

Kpnl Sphl

GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCTCT HG T C I HARD I DGYACRCS

35 89 0 0 724. ' 54

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT HGYTG IRCQHVVLVDYQ R

BamHI 5 TAAGGATCC Ter B. Modified synthetic TGF-VGF hybrids

1. Preparation of pBM11 / NDP / TTV

In this construction, the synthetically modified TTV becomes chimeric. gene expressed as the C-terminal portion of the fusion protein with the first 32 amino acids of the N gene at the N-terminal site. An acid-labile aspartic proline dipeptide separates the two parts of the fusion. The hybrid growth factor contains the amino acid sequence of human TGF in the amino-terminal two-thirds of the gene except for the sequence QEEK that was altered from the natural human TMR sequence QEDK. The carboxy end site was derived from the amino acid sequence of VGF and ended with the order YQR upstream of the natural order PNT 20 a. Preparation of 5kb Ncol pBM11 piasmi the fragment PI asmi the pBM11 / NDP / VGFA was degraded with Ncol and the 5kb Ncol piasmi defragment was gel purified. This fragment has an Ncol overhang at the aspartic proline cleavage site downstream of the sequences encoding the first 32 amino acids of the N gene. The other Ncol site is in the neomycin resistance gene.

b. Preparation of 0.6 kb NcoI-BamHI pBM11 fragment Plasmid pBM11 / N / TTV was digested with Ncol and BamHI and the 0.6 kb NcoI-BamHI plasmid fragment was gel purified. This fragment contains the Ncol overhang in the neomycin resistance gene.

c. Preparation of the 170 bp synthetic TGF / TGF / VGF fragment Plasmid pLEBam / TTV was digested with Ncol and BamHI and the

NcoI-BamHI 17Qbp fragment containing the TGF / TGF / VGF synthetic gene 35 was gel purified. This fragment has the

Ncol overhang at the 5 'end of the gene and the BamHI. overhang at the 3 'end of the gene.

89 0 0724.

55

d. Ligation and isolation of pBM11 / NDP / TTV

The 5kb Ncol and the 0.6 kb NcoI-BamHI plasmid fragments were bound to the 170 bp NcoI-BamHI TTV gene using DNA ligase and the resulting mixture was used for the transformation of competent HB101. The transformants were selected for neomycin such that only colonies with properly reconstructed neomycin resistant genes could survive. The transformants were sorted by restriction analysis with Ncol and nucleotide sequencing using the Sanger-dideoxy technique. Correct build was isolated and designated pBM11 / NDP / TTG.

N gene - *

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA 15 MDA QTRRRERRAEKQAQWK

Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC AANP LLVGVSAKPVR I DP

20 Ncol TGF -

CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT MVVSHFN D CPDSHTQFCF

Kpnl Sphl VGF -

TCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT 25 HGTCRFLVQEEKPACVCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT HG YTGIRCQHVVLVDYQR

30 BamHI

TAAGGATCC Ter

2. Preparation of pBM11 / NDP / VTV ^ In this construction, the synthetically modified VTV

chimeric gene expressed as the C-terminal part of a fusion protein of which the first 32 amino acids of the N gene are located 8900724.

57, located at the N-terminus. An acid labile aspartic proline dipeptide separates the two parts of the fusion. The hybrid growth factor contains the amino acid sequence of human TGF in the central domain, the amino acid sequence QEEK replacing the natural sequence QEDK. The N-terminal and C-terminal domains were derived from the truncated VGF order and start with the order DIPAIR and end with the order YQR located upstream of the natural order PNT, a. Preparation of a 5kb BamHI-Ncol fragment of pBMH 10 Plasmid pBM11 / N / TTV was digested with BamHI and Ncol and the 5 kb BamHI-Ncol fragment was gel purified.

This fragment contained BamHI overhang at the 3 'end of the sequences encoding the first 32 amino acids of the N gene and an Ncol site in the neomycin resistance gene.

15 b. Preparation of a 700 kb KpnI-Ncol fragment from pBM11 / N / TTV Plasmid pBM11 / N / TTV was digested with KpnI and Ncol and the 700 bp KpnI-Ncol fragment was gel purified.

This fragment is made from a portion of plasmid pBM11 containing a portion of the neomycin resistance gene in the Ncol over-Lj hanging. And the C-terminal VGF domain of the TTV synthetic gene in the Kpnl overhang,

c. Ligation and isolation of pBM11 / N / DP / VTV

Qligonucleotides VGF 103a and 104a, the 5kb BamHI-Ncol fragment of pBM11 and the 700 bp KpnI-Ncol fragment of pBM11 / N / TTV 25 were bound together using DNA ligase and then used to transform competent HB101. The transformants were selected for neomycin and sorted by restriction analysis using Clal and nucleotide sequencing according to the Sangerideideoxy technique.

30 N gene -1

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA

mdaqtrrrerraekqaqwk 8900724.

1 1 1 35 - Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC

addlvvsakpvridp 58

Ncol VGF

CCATGGACATCCCGGCTATCCGTCTGTGCGG.CCCGGAAGGCGACGGCTACTGCCT

MDIPAIRLCGPEGDGYCL

Kpnl TGF '- S £ hl VGF -

5 GCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT HGTCRFL VQEEKPACVCS

EcoRI

CATGGCTACACTGGAATTCGTTCGCAGCATGTTGTTCTGGTCGACTACCAGCGT

HGYTGIRCQHVVLVDYQR

10

BamHX

TAAGGATCC

Ter

3. Preparation of pBM16 / NDP / TVV

In this construction, the synthetically modified TVV chimeric gene was expressed as the C-terminal portion of a fusion protein, the first 32 amino acids of the N gene of which are located at the N-terminal portion. The two parts of the fusion are separated by an acid-labile aspartic-proline dipeptide 2Q. The hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain. The central and C-terminal domains were derived from the truncated VGF sequence and end with the sequence YQR. . Also, the synthetic gene has the modification GYACVC in place of GMYCRC.

a. Preparation of 4.3kb NcoI-BgII fragment from pBMi1 / NDP / VGFA Plasmid pBMi1 / NDP / VGFA was digested with Ncol and Bg1II

and the 4.3 kb fragment was gel purified. The Ncol overhang is located at the aspartic acid-proline cleavage site 3Q just downstream of the first 32 amino acids of the N gene.

b. Preparation of the 1.2kb BamHI-Bg1II fragment from pBM11M5 Plasmid pBM11M5 was digested with BamHI and BgtII. and gel purifying the 1.2kb fragment. This fragment differs 33 from the normal pBM11 fragment in that the Ncol. site in the neo-mycine resistance gene has been deleted, with all subsequent vectors lacking this Ncol site designated pBM16.

c. Preparation of the 170bp NcoI-BamHI. TTV synthetic gene S9 00724 ..

59

Plasmid pLEBam / TVV was degraded with Ncol and BamHI and the 17Übp NcoI-BamHI fragment was gel purified. This synthetic gene fragment contains the Ncol site at the 5 'end and the BamHI site at the 3' end.

5 d. Ligation and isolation of pBMl6 / NDP / TVV

The 4.3kb NcoI-BglII fragment of pBM11 / NDP / VGFA and 1 2kb BamHI-BglII fragment of pB1lM5 and the 170 bp NcoI-BamHI TVV synthetic gene fragment were bound together using DNA ligase and the resulting mixture was used for trans-10 formation of competent HB101. The transformants were selected for neomycin and sorted by restriction analysis and nucleotide sequencing using the Sanger dideoxy technique. The plasmid is designated pBM16 to indicate the loss of the Ncol restriction site in the neomycin resistance gene.

15 N gene +

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA

MDAQTRRRERRAEKQAQWK

20 * * * *

Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC

ADDLVGVSAKPVRIDP

Ncol TGF

25 CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT

MVVSHFNDCPDSHTQFCF

/

Kpnl VGF - Sphl TCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT HGTCIHA. RDIDGYACVCS 30

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT

HGYTGIRCQHVVLVDYQR

oc BamHI

TAAGGATCC Ter 8100724.

60

C. Synthetic EGF

1. Preparation of pBM11 / NDP / EGF

In this construction, the human EGF gene is expressed as part of a fusion with the 32 N-terminal amino acids of the N gene 5 located downstream of an Asp-Pro cleavage site.

a. Preparation of a 5kb Ncol fragment of pBM11

Piasmid pBMI1 / DP / VGFA was degraded with Ncol and the 5'-phosphates were removed by treating alkaline phosphatase from calf intestines. The 5kb plasmid fragment was gel purified. This fragment had an Ncol overhang at the Asp-Pro cleavage site downstream of the sequences encoding the first 32 amino acids of the N gene. The other Ncol site is in the neomycin resistance gene.

b. Preparation of a 0.6 kb NcoI-BamHI fragment from pBM11

PI asmi the pBM11 / N / TTV was degraded with Ncol and BamHI

and the 0.6kb NcoI-BamHI plasmid fragment was gel purified. This fragment contains Ncol overhang in the neomycin resistance gene.

c. Ligation and isolation of pBM11 / DP / EGF

The three sets of attached EGF oligonucleotides with an Ncol overhang at the 51-end and a BamHI overhang at the 3 'end, the 5kb Ncol fragment of pBM11 and the 0.6kb NcoI-BamHI fragment of pBM11 were bound together using of T4 DNA ligase and the resulting mixture was used to transform competent E. coli HB101. The transformants were chosen for neomycin in such a way that only colonies of the newly reconstructed neomycin resistance gene would survive.

The transformants were sorted by restriction analysis using EcoRI and BamHI and by DNA sequencing described above.

N gene - * ·

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGCGTCAATGGA

MDAQTRRRERRAEKQAQWK

35 ***

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATCC AA N PLLVGVSAKPVR I DP

89 007 2.4.

61 EGF1 * ECORI EGF2 ^

CATGAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCATGAC M NSDSECPLSHDGYCLHD

EGF3 - 5 Nsil Sphl

GGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTTGTTG GVC MY I E ALDKYACN CVVG

GCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTA

YIGERCQYRDLKWWELR * 10.

