CH681081A5 - - Google Patents

Description

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description

The present invention relates to new polypeptides with growth factor activity, including chimeric polypeptides, and methods of making them using DNA techniques.

A significant number of polypeptides secreted by mammalian cells have been found to have growth factor activity. The sequence of these polypeptides is essentially conserved in a wide range of mammals. The greatest interest is the connection of this connection with the growth of the tumor. Also of interest is the question of how the production of growth factors is controlled and how they control cellular activity.

It has also been found that infection of mammalian cells by viruses leads to proliferation of the growth of the infected cells. Therefore, in the context of the present invention, members of the pox virus, such as variola, vaccinium and viruses which are associated with special diseases, such as the Shope fibroma virus, the Yaba tumor virus and the Molluscum contagiosum virus (MCV) of special interest.

It is also of interest to determine whether viruses that cause cellular proliferation upon infection produce polypeptides that are related to growth factors, either growth factors themselves or growth factor receptors. These compounds could then be used in studies of viral effects, in tests for the presence of viruses, in nutrient media, as mitogens and in the development of therapeutic agents for the treatment of viral infections. It is also of interest to develop compounds which are agonists or antagonists of growth factors, useful for their in vitro use in cell culture cultivation and in the study of mitotic methods and therapy.

Relevant literature

Venkatesan et al., J. Viral. (1982) 44: 637-646 describe the DNA sequencing of a structured, gene-encoding vaccinia virus protein, but it was not found that proteins thereof had growth factor activity. Cooper et al., Ibid (1981) 37: 284-294 report the translation of mRNA encoded within the inverted, terminal repeat of the vaccinium virus genome. Proliferative diseases by members of the poxvirus family have been described for the Shope fibroma virus (Shope, J. Exp. 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 epidemic growth factor (EGF) can be found in Scott et al., Science (1983) 221: 236-240 and Gray et al., Nature (1983) 303: 722-725, The presence of three disulfide bridges in EGF and the transforming growth factor (TGF) are described by Savage et al., J. Biol. Chem. (1973) 248: 7669-7692, see also Doolittle et al., Nature (1984) 307: 558-560 and Australian Patent Application No. 85 006 288. It has been suggested that the EGF- receptor binding region. Molecule is in the loop between the third and fourth cysteine residue (Komoriya et al., Proc. Nati. Acad. Sei. 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. Nati. Acad. Be. USA (1984) 81: 7263-7367. Shope Fibroma Virus Natural Growth Factor (SFGF) was developed by Chang, W.C. et at., in Molecular and Cellular Biol. (1987) 7: 535-540. Shope, J. Exp. Med. (1932) 56: 793-802 describe that infection of adult rabbits by the Shope fibroma virus causes rapid proliferation of fibroblasts, leading to benign fibroma. Infection of newborn or immunosuppressive adult rabbits causes a rapid proliferation of fibroblasts, which leads to an invasive, atypical Fibrosar-com. Allison, A.C., J. Nati. Cancer Inst. (1966) 36: 869-876; Smith, et al., J. nati. Cancer Inst. (1973) 50: 1529-1539; Allison, A.C. J. nati. Cancer Inst. (1966) 35: 859-868.

The natural myxoma virus growth factor (MGF) is described by Upton et al., J. Viral. (1987) 61: 1271-1275. Infection of adult rabbits by the myxoma virus causes a rapidly invasive myxosarcomatic tumor. Strayer, D.S. and Seil, S., J. nati. Cancer Inst. (1983) 71: 105-116.

A DNA sequence capable of encoding a conserved growth factor (natural Molluscum contagiosum virus growth factor (MCGF)) has been discovered. Porter, C.D. and Archard, L.C., J. Gen. Viral. (1987) 68: 673-682. Human infections from the Molluscum con-tagiosum virus indicate benign skin tumors that are characterized by fast-proliferative cells. Brown et al., Sec. Transm. Dis. (1981) 8: 227-234. The Yaba tumor virus causes subcutaneous histocytomas in monkeys and humans, Bearcroft et al., Nature (London) (1958) 182: 195-196, and because of its relationship to the Shope fibroma virus, the myxoma virus and the molluscum contagiosum virus are predicted have a similar growth factor.