BamHI

AGGATCC

Example VI 1S -

Preparation of the polypeptide of interest as a fusion protein with a modified alkaline phosphatase signal sequence

A. Preparation of pBM11 / PAD / EGF

Synthetic oligonucleotides were designed to insert DNA encoding a modified alkaline phosphatase signal peptide and a linker region with 3 cloning sites (Hind III, Smal and BamHI) into the pBM11 expression vector downstream of the P ^ promoter and N -gen ribosomal binding site. The nucleotide sequence was optimized to be as close as possible to the nucleotide sequence of the ami.no end site of lamb days since the 1amb day sequence was developed with that of its ribosomal binding site for efficient ribosome initiation and translation. Also, the second amino acid of the alkaline phosphatase signal sequence, the basic amino acid ly-Sine, was changed to an acidic amino acid, aspartic acid.

30

1. Preparation of Q, 17kb EcoRI-BamHI fragment from EGF

Plasmid pBM11 / NDP / EGF (30 µg) was digested with 30 units of EcoRI and then treated with four units of K] enow fragment of DNA polymerase to create blunt ends. The DNA was finally broken down with 30 units of BamHI and the 0.17 kb fragment of the E6F gene was collected after electrophoresis on agarose gel. The DNA thus purified has a blunt EcoRI site 830072462 at the 5 'end and a BamHI overhang at a 3' end.

2. Preparation of a 0.5 kb Pvu I-HindIII fragment from pBM11 / PAD Plasmid pBM11 / PAD (18 µg) was degraded by 30 units

Hindii and then treated with Klenow fragment to make the ends tacky. The DNA was then degraded with Pvul and the 0.5 kb Pvul-Hind11 (blunt) fragment was collected after electrophoresis on an agarose gel.

3. Preparation of the 5.2 kb PvuI-BamHI fragment from pBMU / PAD Plasmid pBM11 / PAD (18 µg) was digested with 30 units of 10 Pvul followed by 30 units of BamHI. The 5.2 kb fragment was recovered. Electrophoresis on an agarose gel.

4. Ligation and isolation of pBM11 / PAD / EGF

The 0.17kb EcoRI (blunt) -BamHI fragment, the 0.5kb Pvul-HindII (blunt) fragment in the 5.2kb PvuI-BamHI fragment were bound together and the resulting mixture was used for transformation of competent E.coli HB101. The transformants were sorted by DNA sequencing as described above. The desired signal sequence / EGF region had the following sequence: 20 Signal sequence

ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTCACT

MDQSTIALALLPLLFT

EGF

o5 CCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGAC

PVTKANSPSECPLSHD

Nsil

GGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTG

GYCLHDGVCMYIEAL

30

Sphl

GACAAGTACGCATGCAACTGCGTTGTTGGCTACATCGGCGAACGT DKYACNCVV GYIGER

BamHI

or TGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTAAGGATCC

CQYRDLKWWELR * 8900724.

63

The efficiency of foreign protein production in the pBM11 / PAD expression system and the ability to purify functionally active foreign proteins from the fusion product has been illustrated using pBM11 / PAD / EGF as an example. After size exclusion (chromatography / TSK-250), 10.3 mg equivalents of active EGF fusion polypeptide from 23 g (8 liters) of E. coli were de-repressed to express the EGF gene. 40% of the EGF activity was derived from EDF split from the signal sequence.

B. Preparation of pBM11 / PAD / nVGFa

Synthetic oligonucleotides were designed to couple the VGFa synthetic gene with an alkaline phosphatase-modified signal sequence to provide an optimal signal sequence cleavage site by encoding the additional N-terminal 13 residues immediately downstream of the signal sequence cleavage site. the natural VGF, indicated as above as the extreme N-terminus. The nVGFa sequence contains the modified sequences GTC and GYACAC instead of the natural VGF sequences GDC and GMYCSC and ends with sequence YQR upstream of the 20 natural sequence PNT. In this expression system, for the majority of molecules, the signal sequence remains attached to the nVGFa and forms a fusion protein with nVGFa at the C-terminal site.

1. Preparation of Q, 5kb HindlII-PvuI degraded with pBM11 / PAD Plasmid pBM11 / PAD was degraded with HindlII and Pvul and gel purified the 0.5kb fragment. The HindII site is placed at the C-terminal site of the modified alkaline phosphatase signal sequence.

2. Preparation of the 5.2kb pvul-BamHI pBM11 plasmid fragment Plasmid pBM11 / NDP / VGFa was degraded with Pvul and BamHI

3u and the 5.2kb piasmid fragment gel purified.

3. Preparation of the 170bp Ncol (blunt) -BamHI synthetic VGFa gene

Plasmid pBM11 / NDP / VGFa was degraded with Ncol and the 51 overhangs were removed by treatment with S1 nuclease.

This created a blunt sticky end at the first codon of the VGFa truncated synthetic gene. The DNA was then digested with BamHI and the 170bp Ncol (blunt) -BamHI fragment was gel purified.

8900724.! 64 4. Ligation and Isolation of pBMl1 / PAD / nVGFa 01igonucleotides VGF105 and 106, the 0.5kb HindlII-PvuI fragment of pBM11 / PAD, the 5.2kb PvuI-BamHI pBMI'1 fragment and the 170bp Ncol (blunt) -BamHi synthetic VGFa gene were bound together using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected for neomycin and sorted by restriction analysis and nucleotide sequencing using the Sanger -ideoxy technique. A correct structure was isolated containing the modified alkaline phosphatase signal sequence under the nVGFa gene.

Signal sequence

ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA

MDQSTIALALLPLLFTPV T

15 nVGF -

CAAAAGCTGACTCTGGTAACGCTATCGAAACTACTTCTCCGGAAATCACTAACGC KADSGNAIETT SPEITNA

2 0 TACTACTGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGC TTDIPAIRLCGPEGDGYC

Kpnl Sphl

CTGCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCT LHG'TCIHARDIDGYAC RCS

25

EcoRI

CTCATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCG

«W Y T G I RCQHVV L V DYQR BamHI

30 TAGAGGATCC Ter

Example VII

Expression of the polypeptide of interest as a fusion protein with an alkaline phosphatase signal sequence ^ A. Preparation of pBM11 / PAK / nVGFa (alkaline phosphatase signal sequence / nVGFa with natural VGF N-t terminal site and sequences GTC and GYACRC) 8900724.

65 PI asmid pBM11 / PAD / nVGF was mutagenized in vitro (Mori-naga et al., Biotechnology (1984) 2: 636-643) to modify the codons encoding the second amino acid in the signal sequence, i.e., for back-altering of the Asp (D) codon in 5 that for Lys (K), the residue found in the natural order. Mutagenesis also introduced a pVul site in the signal sequence.

Signal sequence:

1 ° ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTeACTCCAGTGACAAAA

mdqstialallpllftpvtk nVGF - gctgactctggtaacgctatcgaaactacttctccggaaatcactaacgctactact adsgnaiettspeitnatt 15

Kpnl gacatcccggctatccgtctgtgcggcccggaaggcgacggctactgcctgcatggt dipairlcgpegdgyclhg 00 sphl L ü acctgcatccatgcacgtgacatcgacggctacgcatgccgttgctaccatggctacactrccgg

BamHI

ggttcgttgccagcatgttgttctggtcgactaccagcgttaaggatcc GIRCQHVVLVDYQR Ter 25

B. Preparation of pBM11 / PAK / EGF

In this expression cassette, the EGF gene is part of a fusion with the alkaline phosphatase signal sequence. Plasmid pBM11 / 3/1 PAD / EGF was mutagenized in vitro to modify the codons encoding the second amino acid in the signal sequence, modifying the Asp (D) codon to that for Lys (K), the residue found in the natural order. This mutagenesis also introduced a Pvul site in the signal sequence.

89 00724.

* 66 Signal sequence - *

ATGAAACAATCTACGATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA MKQSTIALALLPLL F TPVT

5 EGP ♦

CAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCA OPPORTUNITY SECPLSHDGY CLH

Nsil Sphl

TGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTT

Q dgvcmyiealdkya cncv

GTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC

VGYIGERCQYRDLKWWELR

BamHI

GTTAAGGATCC * C. Preparation of TacPak / EGF (alkaline phosphiaia .. signal sequence / human EGF).

. 1. Preparation of Plasmid Fragments

Plasmid p 135.-1 was derived from plasmid pDR540 (Pharmacia) and contained hsfc Cro gene SD and / Bg111 site downstream of the lac SD. pDR540 is an expression vector containing the trp-lac hybrid promoter, pl35-1 was digested with Bg111 and BamHI and treated with bacterial alkaline phosphase.

c O

Plasmid pBMI1 / PAK / EGF was digested with PvuII and BamHI and the approximately 230bp fragment encoding part of the alkaline phosphatasial sequence and human EGF was isolated.

2. Preparation of TacPak 1 and TacPak 2 oligonucleotides 20 Synthetic oligonucleotides TacPak 1 and TacPak 2 were designed with a pendant compatible with the Bg1II site of p135-1 and a PvuII site compatible with the PvuII site in the alkaline phosphatase / EGF PvuII / BamHI fragment. The oligonucleotides were synthesized with an oligonucleotide synthesizer from Applied Biosystems.

BqlII PVUI

~ T “T

TacPakl 5 'GATCTATGAAACAATCTACGAT 3'

TacPak2 3 'ATACTTTGTTAGATGC 5' 8900724.

67 3. Ligation and Isolation of TacPak / EGF Clone

The BgIII-BamHI degraded p135-1, the 230 bp PAK / EGF fragment and oligonucleotides TacPak 1 and TacPak 2 were bound using DNA ligase, transformed into competent HBIüt and a correct structure was isolated by DNA sequencing.

(Hindlll instead of pDRS40)

10 AAGCTTACTCCC

trp-35 (16bp) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG)

mRNA 5 'lacl binding site lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC {AGGA} AACAGGATCACTA

Pvul

croSD (11bp) BglII {AGGA} GGTTCAGATCT

20

Signal sequence *. ->

ATGAAACAATCTACGATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA M KQS T IALALLPLLFTPVT

EGF -> 2 5 CAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCA '

OPPORTUNITY CPLSHDGYCLH

Nsil Sphl

TGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTT DGVCMYIEALDKYACNCV

30

GTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC

VGYIGERCQYRDLKWWELR

BaraHI

GTTAAGGATCC

35 * 6900724.

Μ 68

Example VIII

Expression of a polypeptide of interest as a fusion protein with the alkaline phosphatase signal sequence using an expression cassette comprising a transcription termination region 5

A. Preparation of pTCPt / EGF (rtrp-357l6bp / ac-10) / lacSDJ / l1bp- & TgJ (alkaline phosphatase signal / human EGF / trans.ter.-NEO

This plasmid is designed to access the tac promoter elements and use the cro SD to express human EGF behind the alkaline phosphatase signal sequence. It has a pBR322 background with the neomycin resistance gene.

1. Preparation of the 420bp HindIII (blunt) -BamHI fragment of TacPak / EGF

TacPak / EGF was degraded with HindII and then treated with the Klenow fragment of DNA polymerase to create blunt ends. The DNA was then degraded with BamHI and the 420 bp fragment containing the tac promoter elements and the coding region for the alkaline phosphatasial sequence and human EGF 20 was isolated by agarose gel electrophoresis.

2. Preparation of the 2.8kb EcoRI (blunt) -BamHI fragment from pBM16t / NDP / VGFa pBM16t / NDP / VGFa was digested with EcoRI and then treated with Klenow to create blunt ends. The DNA was then digested with BamHI and the 2.8 kb fragment isolated. This DNA fragment contains the pBR322 origin, the neomycin resistance gene from which the Ncol site has been removed, and the 322-like transcription terminator downstream of the BamHI site.

3. Ligation and isolation of pTCPt / EGF

The 2.8 kb EcoRI (blunt) -BamHI fragment of pBM16t / NDP / VGFa was bound to the 420bp HindIII (blunt) -BamHI fragment of TacPak / EGF and the resulting DNA was used to transform competent JM109 (lac1q). Correct build-up was isolated due to its resistance to neomycin and DNA sequencing.

89 0 072 4, "69 (Hindlll instead of PDR54 °)

AAGCTTACTCCC

trp-35 (16bp) lac-10 5 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG)

mRNA 5 'lac1. binding site lacSD

tgtgg / aattgtg agcggataacaatttcacac {agga} aacaggatcacta 10 croSD (Hbp) lgTlI s1 «« Ml order -,> {agga} ggttcagatct atgaaacaatctacgatcgccctcgcacttctcc MKQS TIALALLP EG F ->

CACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGTC

llftpvtkansdsecpls 15

Nsil

TCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGAC

HDGYCLHDGVCMYIEALD

20 Sphl

AAGTACGCATGCAACTGCGTTGTTGGCTACATCGGC KYAC NCVVGYIG

BamHI

GAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGA „ERCQYRDLKW WELR * 25

Trans. Term.

CTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATC

3ü B · Preparation of pTCPt / nVGFa (ftrp ~ 35j 16bp / ïac-1 oli ac $ pj (croSQ? Hbp / ATGj / alkaline phosphatase signal / n-terminal VGFa with the order GTC and GYACRC) /trans.term.-NEP

This plasmid contains the tac promoter elements and uses the cro SD for expression of the modified VGF gene with the N-terminal extension downstream of the alkaline phosphatase 1, 10 val. order. The plasmid has a pBR322 background with the neo-8900724.

70 * mycine resistance gene.

1. Preparation of the 350bp PvuI-BamHI fragment from ρΒΜΊΤ / ΡΑΚ / nVGFa

Plasmid pBM11 / PAK / nVGFa was digested with Pv.ul and BamHI 5 and the 350bp fragment was isolated by gel electrophoresis. This fragment contains the majority of the alkaline phosphatases and the nVGFa gene.

2. Preparation of the 2.8kb Pvul-BamHI fragment from pTCPt / EGF Plasmid pTCPt / EGF was digested with Pvul and BamHI and the 2.8kb fragment was isolated by gel electrophoresis.

3. Li gation and isolation of PtCPt / nVGFa

The 2.8kb fragment and the 350bp fragment were joined together using DNA ligase and the DNA was used to transform competent JM109 (lac1q). Correct construction was isolated by restriction analysis.

(HindlII instead of pDR540) AAGCTTACTCCC

20 trp-35 (16bp) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG)

mRNA 5 'lacï binoinase site lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC {AGGA} AACAGGATCACTA

25 Pvul croSD (llbp) BglII .signal sheet no ^ da -> {agga} ggttcagatct atgaaAcaa ^ ctacgatcgccctcgcacttctccc

Q K Q S T I A L A L L P

nVGF -> 30 actgctgttcactccagtgacaaaagctgactctggtaacgctatcgaaactact

LLFTPVTKADSGNAIETT

tctccggaaatcactaacgctactactgacatcccggctatccgtctgtgcggcc

SPEITNATTDIPA IRLCGP

Kpnl

CGGAAGGCGACGGCTACTGCCTGCATGGTACCTGCATCCATGCACGTGACATCGA egdgy CLHGTC IHARD I D

09 00 72 4; * 71

SphI EcoRI

CGGCTACGCATGCCGTTGCTCTCATGGCTACACTGGAATTCGTTGCCAGCATGTT GYACRCSHGYTGIRCQHV

BamHI Trans.

5 GTTCTGGTCGACTACCAGCGTTAAGGATCCGTGACTAATTGGGGACCCTAGAGGT VLVDYQR *

Term.

CCCCTTTTTTATTTTAAAACGATC

10 C. Preparation of pTNPt / EGF (Ztrp-357l7bp / lac-10 / AnSPjobpATGj7 / alkaline phosphatase signal / human EGF / trans.term.-ΝΕΟ

This plasmid is designed to have access to the tac promoter elements and to use the N gene SD for expression of human EGF behind the alkaline phosphatase signal sequence. It has the pBR322 background with the neomycin resistance gene.

1. Preparation of 2.8kb PvuI-BamHI pTNPt

Plasmid pTNPt was digested with Pvul and BamHI and the 2.8 kb fragment was isolated by gel electrophoresis.

2. Preparation of 3QQbp Pvul-BamHI fragment from pBMI1 / PAK /

EGF

Plasmid pBM11 / PAK / EGF was digested with Pvul and BamHI and the 300bp fragment was isolated.

25 3 · Ligation and isolation of pTNPt / EGF

The 2.8 fragment and the 300bp fragment were bound together using DNA ligase and the DNA was transformed into competent JM109 (lac1q). Correct construction was isolated by restriction analysis and by DNA sequencing.

30 (EcoRI instead of P ^ Mll)

GOESTACTCCCCATCC

35 SstI

trp-35 (17bp) lac-10 5'lac

CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG) TGTGG / AATTG

8900714. "72

BsmI

mRNA-> * n mRNA->

TGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCTTTGGGGTGTGT

GATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGCTGCCAATGTGC

5

CAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACACACCACCAAAGCTAA

Pvul nSD (8bp) signal passed _>

10 CTGAC {AGGA} GAATCCAG ATGAAACAATCTACGATCGCCCTCGCACTTCTC

MKQSTIALALL

EGF ->

CCACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGT PLLFTPV TKAN SD SECPL S

15

Nsil

CTCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGA HDGYCLHDGVCMY I EALD

Sphl

2 o CAAGTACGCATGCAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCAGTACCGT

KYACN CVVGY IGE RCQYR

BamHI BamHI

GACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGACTAATTGGGGA D L K W W E L R * 25 (BamHI instead of pBMll)

Trans. Term. I

CCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC

Example IX

Combination of growth factor genes for expression in eukaryotic cells

A. Preparation of piasmi de pRSV / VGF

Transfection of this plasmid into Chinese hamster ovary cells resulted in the production of natural VGF,

(a) Preparation of HindII (blunt) -BglII (blunt) pRSV

89007247 * 73

Plasmid pRSV was digested with HindII and BgtII and the 4kb fragment was gel purified. The protruding ends were blunted with the Klenow fragment of DNA polymerase, after which the 5'-phosphates were removed with calf intestinal alkaline phosphatase.

(b) Preparation of Ddel fragment containing the VGF gene

A subcloned gonomic fragment of vaccinia DNA was digested with Ddel, the protruding ends blunted with the Klenow fragment, and a 550bp dDel fragment was subjected to gel-10 purification.

(c) Ligation and Isolation of pRSV / VGF. .

The 4kb HindII (blunt) -BglII (blunt) fragment of pRSV and the 550bp Ddel (blunt) fragment of vaccinia DNA were bound together using DNA ligase and the transformants were selected with ampicillin and sorted by restriction analysis.

A correct build-up was designated as pRSV / VGF.

(d) Transfection of CHQ cells by pRSV / VGF

Plasmid pRSV / VGF was co-transfected with a plasmid 20 pRSV by calcium phosphate precipitation in Chinese hamster ovary (CHO) cells. The transfectants were sorted by Southern dot blot hybridization and a positive clone designated pRSV / VGF 52 was isolated.

B. Preparation of plasmid pAC / VGF

An additional expression system used to express the VGF recombinant protein is an insect system. See Maeda et al and Carbonell et al. » supra. In such a system, Autographa californica nuclear polyhydrosis virus 30 (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells.

The envelope gene can be donated in non-essential regions (e.g., the poly-hedrin gene) of the virus and is placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

Successful insertion of the VGF gene construction results in the inactivation of the polyhedrin gene and the production of unoccluded recombinant virus (i.e. virus containing the protein-like coat 8900724.

ύ 74 which is encoded by the polyhedrin gene is missing). These recombinant viruses are then used to infect Spodoptera frugiperda cells expressing the introduced gene. The VGF gene of interest was placed under control of a suitable insect cell system promoter. PI asmi de pAc610 contains the polyhedrine gene donated in a plasmid vector which has an ampicillin resistance marker. Poly couplers are introduced into this gene which are present 50 bases downstream of the transcription start site of the polyhedrin gene and 7 bases before the first ATG 10. The VGF gene is donated to a suitable poly coupler site so that it is under the control of the polyhedrin promoter. The ATG initiation methionine codon and the translation term codons are those of the VGF gene; the transcription start sites and polyadenylation signals are those of the polyhedrin gene.

(A) Preparation of Smal-BamHI pAc610.

PlaStfide pAc610 was digested with Sma I and BamHI.

(b) Preparation of HindIII (blunt) -BglII fragment of the VGF gene.

Plasmid pRSV / VGF was digested with HindII and the protruding ends blunted with the Klenow fragment. The DNA was then digested with BglII and the 560bp fragment gel purified.