Mice EGF (mEGF) Scott et al., 1983, supra; Gray et al., 1983, supra) and human EGF (hEGF)

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(Gregory et al., Int. J. Pept. Protein Res. (1977) 9: 107-118) are known to be homologous to human mouse and rat TGF (Marquardt et al., Proc. Nati. Acad Sci. USA (1983) 80: 4684-4688) and residues 45 to 85 of a 140 residue polypeptide, vaccinia growth factor (VGF) are encoded by the vaccinia virus genome (Venkatesan et al., 1982, supra).

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

Reference is expressly made to these articles.

The present invention relates to the novel polymer peptides defined in claim 1. You can find analogies in fragments of viral proteins, which compositions act as mitogens and are used as nutrient media, as reagents for the detection of growth factor receptors or the presence of growth factors and as competitors for transforming growth factors and epidermal growth factors. Compositions containing such peptides find therapeutic use, for example, to promote epithelization and healing of burns and wounds. The new peptides can be synthesized. The main peptides can be formed as oligopeptides or as fused proteins, whereby recombinant techniques can be used in a wide variety of hosts. The present invention also relates to the DNA sequences which encode the new polypeptides.

Brief description of the drawings

Figure 1 is a comparison of an amino acid sequence of a vaccinaia virus protein with a known growth factor, wherein the initial amino acid of VGF aspartic acid (D) at position 20 of methionine is which position aa ~ 24 of the disclosed peptides;

Figure 1 shows the proposed structure of mature VGF with asparagine (N) replacements. The potential glycosylation sites in MGF and SFGF are indicated by stars.

New compositions that can serve as agonists or antagonists of epidermal growth factor (EGF) or transforming growth factor (TGF) are made available and are for a large variety of applications in cell culture, diagnosis, in vivo therapy and in combination with others Peptides useful in the formation of hybrid polypeptides as immunogens for the production of antibodies. Polynucleotide sequences can be isolated or prepared which can be inserted into an expression vector to express the main compositions.

The compositions find analogies in fragments of viral proteins, particularly smallpox virus proteins. The VGF possesses uncharged and hydrophobic residues near the N-terminus between residues 5 and 15 and near the C-terminus between residues 100 and 124. It can be concluded from their analogy to integral membrane glycoproteins that they have an N-terminal hydrophobic signal sequence which is proteolytically removed during or immediately after translation, and a C-terminal transmembrane sequence which serves as an anchor for 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 release a membrane-pooled protein after removal of the 1 g amino acid sequence. A soluble growth factor peptide with about 57 residues is obtained by the intracellular method. Cell (1985), 42: 383-393. The comparison of the myxoma growth factor (MGF) and the Shope fibroma growth factor (SFGF) with other members of the growth factor family (see FIG. 1) shows various remarkable differences. MGF and SFGF, which are synthesized as precursor molecules, have a signal sequence at the N-terminus, but unlike the other growth factor members, they have no transmembrane region at the C-terminus, which indicates that they are secreted molecules. Sequence analysis shows a significant use of the amino acid asparagine in both MGF and SGF. In the 6 positions, both MGF and SFGF have preserved asparagine, while the other members have no asparagine. At these sites, the asparagines are replaced by 1 highly conserved glycine residues at sites 8 and 31, with the first cysteine in position 2. Both of these glycine residues occur in combination with highly preserved tyrosine residues in positions 9 and 32. Because of the highly conserved nature of the amino acids on this side of the molecule, it is believed that the cysteine loops present on this part 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 turning potentials (Chou and Fassman, Ann. Rev. Biochem. (1978) 47: 251-276). As a result, the preservation of this turning potential seems extremely important for the activity of a growth factor. The growth factors, which are different from Myxom and Shope, use glycine almost exclusively in important turning regions of the molecule. In MGF and SFGF, almost all of these glycins have been converted to asparagines (see FIG. 2). This indicates the important biological function, which is based on the replacement of the highly conserved glycine with the asparagine. There is a possibility that the replacement of glycine in the conserved turning regions in the growth factor structure

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leads to increased functionality of the growth factor by asparagine, either by increasing the binding to the EGF receptor or to a related receptor, by improving the direct effect of the growth factor in damaged cells or by increasing the stability of the molecule.