(c) Ligation and Isolation of pAc / VGF

The Smal-BamHI fragment of pAc610 and the 560bp fragment of pRSV / VGF containing the VGF gene were joined together using DNA ligase and the transformants were sorted by restriction analysis. A short build-up called pAc / VGF was isolated.

(d) Transfection of Sf9 cells with pAc / VGF

3U Plasmid pAc / VGF was co-transfected with AcNPV wild-type bacilovirus DNA in Sf9 insect cells (Spodoptera frigiperda).

The transfectants were sorted for occlusion negative phenotype in plaque assays. An occlusive negative plaque was isolated, and after 5 rounds of successful plaque purification, a high titer recombinant virus stock was prepared.

C. Preparation of pAc / SFGF

69 0 0 7 2 k; β 75

Baculovirus expression of the natural Shope fibroma virus growth factor.

(a) Preparation of BamHI degraded pAc610.

PI asmide pAc610 was degraded with BamHI and Smal. The Sma I site in this plasmid is located downstream of the baculovirus poly-hedrin gene promoter.

(b) Preparation of a 430bp HindII-Sau3a fragment of the Shope fibromavirus genomic DNA

The 3.7kb BgtII fragment of the 19kb BamHL C fragment from 1µ Shope fibromavirus genomic DNA was donated into a BamHI degraded SP64 plasmid and designated SP64 / 3.7. Plasmid SP64 / 3.7 was digested with Hindi and the ikb Hindi fragment was gel purified. The Ikb fragment was then digested with Sau3a and the 43Gbp HincII-Sau3a fragment was gel purified.

(c) Ligation and Isolation of pAc / SFGF

The BamHI-Narrow degraded pAc610 was bound to the 430bp HincII-Sau3a fragment containing the SFGF gene and the resulting mixture transformed into competent E.coli HB101. The transformants were sorted by restriction analysis and a correct structure was isolated and indicated as pAc / SFGF.

Example X.

Preparation of polypeptides A. Solid phase synthesis of VGF and TGF

1. Synthetic VGF fragment.

The order corresponding to truncated VGFe starting with the order DIPAIR and ending with the order LVDY was combined with a peptide synthesizer from Apllied Biosystems Model 43GA 30 using the standard protocol. Treatment of the final peptide resin with liquid HF under standard "low-high" conditions gave the crude peptide without a protecting group. The peptide was subjected to chromatofocusing on PBE 94 (

Pharmacia). The partially purified peptide was eluted at pH 6.5. Brief treatment with 0.2 N sodium hydroxide removed the cysteine protecting groups (ethyl carbamoyl) and the peptide was oxidized in the presence of oxidized and reduced 8900724.

76 glutathione. Purification by gel filtration in HPLC gave highly purified VGF in the following order;

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSHGYTGIRCQHVVLVDY

5 2. Synthetic human and rat TGF-tx

Human and rat TGF-c * were chemically synthesized, essentially as described above for VGF, and supplied by Penninsula Labs.

B. Isolation of recombinant polypeptides prepared in prokaryotic cells 1. Growth factors cultured in pBM-based vectors using the PL promoter and the ts Cl respressor 15 E. Coli (HB101) containing the pBMU / NDP / growth factor p1 asmins. grown in Luria broth at 30 ° C. The culture density was measured at 550 nm and after the density reached an absorbance of 0.7 to 0.9, the synthesis of the growth factor fusion protein was initiated by raising the temperature to 42 ° C.

The culture was incubated at this temperature for 5-20 hours after which the bacteria were isolated by centrifugation and frozen at -70 ° C until use.

To isolate the recombinant protein, the cells were thawed in buffer containing 0.05 M NaH 2 PO 4 pH 7.2, 1.5 M NaCl, 0.01 M EDTA. One hundred and fifty ml of the buffer was used to prepare 50 g of bacteria. The cells were disrupted by sound waves on ice for 15 minutes using a 6mm probe, 50 bij pulse at 60 watts of power. After cell disruption, the insoluble protein was collected by centrifugation at 12,000 rpm in a GSA rotor for 90 minutes.

The cake containing the insoluble protein was then resuspended in 50 ml of 6 m guanidine hydrochloride. The insoluble material was collected in a Beckman type 30 ultracentrifuge rotor 35 by centrifugation for 2 hours at 25000 rpm. The top layer was collected and stored at -20 ° C until further consumption.

Purification of the fusion protein was performed with either a Sephacryl S300 or Fractogel HW-55 column equilibrated 0900724.

77 brought with 1 H guanidine hydrochloride. Fractions containing the fusion protein were identified as those containing a polypeptide having a molecular weight corresponding to the molecular weight of the polypeptide encoded by the synthetic gene 5 as determined on a 15% polyacrylamide urea gel. contain.

To obtain an active form of the recombinant growth factor, the fusion protein was refolled by placing it in 50 mM Tris-HCl buffer, pH 8.7, containing 1 M guanidine hydrochloride.

Incubate 1.25 mM reduced glutathione and 0.24 mM oxidized glutathione at 4 ° C for 3-10 days. The growth factor biological activity was monitored by a competitive receptor binding assay as described above (see Example I.C.). After a maximum level of activity was reached, the protein was calcined against distilled water and dried by freeze drying.

If it was desired to remove the main order, the protein was cleaved either by resuspending in 70% formic acid and incubating at 40 ° C for 3 days or by overnight in a 100-fold molar excess of cyanogen at room temperature incubate bromide. The cleaved product was dialized against distilled water and dried by freeze drying.

To further purify the recombinant growth factor, the growth factor was resuspended in 40% acetonitrile, 0.1% TFA and purified by HPLC using a BioRad TSK-250 column. Fraction containing the growth factor was pooled and further purified using reverse phase HPLC, either Waters y Bondapak C-18 or Rainin Dynamax C-8. The eluent was a linear gradient of 20-40% acetonitrile containing 0.1% TFA. Fractions containing receptor binding activity were pooled, lyophilized and stored at -20 ° C until use.

(A) T6F and modified TGF

(i) N / TGF

Recombinantly modified human TGF was produced from piasmi the pBM11 / N / TGF and contained 13 amino acids of the N gene at the N-terminal site and the sequence modification QEEK instead of the natural human sequence QEDK.

(b) Modified and truncated VGF

(i) PAD / nVGFa 8900724.

"78 o

Recombinantly modified VGF was produced from plasmid pBM11 / PAD / nVGFa containing the extreme N-terminal sequence of VGF and the modified sequence GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC. The nVGFa fragment was expressed as a fusion protein with a modified alkaline phosphatase signal sequence at the N-terminus and truncated at the order YQR at the C-terminus.

(ii) NDP / VGFa

Recombinantly modified VGF was produced from plasmid pBM11 / NDP / 10 VGFa starting in the D1 PAIR sequence and ending in the YQR sequence in VGF. It contains the modified sequences GTC and GYACRC instead of the natural VGF sequence GDC and GMYCRC.

The VGFa fragment was expressed as a 32 amino acid fusion protein of the N gene at the N terminus and the acid labile dipeptide aspartic proline.

(iii) VGFa

The VGF fragment was prepared as described under 2.b as above and after cleavage from the fusion protein by acid treatment, it was then further purified by HPLC.

(Iv) NDP / VGFA

Recombinantly modified VGF was produced from plasmid pBM11 / NDP / VGFA starting with the DIPAIR sequence and ending with the YQR sequence in VGF and with the modified sequences GTC and GYACVC instead of the natural VGF sequence GDC and GMYCAC. The VGFA 25 fragment was expressed as a 32 amino acid fusion protein of the N gene at the N-terminal site and the acid-labile dipeptide aspartic proline.

(c) Chimeric TGF / VGF hybrids (i) N / TTV (TGF / TGF / VGF) 30 Recombinantly modified TTV was produced from pBMH / N / TTV and contained the amino acid sequence of human TGF in the amino terminal two-thirds of the gene except of the order QEEK that was changed from the natural human order QEDK. The carboxy end site was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT. The TTV fragment was expressed as a 33 amino acid fusion protein of the N gene at the N-terminal site.

89 007 I.

79

(ii) NDP / TTV

Recombinant TTV was produced from plasmid pBMI1 / N / TTV and modified as described in (a) with the exception that the TTV fragment was expressed as a 32 amino acid fusion protein of the N gene at N terminus and the acid labile dipeptide aspar -Gic acid proline.

(iii) NDP / VTV

Recombinant modified VTV was produced from piasmi the pBM11 / NDP / VTV and contained the amino acid sequence of human TGF 10 in the central domain where the amino acid sequence QEEK replaced the natural sequence QEDK. The N-terminal and C-terminal domains were derived from the truncated VGF sequence and started with the order DlPAIR and ending with the order YQR. The VTV fragment was expressed as a 32 amino acid fusion protein of the 15 N gene at the N-terminal site and the acid-labile dipeptide aspartic acid proline.

(iv) NDP / TVV

Recombinantly modified VTV was produced from plasmid pBM11 / NDP / TVV and contained the amino acid sequence of human TGF in the N-terminal domain of the gene. The central C-terminal domains were derived from the truncated VGF sequence and ended with the sequence YQR. The synthetic gene also had the modification GYACVC for GMYCRC. The TVV fragment was expressed as a 32 amino acid fusion protein of the N gene at the N-terminal site and the acid labile dipeptide aspartic proline.

2. Growth factors produced in vectors comprising the tac or lac promoter

Bacterial hosts containing expression cassettes containing the. tac or lac promoters were grown at 30 to 37 ° C to an optical density of A600 = 0.2 to 0.8 in either LB broth or a chemically defined medium such as M9 medium supplemented with thiamine and glycose. A suitable antibiotic was included in the growth medium for the selection of hosts containing the expression cassette. The bacterial cultures were induced at a concentration of 100 to 1000 mM IPTG and es 0 0 72 4.

80 grown at 30 ° C for 16-24 hours. For the expression cassettes lacking a Tac I gene, the bacterial hosts carried an F factor with the lac ql gene, such as JM109, XL1, JM103, etc. For expression cassettes carrying the lac I gene, examples of bacterial hosts HB 101, DH1, DH5 etc. In case the bacterial host has a functional lac operon (lac +), the expression cassette can be induced with 1% lactose. After the induction period, the growth factors can be isolated from either the medium or the cell level.

10 (a) PAH / EGF

Human EGF produced from the expression cassettes TacPak / EGF was isolated from the medium in an inactive form with the alkaline phosphatase signal sequence removed. Approximately 85% of the active EGF was found in the medium with the remainder associated with the cell pellet. These expression cassettes provided 4 mg / l active EGF. The cells were removed from the medium by centrifugation and the medium was passed through Amicon SY30, 30000 Mr cut-off spiral filter and then passed through a Q-Sucrose column while eluting the highly purified human 20 EGF in 20mM NaPO ^ pH 7 with a 0- 0.5 M NaCl gradient. Optionally, the growth factors can be isolated from the cell pellet by osmotic shock or sonication and purified according to essentially the same procedure as described above.

(b) PAK / NVGFa Recombinant nVGFa was produced from the expression cassette pTCPt / nVGFa. The nVGFa was isolated from the cell pellet by sonication and was shown to make up approximately 40% of the total bacterial protein.

C. Isolation of VGF and SFGF produced by eukaryotic expression of the natural genes 1. Preparation of VGF in monkey kidney cells

Natural VGF was produced by infection of monkey kidney cells with vaccinia virus. Cercopithecus monkey (BSC-1) cell mono-35 layers were infected with 15 plaque forming units (PVU) per cell purified VV (strain WR grown in Hela cells and purified by sucrose density gradient sedimentation) (6. Mosse, 1981 in 8900724.

81- Λ

Gene Amplification an Analysis, eds. Chirickjian, J.G. and Papis, T.S. (Elsevier (North Holland. NY), part 2, pp. 253-266). The infected cells were supplemented with 2% fetal calf serum per 2 x 10 5 cells at 37 ° G with an approximately 1ml Eagle 6 basal medium. Pseudo-infected cells were treated identically. Cell culture supernatants were clarified by low speed centrifugation and lyophilized. The residue was then resuspended in 1 M acetic acid and intensively visualized against 0.2 M acetic acid. Insoluble material was removed by centrifugation and the top layer was lyophilized and resuspended in 1 / 100th of the original volume of 1 M acetic acid and stored at 4 ° C.

2. Preparation of VGF in CHO cells

Natural VGF was also produced by transfection of the plasmid pRSV / VGF containing the VGF gene fragment into Chinese hamster ovary cells. The transfected cells were expanded and the media and cell pellet examined for the presence of VGF by immunoprecipitation using an antiserum against an N-terminal VGF peptide.

3. Preparation of VGF in Silkworm, Using Baculovirus Promoter (a) Transformation

The host cell for AcNPV is Spodoptera frugiperda (sf9).

In order to produce a recombinant virus stock, the VGF-25 containing plasmid is mixed with AcNPV DNA and transfected into sf9 cells using the calcium phosphate technique. Recombinant viruses are purified from the medium and plaque over sf9 cells.

Isolated Recombinant plaques are identified by hybridization using radiolabeled VGF DNA as a probe.

30 (b) Expression

Once recombinant virus has been identified, it is expanded on sf9 cells. The recombinant virus material is then used to infect sf cells, the cells are lysed and the supernatants sorted for production of VGF protein, which is purified as described above for vaccinia virus-infected mammalian cells.

0900724.

82

The predicted sequence of VGF produced in insect cells is: DSGNAIETTSPEITNATTDIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSHGYTGIR CQHWLMYQRSENPNTTTS YIPSPGIMLVLVG1111TCCLLSVYRFTRRTKLPI

A potential glycosylation site occurs at the asparagine at position 15 from the N-terminal end of the polypeptide.

This 121 residue sequence starts with the N-terminal sequence which is purified directly for VGF from VV-infected apecells (i.e.

10 at residue 20 of the VGF open reading frame) has been determined and proceeds to the last residue of the open reading frame. Thus, it lacks the signal peptide (open reading frame residues 1-19) but includes the transmembraneous sequence (YIPSPGIMLVLVGIIIITCCLLSVY).

The predicted molecular weight of the 121 residue peptide is 13,304, not counting carbohydrate. The apparent molecular weight of the baculovirus-produced VGF 17000 is significantly smaller than that obtained from VV-infected cells, indicating that the two forms have probably been processed differently. The baculovirus-produced VGF may lack an added portion of the N-terminal sequence or a portion of the C-terminal sequence or both and / or may differ in the type and degree of glycosylation.

4. Preparation of SFGF in silkworm using baculovirus promoter

(A) Transfection of Sf9 cells with pAc / SFGF

Plasmid pAc / SFGF was transfected into Sf9 insect cells (Spodopters frugi-perda) together with AcNPV wild-type baculo virus DNA. The transfectants were sorted for an occlusion negative phenotype in plaque assays. An occlusion of negative plaque was isolated and / 5 rounds of consecutive plaque purification, a high titer of recombinant virus material was prepared.

The recombinant virus appears to contain the SFGF gene according to Southern analysis.

^ D. Natural EGF

1. Natural EGF was isolated from mouse salivary glands or purchased from Collaborative Research.

Ö9D0 7 2 4.1 83

Example XI Activity determinations A. Mitogenic determination

Mitogenesis assays were performed as follows: 5 diploid human fibroblasts obtained from neonatal transplanted foreskin were inoculated at a density of 3 x 10 6 cells / cup (96 cup plates, Nuclon, Roskil de, Denmark) and were grown to confluence in Dulbecco's Eagle's medium (GIBCO) / 10% newborn calf serum. The cultures were then placed in a medium containing 0.2% newborn calf serum and two days later the EGF to be tested (10 mg / ml) or the growth factor (10 ng equivalent EGF / ml) was added. After 8 hours, the cultures were labeled with 5- (I) iodo-2'-deoxyuridine (Amersham, 10 µCi / ml, 5 Cl / mg; 1 Ci = 37 GBq) and the amount of isotopes incorporated in TCA insoluble material was determined as (Twardzik et al., Proc. Natl. Acad. Sci. USA 1985) 182: 5300-5304).

B. Gentle Agar Colony Growth Stimulating Assay

A 0.5 ml base layer of 0.5¾ agar (Agar Noble; Difco 20 Laboratories, Detroit Michigan) in growth medium was added to 24-well Costar tissue culture plates. Half a ml of 0.3% 4 agar in growth medium containing 1-1.5 x 10 cells / ml NRK cells or another cell line of interest and various concentrations of the factor to be determined was applied to the base layer of agar .

The plates were incubated at 37 ° C in a humidified atmosphere of 5% CO2 in air and refed after 7 days by adding 0.5 ml 0.3% agar in growth medium to obtain the same concentration of the factor to be determined. The colonies were not fixed and counted not colored. The number of colonies with more than 6 cells was noted.

C. EGF receptor binding inhibition test

The radio receptor assays were performed as follows: Binding of I-labeled EGF (0 I-EGF) to its receptor on mono-35 layers of A431 cells was modified from the procedure described by Cohen and Carpenter, Proc. Natl. Acad. Sci. USA (1975) 72: 1317-1321. The cells (1 x 10 ^ per cup) were run at 24-89 0 0 72: 4.

84 cup plates (Linbro, Flow Laboratories) fixed with 1.0¾¾ forma line in phosphate buffered brine prior to assay. Formalin-fixed cells do not release from plates as easily as the unfixed cells, and thus replicate values were more uniform.

c '125 10

Under these assay conditions, I-EGF (1 x 10 cpm / nmol) saturates the binding assay at 3 ntf; the determinations were made at 10¾ of the saturation value. Growth factor concentrations are expressed as ng equivalents of EGF / ml, i.e. the amount of 125 needed to produce an inhibition of I-EGF binding equivalent to that produced by a known amount of EGF.

D. Radioimmunoassay

Each 50 muliter reaction mixture contained the following: 20mM sodium phosphate at pH 7.4, 200mM NaCl, 40mM dithiothreitol, 0.1¾ 15 bovine serum albumin, 0.1¾ NaNg, ^ sl-labeled peptide (2 x 10 ^ cpm) corresponding to a 17 carboxyl-terminal residues of ocTGF (Linsley et al., Proc. Natl. Acad. Sci. USA (1985) 82: 356-369), antiserum in a final dilution of 1: 5000 and other additives as indicated. The reaction was initiated by the addition of antiserum 20 and continued at 23 ° C for 90 minutes. An equal yolurne 10¾ formalin-fixed S. aureus (Pansorbin, Calbiochem) was added and the incubation was continued for an additional 30 minutes at 23 ° C. The immunoadsorbents were removed by sedimentation and the amount of I-bound. labeled pep-tide was measured. The amount of bound peptide was corrected for nonspecific binding measured in the absence of antibody (less than 5% of the total) and expressed as a percentage of the maximum binding.

30 E. Wound Healing 1. Milddendermal burns

Mid-dermal burns were applied to the dorsal thorax of anesthetized Yorkshire xsugên (30.5 kg) where the ridges were shaved and depilated with commercially available hair removal cream. A brass mold plate (3 x 3cm, 147g) was equilibrated in a 70 ° C water bath and placed in tight contact with the skin for 10 seconds. The result 89 0072.4. " 85 blister was then removed. 5 mid-dermal wounds were applied to each side of the spinal cord approximately 2.5 cm apart. The burns were treated twice daily with approximately 3 ml of carrier cream (Silvadene) 5 alone or with growth factor present or left untreated.

After 9 days or 10 days of treatment with natural VGF, the pigs were anesthetized and the burn scar removed.

A biopsy was performed of each burn from the re-epithelialized areas.

10 2. Mid-dermal donor graft wounds

A 5 month old 20.5 kg piglet was anesthetized with 20 mg / kg ketamine and 2 mg / kg Rompum. The dorsal thorax was shaved, prepared with Betadine and rinsed thoroughly with brine. A series of six 5 x 5 cm donor sites were placed on each side of the dorsal thorax with a 50/1000 inch Padgett skin graft knife by making two strokes at 30/1000 inch. The topical therapy included 1 ml of brine in 20 mg of Silvadene evenly distributed between the six wounds on the left. The right side was treated with 1 ml of the growth factor to be tested, in 20 mg

Silvadene evenly distributed between the six wounds. All wounds were covered with a large burn dressing, and other common coverings. The animal was anesthetized as described above on days 1, 2, 3, 4, 7, 8, 9, 10 and 11 after surgery. The bandage was removed. The wounds were gently wiped with betadine and rinsed thoroughly with brine. The appropriate agent was applied and the wounds were repaired as described above.