Two new asparagines in MGF and SFGF lead to potential N-linked glycosylation sites. Furthermore, these sites are present in two cysteine loops, which allows a variable number of amino acids. As a result, the new occurrence of asparagine can lead to several advantages of the growth factor in the improved functionality. One of these could be the stabilization of the protein in vivo by protection against degradation or the immunogenic sites by glycosylation.

The main polypeptides are characterized by having at least one loop or circle, usually three loops or circles, as a result of cysteines which form bridges, the physiologically active part of the molecule which contributes the growth factor activity, about 12 to 65 amino acids, usually has 15 to 60 amino acids. Of these three loops, two of the loops have 12 to 15 amino acids (14 to 17 ring-shaped amino acids) excluding the cysteine bridge, in particular one of them has 12 or 13 amino acids (14 or 15 ring-shaped amino acids) and the other has 15 amino acids (17 ring-shaped amino acids), whereby the N-terminal proximal loop normally has 12 to 13 amino acids, in particular 12 amino acids and the middle loop has 15 amino acids.

The third loop or C-proximal loop has 8 amino acids (10 ring-shaped amino acids) and comprises flanking amino acids and has the following formula:

PP3- (aa25-C-aa27) m - C-aa29-aa30-G-Y

PP4- (aa40-aa39-aa38) n-C- R- aa35-G-aa33

wherein the one-letter symbols for the amino acids have the conventional meaning, wherein C is cysteine, G glycine, N asparagine, Y tyrosine and R is arginine;

aa25 is a neutral amino acid, which can be aliphatic, in particular with 3 to 4 carbon atoms or aromatic, in particular 9 carbon atoms and has 0 to 1 hydroxyl group, e.g. Alanine, serine, threonine, tyrosine or phenylalanine;

aa27 is a neutral or basic amino acid which has in particular 3 to 6 carbon atoms and, if it is neutral, has 0 to 1 carboxamide group, for example arginine, alanine, valine, leucine, isoieucine, asparagine or glutamine;

aa £ 9 is a neutral or basic amino acid which is aliphatic or aromatic, the aromatic being, for example, histidine and the aliphatic having 2 to 6, usually 3 to 6 carbon atoms and having 0 or 1 hydroxyl group, and for example serine, threonine, leucine, Is valine, isoieucine or arginine;

aa30 is a neutral amino acid that is aliphatic or aromatic, the aromatic e.g. Is histidine and has aliphatic 3 to 6 carbon atoms and has 0 or 1 hydroxyl group and is in particular serine, isoieucine and valine;

aa33 represents a neutral, aliphatic amino acid of 2 to 6, usually 3 to 6 carbon atoms with 0 or 1 hydroxyl group, in particular it is represented by serine, threonine, valine, leucine or isoieucine or it can be an acidic amino acid such as glutamic acid or Aspara -gic acid;

aa35 is a neutral or acidic amino acid with 3 to 6 carbon atoms, wherein the neutral one is for example alanine, valine, leucine, isoieucine and serine and the acidic one is for example aspartic or glutamic acid;

aa38 is an aliphatic, neutral, substituted amino acid, wherein the substituent is carboxamide and has 4 to 5 carbon atoms or is an acidic amino acid such as asparagine, glutamine, aspartic acid or glutamic acid;

aa39 is an aromatic amino acid or a neutral aliphatic amino acid having 3 to 4 carbon atoms, usually with a hydroxyl substituent, preferably it is aromatic and includes phenylalanine, histidine, tyrosine, serine or threonine;

aa40 is a neutral, substituted or unsubstituted, aliphatic amino acid of 3 to 6 carbon atoms or a basic amino acid such as alanine, valine, isoieucine, glutamine or arginine;

m and n are 0 or 1;

pp3 can represent a polypeptide chain of no more than 1000 amino acids in total and pp4 can be hydrogen and form the end of the polypeptide.