Example XII

Biological activity of recombinant growth factors prepared in prokaryotic cells A. EGF receptor binding

This analysis determines the ability of the molecule to bind to the EGF receptor as measured by its ability to prevent EGF from binding to its receptor. All growth factors and chimeric growth factors, whether modified or truncated, are 89 00724.

»86 to date have been isolated were active in the EGF receptor binding inhibition assay» A summary of these results is given below:

5 TABLE

EGF receptor binding of recombinant growth factors Peptide Expression Binds to EGF

_ cassette iu.i government receptor 10 N / TGF - modified truncated fusion pBM11 / N / TGF 95% Yes PAD / nVGFa - modified truncated fusion pBM11 / PAD / nVGFa 95% Yes 15 NDP / VGFa - modified truncated fusion pBM11 / NDP / VGFa 95% Yes NDP / VGFA - Modified 20 Truncated Fusion pBM11 / NDP / VGFA 95% Yes N / TTV-Modified Truncated Chimeric Fusion pBM11 / N / TTV 95% Yes 25 NDP / TTV - Modified Truncated Chimeric Fusion pBM11 / NDP / TTV 95% Yes NDP / VTV - Modified Truncated Chimeric Fusion pBM11 / NDP / VTV 95% Yes 30 NDP / TVV - Modified Truncated Chimeric Fusion pBM11 / NDP / TVV 95% Yes ΡΑΚ / EGF pBM11 / PAK / EGF 95% Yes EGF pBM11 / PAH / EGF 95% Yes 8900 724.

35 Λ 87 PAD / EGF pBMI1 / PAD / EFD 95% Yes EGF pBMIt / PAD / EGF 95% Yes 5 ΡΑΚ / EGF TacPak / EGF 95% Yes EGF TacPak / EGF 95% Yes ΡΑΚ / EGF pTCPt / EGF 95% Yes 10 EGF pTCPt / EGF 95% Ja jg A comparison of the binding inhibition curves for natural rave EGF and the bacterially expressed recombinant chimeric growth factor N / TTV (a polypeptide fusion of the 32 N-terminal amino acids of the lambda N gene and the modified and truncated TGF / VGF hybrid) suggests that there were no differences in the 2q binding activity.

B. Mitogenic activity

The activity of several of the purified growth factors was investigated and the activity determined was similar in all cases to the effect induced by EGF in 2g.

The compounds investigated are indicated below:

TABLE

3Q Mitogenic activity of growth factors

Peptide Mitogenic Activity * TTV - Modified Truncated Chimeric Yes N / TVV - Modified Truncated Chimeric Fusion Yes o 125 * Measured by incorporating H-thymidine or I-IdU 8900724.

00 88 C. Wound healing 1. Mid-dermal injuries

The effect of natural or synthetic TGF, EGF and VGF as well as recombinant growth factors on mid-dermal lesions was determined as described in Example F1. The percentage of the original burnt area that had healed was measured by computer telemetry and the percentage of wound re-epithelialization was determined. Untreated wounds were approximately 15% re-epithelialized. Treatment with Silvadene 100 alone or with Silvadene EGF resulted in approximately 50% re-epithelization, while treatment with synthetic TGF or natural VGF resulted in approximately 90% re-epithelization. optimal concentration to promote re-epithelization of EGF was 1-10 mug / ml, while synthetic TGF and natural VGF produced a maximum response at 0.1 mug / ml.

Experiments similar to those described above were performed to examine / effect on wound healing of either TGF, a modified truncated form of VGF (VGFa), or a modified truncated chimeric fusion of TGF and VGF (TTV), all of which had been produced in bacteria by recombinant technology.

These recombinant growth factors and hybrid growth factors accelerated wound healing to the same degree as either synthetic TGF or natural VGF, at an optimal concentration of 0.1 mug / ml.

2. Middermal Donor Graft Injuries Modified Truncated VGFa was investigated for its ability to accelerate wound healing in a donor graft model. The treatment regimen was as described above (see Example I) using 1 ml VGFa, 5 mosquito / ml, in 20 g Silvadene.

Photos were taken daily. A summary of the results is given below.

Table

Effect of recombinant VGFa on wound healing

Wound condition 33 Treatment * P0D ** 7 POD 8 POD 9 POD 10

Brine Fully open Open with some virtually healed λ λ λ λ «» λ, cure cured 89 00 724 «£ 89 TABLE CONTINUED_Wound condition _

Treatment * POD ** 7 POD 8 POP 9 POP 10 VGFa - modified Open near cure cure g truncated clear cure epithelization * Silvadene is the carrier ** POD = day after surgery 10 ---

The modified truncated VGFa accelerated wound healing compared to the carrier control.

Photos (not shown) at POP 8 showed significant differences between brine and VGFa.

Example XIII

Biological activity of VGF prepared in eukaryotic cells A. VGF prepared in monkey (BSC-1) cells.

1. The medium derived from BSC-1 cells 24 hours after infection with 125 VV was examined for the presence of material that could compete with I-EGF for binding to EGF receptor-rich human epidermal carcinoma cells (A431). VV-infected cells released a potent activity, which competed with EGF, 2g which activity is referred to as VGF.

Control medium full of urn from pseudo-infected. BSC-1 control cultures contain minimal activity for competition with EGF. Increased levels of VGF in the culture medium were observed when monitoring the VGF production at the earliest time observed, 2 hours after infection. After 12 hours, maximum amounts of this activity were found in the culture supernatant, with only a slight increase noted after 24 hours. The level of VGF production was found to be a function of the multiplicity of virus infection as demonstrated by the following Table 35.

8300724.

90

TABLE

Effect of multiplicity of infection on VGF delivery

Virus Mutuality Issued V6F

5 pfu per cell ng.equiv. of EGF per ml 0 -. 10 2.7 20 4.5, 0 40 6> 1 80 10.1

Cultures of BSC-1 cells were infected at the indicated pfu-to-cell ratio, and the supernatants (approximately 1 ml / 2 x 10, c cells) were collected 24 hours after infection. Samples from each lo thereof were acidified, lyophilized and assayed in duplicate radio receptor assays for EGF as described by Delarco and TOdaro, Proc. Natl. Acad. Sci. USA (1978) 75: 401-405). ng. equivalent = nanogram equivalents.

20

2. Partial purification of VGF

The competition activity with EGF found in VV-infected BSC-1 cells was partially purified from acid-extracted culture supernatants 24 hours after infection as described above. Acid-solubilized polypeptides (10.5 mg) from the top layers were applied to a Bio-Gel P10 column equilibrated with 1 M acetic acid and samples of each fraction 2Q were tested for activity in competition with EGF. The major active peak (fraction 42) was eluted slightly after the M 29000 carboxylic acid anhydrase marker with an apparent MD of 25000.

The molecular weight was confirmed using tendonically coupled Bio-Sil TSK 250 HPLC sorting columns, eluting the total 2g activity as a major peak in the region of the M ^ 25000 protein marker. 1 Micorgram of partially purified VGF was equivalent to 90 ng of EGF in the radioreceptor competitor assay.

8900724.

91

3. Further purification of VGF

Pooled fractions from the gel filtration column (25-35) were concentrated by vacuum centrifugation, resuspended in 0.05% trifluoroacetic acid (TFA), clarified and injected into a 3.9 mm x 30 cm muBondapak Cg column (Waters, Milford MA ).

Peptides were eluted with a linear 20-60% gradient of acetonitrile in 0.05% TFA at a flow rate of 1.0 ml / minute at 22 ° C. Aliquots of each fraction were analyzed for EGF competitive activity in a radio receptor analysis. The peptide corresponding to the peak activity was collected and diluted with 0.05% TFA and reinjected into a muBondapak column and eluted under isocratic conditions.

The acetonitrile concentration was about 22-25%.

A comparison of the levels of VGF produced by 15 BSC-1 cells infected with 15pfu VV per cell with those of c * TGF produced by retrovirus transformed cells is shown in the following table.

Table

Comparison of the biological activity of VGF with oCTGF

and EGF

EGF receptor Induction of DNA Stimulation of compound2 synthesis3 anchor-independent cell growth - - ™ - -

Growth eq, EGF / I IdU incorporated Soft agar colonies / factor ml of medium (cpm / dish) _ plate_

None - 1.779 <20 VGF 2.3 3.760 108 30 TGF-oi 0.2 NT 294 EGF - 8.482 346 NT = not tested ^ 1. VGF (90 ng equivalents (eq) EGF / mosquito protein) was purified by gel filtration followed by elution from a C18 muBondapak (Waters Associates) column. TGF was purified from Snyder-Theilen cat sarcoma virus-transformed Fisher rat embryo cells as 83 0 0724.

I 92 described by Marquart et al, Proc. Natl. Acad. Sci. USa (1983) 80: 4684-4688; EGF was purified from mouse submaxillary gland (Cohen et al., J. Biol. Chem. (1980) 255: 41834-41842.

2. The quantification of EGF equivalents was based on a 125 standard I EGF binding competition curve as described.

3. Mitogenesis assays were as described; quiescent cultures of diploid human fibroblasts were treated with 10 ng of purified EGF / ml or the same number of EGF equivalent of VGF / ml. The values for (^ I) iodo-deoxyuridine [{^ 1) 1duj included indicate the average of triplicate determinations.

4. The number of soft agar colonies represents the average number of colonies containing a minimum of 20 NRK cells / 6 random low power fields 10 days after inoculation (1.5 x 10 cells / ml) with purified EGF (5 ng / ml) or the same number of EGF equivalents of VGF / ml and 2.0 ng TGF-1 / ml purified from human platelets as described in Assoian et al., J. Biol. Chem, (1983) 258: 7155-7160.

Pictures of NRK cells treated only with TGF- gave no colonies.

B. VGF prepared in CHO cells

The biological activity of VGF prepared in CHO cells was evaluated using the EGF receptor binding assay.

Culture growth medium was used without further purification. The growth factor bound to the EGF receptor.

C. VGF prepared in silkworm

The biological activity of partially purified (approximately 50%) VGF prepared in silkworms was evaluated using the EGF receptor binding assay and was found to bind to the EGF receptor.

8900724.

93

Table

Percentage of epithelialization of wounds after growth factor treatment

Pig 1 5 (9 days after far branding)

Right side Silvadene Untreated Natural VGF mosquito / ml

10 15¾ 0% OjX

70% 70% H-FGF mosquito / ml

Left side Silvadene Untreated 0.1 0.1 0.1 75% 55% 60% 70% 30% 15

Pig 2 (10 days after burning) VGF * mosquito / ml

Right side Si 1vadene Untreated 0.1 0.5 1.0 2Ü 50% 0% 95% 95% 60%

Left side Silvadene Untreated _0J_ £ ^ 5 y ^ O

50% 0% 60% 75% 40%

Pig 3 25 (9 days after burning) right side Silvadene Untreated 0.1 1.0 10 20% 25% 90% 65% 90%

Human TGF mosquito / ml

Left side Silvadene Untreated M. JU.

25% 5% 90% 85% 65% * VGF: Natural VGF prepared from vaccinia virus infected cells.

35 8900724.

* 94

D. Immunological comparison of VGF and TGF

In the radioimmunoassay described above, a 50 µ displacement of anti-antibody from antibody to the carboxyl-terminated 17 amino acids of rat TGF molecule was observed at an anti-concentration of approximately 0.2 to 0.2 ng 125. equivalents EGF, where I-labeled oi TGF competes with f * TGF.

No competition was observed in studies of VGF at equivalent concentrations. In a competing radioimmunoassay for native EGF, EGF preparations, even when tested at 50 ng EGF equivalents / ml, show minimal displacement (<10%) 125 of I-EGF from a polyclonal antibody to native EGF.

It is clear from the above results that the VGF is a potent epithelizing agent which is satisfactorily comparable to other growth factors previously investigated and demonstrated to have mitogenic activity.

The subject polypeptides can be used with various hosts to induce an immunogenic response, enhance cellular proliferation, wound healing, and the like. For wounds, epithelization and vascularization are observed, as well as a rapid recovery of wound strength, ie, resistance to tearing or tearing. Hosts include mammals, such as rodents, pets, primates, and humans.

According to the present invention, new compositions are provided with a wide range of possibilities in various fields, such as in diagnostics, in vitro and in vivo effects on cells, activity as mitogens, additives in nutrient media, use as agonists and antagonists for EGF and TGF, acting as immunogens, acting as therapeutic agents, accelerating wound healing, etc.

By further providing a small oligopeptide with binding activity, the oligopeptide is useful alone or in combination with other polypeptides fused to the other polypeptides to vary the properties of the other polypeptides, resulting in binding of the polypeptide to cells with 35 growth factor receptors. Thus, one can reversibly bind different polypeptides to cells with growth factor receptors, thereby influencing the properties of the cells in a predetermined manner.

0900724.

4 95

GROWTH FACT QR FAMILY

MGF Myxoma virus growth factor SFGF Shope fibroma virus growth factor 5 VGF Vaccinia growth factor

hTGF Human TGF

rTGF Ratte TGF

mEGF Muize TGF

rEGF Ratte EGF

10 gpEGF Guinea pig EGF

i *

«F KVPKDLVATLLCAKCIVDA ......... T H P S L D Η Y l T I I K A I K L

SFGF NArRKLV-ASLLCIXYAVHA ------------- N X fl Y L Υ I V K Η V K V

15 VGF KSNKYLNLLFAAMI IRSFA-DSBNA1ETTSPEITNATTBIPA I RL

/ hTGF KVPSAG0LALPALBIVLAACQAENSTSPL5ABPPVPAAVVSHFNB

rTGF KVPAA60LALIALBILVAVCBAENSTPPLSRPSPVAAAVVSHFNK

hEGF NSDSE

* Br f HSrPB

rEGF HSYPB

fpEGF * 0 B A P G

& vt ^ veayv '<. epÊij> KTesei4Ts "> K.PK <kKK.vu; c 2 5 1 0 15 20 25 30 35 40 45 2Q * F CNBOYXXYCLNNeTC'FTVALttNVSLNPFCACH'IHYVBSRCBFIN.LITIK · SFGNNVCH-FTGNNVCH-FTGNNVCF

VGF C6PE6D6YCLH-6DCIH - AR0ID - GNYCRCSH6YTBIRCeHVVLVDYBRSENPHTTTS

hTGF CPBSHTQFCFH-GTCRFLVOEDK ---- PACVCHSGYVGARCEHADLLAVVAASQKKOAIT

rTGF CPBSHrOYCFH-BTCRFLVeEEK ---- PACVCHSGYVGVRCEHADLLAVVflflSOKKOA I T

hEGF CPLSHD6YCLHB6VCKYIEALBK ---- YACKCVVBYIBERCOYROLKUWELR

CPSSYBGYCLN6BVCMHIESLBS ---- YTCNCVI6YS6BRC0TRDLRUHELR

rEGF CPPSYB6YCLN66VCMYVESVDR ---- YVCNCVlBYIGERCDHRDLR

IPEGF CPPSKBBYCLHBBVCHHIESLNT ---- YACNCVI6YV6ERCEHQBLBLVE

CT4 Mfaii * * * Citt »4EE <7i.trtueK vTcKCQ ---- <AP-7 * ^ Ce CCST <* -.. Cb VGF ΥΓΡΒΡ6 INLVLV6I II ITCCLLSVYRFYRRT KLTXA f4 £ \ A / f JFE / hTGF ALVVVSIVALAVLIITCVLIHCCeVRKHCEHCRALlCRHEKPSALLKGRTACCHSETVVt rTGF ALVVVSIVALAVLIITCVLIHCCOVX. KHCEHCRALVCRHEKPSALLKGRTACCHSETVVi 3Ü * Numbers correspond to the amino acid residues in VGF as indicated in the description.

35 FIGURE 1 8900724.

♦ 96 v ^) @ ® @ ®e®®®®®® # - + In SFGF this is aspartic acid * Potential glycosylation site in SFGF and / or MGF N Indicated amino acid has been replaced by asparagine.

FIGURE 2 «S0O72U.

Claims (37)