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pp3 and pp4 can also represent the same or different polypeptide chains of no more than 1000 amino acids in total. The polypeptide chains normally have no more than about 500 amino acids, preferably at least 90% of the amino acids, in particular at least about 95% of the amino acids present, are present in one of the two polypeptide chains; in some cases the chain can consist of only one amino acid and comprise no more than 100 amino acids, often no more than about 50 amino acids, depending on the use of the polypeptide and the role of the extended chain; the polypeptide chains may be related to naturally occurring polypeptides associated with naturally occurring growth factors and smallpox virus proteins, or may be different from naturally occurring chains or fragments thereof which may be different from the polypeptide chain specifically represented in the formula, normally unrelated, with m, n, and pp4 selected so that the third region has at least 14 amino C-terminal amino acids that begin at amino acids aa25 through aa29 and end at aa4 "through aa53.

The definitions of the amino acids are set out below.

Neutral (Ne)

aliphatic (AI)

unsubstituted G, A, V, L, I, P substituted oxy S, T

thio C, M

amido N, Q aromatic (Ar)

unsubstituted F

substituted Y

heterocyclic H, W

Loaded (at pH 6.0)

basic (Ba) K, R

acidic (Ac) D, E

The abbreviations in brackets refer to special amino acid groups. The unsubstituted hetero substituents should not be present other than the carboxy and amino groups present in the glycine. The amino acids are naturally occurring L-amino acids.

The neutral amino acids can also be described as those with non-polar or polar (but uncharged) R groups. The amino acids that fall under these definitions are as follows:

non-polar amino acids A, V, L, I, P, M, F, W polar amino acids G, S, T, C, Y, N, Q

The loop between the cysteines at positions 28 and 37 in the above formula has 0 or 1 acidic amino acid (S). The amino acids directly flanking the cysteines (i.e., amino acids 27 and 38) outside the loop can include at least one charged amino acid and at least one substituted aliphatic amino acid. Of the amino acids of the loop (28 to 37), 4 to 6, usually 5 amino acids are neutral, aliphatic amino acids and no more than two, usually one, are aromatic amino acids.

Of particular interest are compounds in which the polypeptides have less than 130 amino acids, in particular less than 55 amino acids and at least 44 amino acids, preferably at least about 53 amino acids. Of particular interest are polypeptides which contain amino acids 44 to 88 and fragments thereof from VGF (aa1 to aa47, see FIG. 1).

Preferably aa29 is a substituted or unsubstituted aliphatic amino acid having 3 to 6 carbon atoms with 0 to 1 hydroxyl group, in particular serine or valine, or an aromatic amino acid, in particular histidine.

aa30 is preferably histidine, serine or an unsubstituted amino acid of 5 to 6 carbon atoms, especially isoieucine;

aa33 is preferably a substituted or unsubstituted aliphatic amino acid with 0 to 1 hydroxy groups and with 3 to 6 carbon atoms;

aa35 is preferably an aliphatic amino acid having 3 to 6 carbon atoms;

aa38 is preferably glutamine or glutamic acid;

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aa39 is preferably aromatic, especially histidine or phenylalanine;

aa40 is preferably a neutral or basic amino acid.

Of particular interest is the presence of two loops, the C-proximal loop being connected to the second loop, the amino acids of the second loop being able to vary widely. The second loop preferably has 14 to 16 amino acids, excluding the cysteine bridge, in particular about 15 amino acids. Of the amino acids, 6 to 9, preferably 7 to 9, in particular about 8, aliphatic amino acids, which can be substituted or unsubstituted, preferably not more than about three of the amino acids are substituted, in particular 1 to 2 amino acids are substituted; there are 2 to 4 aromatic amino acids, in particular three aromatic amino acids, histidine and tyrosine are desired; there are 2 to 4 acidic amino acids, preferably 3 acidic amino acids, in particular aspartic acid and there are 0 to 2, normally 1 basic amino acid, in particular arginine. Desirably, the cysteine, which is the main loop closest to the cysteine of the other loop, is separated by 0 to 2, usually 0 to 1, especially arginine.