1. DNA sequence encoding a polypeptide characterized in that it (1) is capable of binding to an E6F receptor, (2) substantially the same loop structure as a naturally occurring growth factor capable of binding to an EGF receptor ("natural ligand") and (3) has at least about 30% homology to a natural ligand, said polypeptide comprising three domains, each of which has an amino acid sequence of at least about 30% homology to the fragments of the same or different natural ligands, and which have the same relative position as said fragments of said natural ligands, which domains are defined as: a first domain of at least about 1! N-terminal -f 1 amino acids starting at about amino acid aa to aa 11 15 and ending at about amino acid aa to aa; a second domain of at least about 11 central fragment 11 15 amino acids starting at about amino acid aa to aa and Λρ ΛΛ ending at about amino acid aa 0 to aa ^ y; A third domain of at least about 14 amino C-terminal amino acids starting at about amino acid aa to 29 27 35 aa and ending at about aa to aa, and optionally named. DNA sequence further encodes a fourth domain of at least about six extremely N-terminal amino acids starting at about aa "^ to" ^ and ending at about amino acid aa "^ to aa" 7, optionally having an amino acid sequence that is can be selectively cleaved when the carboxy end site of said fourth domain is introduced, linked to the amino end site of said first domain provided that when said polypeptide 30 has an amino acid sequence identical to a natural ligand, said DNA sequence encoding for said fourth domain is present and encodes other than the extremely N-terminal amino acids of the natural ligand. DNA sequence according to claim 1, characterized in that said natural ligand is VGF, TGF- *, MGF, EGF, SFGF or amphiregulin. 88 0 0 724 .. DNA sequence according to claim 1, characterized in that said DNA sequence further encodes a major sequence, optionally having an amino acid sequence selectively cleaved at the carboxy end site of said major sequence, coupled to the amino sequence. end site of said first domain, or when said fourth domain is present, at the amino end site of said fourth domain. DNA sequence according to claim 3, characterized in that said main sequence is an N-terminal fragment of a lambda DNA sequence according to claim 3, characterized in that said main sequence is a signal sequence or a mutant thereof. DNA sequence according to claim 5, characterized in that said signal sequence is an alkaline phosphatase signal sequence, a human monkey TGF-γ51 signal sequence or a K-lactis killer toxin signal sequence. DNA sequence according to any one of claims 1-6, characterized in that at least one of said domains is at least about 30% homologous to a fragment of VGF, TGF-tf, EGF or SFGF. DNA sequence according to any one of claims 1, 4 and 6, characterized in that said DNA sequence encodes VGF. DNA sequence according to any one of claims 1, 4 and 6, characterized in that said DNA sequence encodes TGF. DNA sequence according to any one of claims 1, 4 and 6, characterized in that said DNA sequence encodes EGF. 10 is N protein, lambda cro protein, or amphiregulin. DNA sequence according to claim 1, characterized in that said DNA sequence encodes SFGF. 12. DNA sequence according to claim 1, characterized in that said sequence comprises at least one change from the sequence / coding for a natural ligand, which results in the following changes in the amino acid sequence: HGD in HGT; GMYC in GYAC; RCS in VCS; COHGDC in COHGTC and GMYCRC in GYACVC. 13. DNA sequence encoding a polypeptide of interest and a major sequence of about 8 to 45 N-terminal amino acids, including modifications thereof, of a bacteriophage lambda N protein or cro protein, amphiregulin, T4 gene 32 or a 8900724. 99 * bacterial alkaline phosphatase signal sequence, linked to the amino terminal site of said polypeptide of interest, optionally via an amino acid sequence that can be selectively cleaved, which polypeptide of interest comprises three domains, the order of each domain substantially corresponding to the sequence of a fragment of a naturally occurring growth factor capable of binding to an EGF receptor ("natural ligand") and having the same relative position as said fragments of said natural ligand, which domains consist of at least about 10 11 N-terminal amino acids begin at about amino acid-fi 11 m aa to aa and end at about amino acid aa to aa; a second domain of at least about 11 central fragment amino acids, starting at about amino acids aa to aa and ending at about amino acids aa to aa; a third domain of at least 15 about 14 amino C-terminal amino acids starting at about 25 29 40 47 amino acid aa to aa and ending at about aa to aa DNA sequence according to claim 13, characterized in that said main sequence consists of about 32 or 33 N-terminal amino acids of said N gene. DNA sequence according to claim 14, characterized in that said polypeptide of interest is TGF-VGF, VGFA, VGFa or EGF. DNA sequence according to claim 13, characterized in that said main sequence is an alkaline phosphatase signal sequence or a modified alkaline phosphatase signal sequence. DNA sequence according to claim 16, characterized in that said polypeptide of interest is VGFa, nVGFa or EGF. DNA sequence according to claim 14, characterized in that said polypeptide of interest has the following amino acid sequence: DNA sequence according to claim 14, characterized in that said bubble polypeptide has the following amino acid sequence: MDIFAIRLCGPBGDGYCLHG TCRFLVQE2KPACVCSHGYT GIRCQHVVVVYQR. 8000724. t * DNA sequence according to claim 14, characterized in that said polypeptide has the following amino acid sequence: MVVSHFNDCFD8HTQFCFHGT CXHARDXDGYA CV CSHGYTGI 5 RCQHVVLVDYQR, DNA sequence according to claim 13, characterized in that said main sequence consists of about 21 N-terminal amino acids of said cro protein. DNA sequence according to claim 19, characterized in that said polypeptide of interest has the following amino acid sequence b6Zlt: KVVSHFNDCFDSHTQFCFHG IRFLVQEEKFACVCSHGYT GIRCQHVVLVDYQR. DNA sequence according to claim 13, characterized in that said main sequence consists of approximately 8 N-terminal amino acids of T4 gene 32. DNA sequence according to claim 23, characterized in that said polypeptide of interest has the following amino acid sequence: V V 8 B F N D C P D S Η T Q F C F H Q T CRFLVQ ΕΕΠ ACVC SH4Ï T G IRCQHVVIiDDYQR » 25. Expression cassette comprising in the transcription direction a transcription and translation initiation regulatory region functional in a host cell; a DNA sequence encoding a polypeptide characterized in that it (1) is capable of binding to an EGF receptor, (2) substantially the same loop structure as a naturally occurring growth factor capable of binding to an EGF receptor ("natural ligand ") and (3) has at least about 30% homology to a natural ligand, said polypeptide comprising three domains, each of which has an amino acid sequence with at least about 30¾35 homology to fragments of the same or different natural ligands, and with the same relative position as said fragments of said natural ligands, which domains are defined as: 89 0 0 724. 101 * i »a first domain of at least about 11 N-terminal -61 amino acids starting at about amino acid aa to aa and ΛΑ Λ Γ ending at about amino acid aa to aa; a second domain of at least about 11 central 11 15 5 fragment amino acids starting at about amino acid aa to aa 'ic OQ and ending at about amino acid aa to aa; a third domain of at least about 14 amino C-terminal amino acids starting at about amino acid aa to aa and ending at about aa40 to aa53; and 10 said DNA sequence optionally further encodes a fourth domain having at least about 6 extremely N-terminal 4 amino acids starting at about aa to aa and ending at -45 -7 about amino acid aa to aa, optionally having an amino acid sequence that is selective can be cleaved when the carboxy-15 end site of said fourth domain is introduced, linked to the amino end site of said first domain provided that when said polypeptide has an amino acid sequence identical to a natural ligand, a DNA sequence encoding said fourth domain is present and encodes other than the extreme N-terminal amino acids of the natural ligand and a transcription and translation termination regulatory region functional in said host cell. 26. Host cell comprising an expression cassette comprising in the transcription direction a transcription and translation initiation regulatory region functional in a host cell; a DNA sequence encoding a polypeptide, characterized in that it (1) is capable of binding to an EGF receptor, (2) substantially the same loop structure as a naturally occurring growth factor capable of binding to an EGF receptor ( "natural ligand") and (3) has at least about 30% homology to a natural ligand, said polypeptide comprising three domains, each of which has an amino acid sequence with at least about 30% homology to fragments of the same or different natural ligands and having the same relative position as said fragments of said natural ligands, which domains are defined as: 83 0 0 72 4. k Η a first domain of at least about 11 N-terminal _r λ amino acids starting at about amino acid aa through o. and 11 ending at about amino acid aa through aa; a second domain of at least about 11 central fragments of mentamino acids starting at about amino acid aa to aa 29 and ending at about amino acid aa 'to aa; a third domain of at least about 14 amino C-terminal 29 amino acids starting at about amino acid aa to aa 40 53 and ending at about aa to aa and said DNA sequence optionally further encodes a fourth domain of at least about 6 extremely N-terminal amino-fi -1 acids starting at about aa to aa and ending at about -45 -7 amino acid aa to aa, optionally having an amino acid sequence selectively cleaved at the carboxy end site of said fourth domain is inserted, linked to the amino end site of said first domain, provided that when said polypeptide has an amino acid sequence identical to a natural ligand, a DNA sequence encoding said fourth domain is present and other than the extreme N - terminal amino acids 20 of the natural ligand encode and a transcription and translation termination regulatory region functional in said host cell. 27. A method of producing a polypeptide characterized in that it (1) is capable of binding to an EGF receptor, 25 (2) substantially the same loop structure as / naturally occurring growth factor capable of binding to an EGF receptor ("natural ligand") and (3) has at least about 30¾ homology to a natural EGF receptor binder, said polypeptide comprising three domains, each of which is an amino acid sequence with at least about 30% homology to fragments of the same or different natural ligands and having the same relative position as said fragments of said natural ligands, which domains are defined as: a first domain of at least about 11 N-terminal -f 1 amino acids starting at about amino acid aa to aa and 11 ending at about amino acid aa to aa; 8900724. X 103 5. i it »a second domain of at least about 11 central 11.c fragment amino acids starting at about amino acid aa to aa 25 29 and ending at about amino acid aa to aa, a third domain of at least about 14 amino c terminal 25 29 3 d amino acids starting at about amino acid aa to aa and ending at about aa4 ^ to aa53; and optionally a fourth domain of at least about 6 extremely N-terminal amino acids starting at about aa "7 to -1 -45 -7 aa and ending at about amino acid aa to aa, 10 optionally having an amino acid sequence selectively cleaved at the carboxy end site of said fourth domain has been introduced, linked to the amino end site of said first domain provided that when said polypeptide has an amino acid sequence identical to a natural ligand, said fourth domain is present and is different from the extreme N-terminal amino acids of the natural ligand; and a transcription and translation termination regulatory region functional in said host cell, said method comprising: culturing a host cell containing an expression cassette, which, in the direction of transcription, a transcription and translation initiation regulatory region functional in said host cell, a DNA sequence encoding geno emd polypeptide and a transcription and translation termination regulatory region, in an appropriate culture medium expressing said polypeptide and isolating said polypeptide. 28. Plasmid comprising functional domains of. ρΒΗΠ / ïïDP / BÖF; pBMil / PAD / EG? / pBMll / FAK / EGF; pBMll / N / TGFf pBMll / NDP / VGFA> pBMll / NDP / VGFiJ 30 pBXll / PAD / nVGFij pBMll / PAK / nVGFa / pRSV / VGF; pAe / SFGF; pAc / VGFj pfac / TTV? TacPlk / EGFj pTCPt / BGFj pTCPt / nVGF * j pBXH / N / TTVj pBMXl / NDP / TTVj pBMll / NDP / VTVj Ot paais / NDF / TW. 29. Polypeptide characterized in that it (1) is capable of binding to an EGF receptor, (2) substantially the same loop structure as a naturally occurring growth factor capable of binding an EGF receptor ("natural ligand") and has 8900724. fh & X 9 (3) has at least about 30¾ homology to a natural E6F receptor binder, said polypeptide comprising three domains, each of which has an amino acid sequence with at least about 30% homology to fragments of the same or different natural ligands and having the same relative position as said fragments of said natural ligands, which domains are defined as: a first domain of at least about 11 N-terminal -f 1 amino acids starting at about amino acid aa to aa and ending i 1 ie 10 at about amino acid aa to aa, a second domain of at least about 11 central fragment 11 15 amino acids starting at about amino acid aa to aa and 25 29 egg denoting about amino acids aa to aa; a third domain of at least about 14 amino C-terminal 25 29 15 amino acids starting at about amino acid aa to aa and ending at about aa ^ to aa33; and optionally a fourth domain of at least about 6 extremely -7 -1 N-terminal amino acids starting at about aa to aa -45 -7 and ending at about amino acid aa to aa, optionally having an amino acid sequence selectively cleaved at the carboxy end site of said fourth domain has been introduced, linked to the amino end site of said first domain provided that when said polypeptide has an amino acid sequence identical to a natural ligand, said fourth domain is present and has the order other than that of the extreme N-terminal amino acids of the natural ligand. 30. Polypeptide comprising the following amino acid sequence or a fragment thereof of at least 37 amino acids comprising at least about amino acids 1-37: on M -24 -20 -15 -10 -5 -1 DS.GHAIETTSP1ITNATTDIFAIR 15 10 15, 20 l CG ί EQDG y C 1 H 6 DC 1 B X2 ARD 25 30 35 40 IDGX3NYCRCSBGVTG IRC O SVV 35 45 50 53 LVDYQRSSNPNT wherein X is a bond or an amino acid; X2 is a bond or 1-2 8900724 .. 105 * f f amino acids; X3 is a bond or 1-4 amino acids and X3 X2 and X3 may be the same or different. 30 KVVSHFNDCPDSHTQFCFHG TCRPLVQB2KPACVCSHGYT GIRCQHVVLVDYQR. Polypeptide according to claim 30, characterized in that 14 24 is at least one of said aa threonine; said aa is tyrosine 27; said aa is alanine and said aa is valine. Polypeptide according to claim 30, characterized in that c 7 comprises said amino acid sequence aa to aa. Polypeptide according to claim 32, characterized in that 14 24 is said aa threonine, said aa is tyrosine and said aa25 is alanine. 34. The polypeptide according to claim 13, characterized in that -fi 1Q said aa to aa of said amino acid sequence are replaced by the following; êA “6 5 10 13 VV8HFNDCPDSHTQFCFHG A polypeptide according to claim 33, characterized in that said aa ^ to aa27 of said amino acid sequence are replaced by the following; 11 14 20 25 27 TCRFLVQEDK9ACV The polypeptide according to claim 35, characterized in that said aa is replaced by glutamic acid. A DNA sequence encoding a polypeptide having a sequence according to any one of claims 30-36. 8900724Γ