Compounds of the following formula are of particular interest for some applications:

Y L

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

N

H- (aa12a) p-G-aa14-C-aa16-Ar1- (aa17a) p- (aa17b) p aa18_aa19_aa20-.aa21_aa22_ (aa22a) (aa22b) p_aa23

N

aa24- (aa25-C-aa27-C-aa29-aa3 ° -G-Y-aa3 3-G-aa35 Q

R-C-E-aa39-aa4 0) -aa4 i-L-aa43-aa44-pp2

wherein the symbols aa25 to aa4 ° have the above meaning; pp1 and pp2 are the same or different and can represent hydrogens which are the terminal part of the specified polypeptide or can be polypeptides with a total of up to 1000 amino acids, normally up to about 500 amino acids and can have only one amino acid in total or can be individual or separate polypeptides be from 1 to 100 amino acids, usually from 1 to 75 amino acids, especially about 5 to 50 amino acids; these polypeptides have specific applications for changing the specifically described sequence for a predetermined purpose; pp1 and pp2 can be the same or different from natural smallpox virus polypeptide, usually different;

aa1 can be any amino acid, especially an aliphatic amino acid, a basic amino acid or an acidic amino acid, preferably an unsubstituted aliphatic amino acid with 2 to 6 carbon atoms, including glycine, leucine, valine, lysine, glutamic acid and aspartic acid;

aa3 is a neutral amino acid, in particular with 2 to 4 carbon atoms, in particular asparagine, glycine and proline;

aa4 can be any amino acid, aliphatic or aromatic, especially 2 to 6 carbon atoms, which can be neutral or acidic, especially 3 to 6 carbon atoms, including proline, asparagine, aspartic acid, histidine, serine and leucine;

aas is an acidic or neutral, substituted, aliphatic amino acid, especially glutamic acid or aspartic acid or a hydroxy-substituted amino acid with 3 to 4 carbon atoms, especially serine;

aa6 is an unsubstituted, aliphatic amino acid with 2 to 6, usually 2 to 3 carbon atoms or an aromatic amino acid, especially glycine, histidine or tyrosine;

aa7 is an acidic, basic or neutral amino acid, in particular with 3 to 6 carbon atoms, such as glutamic acid, aspartic acid, lysine and threonine;

aa8 is a neutral, unsubstituted, aliphatic amino acid with 2 to 3 carbon atoms or a carboxamide substituted amino acid with 4 to 5 carbon atoms, e.g. Asparagine, glutamine or glycine;

aa11 is a neutral amino acid, either an aliphatic or aromatic amino acid, in particular an aliphatic, preferably having 5 to 6 carbon atoms and particularly preferably leucine or phenylalanine;

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aa12 is an aromatic amino acid or a carboxamide-substituted, aliphatic amino acid with 4 to 5 carbon atoms, especially histidine or asparagine;

aa12 is a neutral, aliphatic amino acid or an acidic amino acid, in particular glycine, asparagine or aspartic acid aa14 is an acidic amino acid or a neutral, aliphatic amino acid which is substituted or unsubstituted with 4 to 5 carbon atoms with 0 or 1 hydroxyl group, in particular aspartic acid, threonine or Vaiin;

aa16 is a bond or a neutral or basic aliphatic amino acid, either substituted or unsubstituted with 4 to 6 carbon atoms, in particular isoieucine, arginine or methionine;

Ar1 represents an aromatic amino acid which may have a carbocyclic or heterocyclic ring and which includes histidine, phenylalanine or tyrosine;

aai7a is a neutral, aliphatic amino acid, either substituted or unsubstituted with 3 to 6, usually 3 to 5 carbon atoms with 0 to 1 hydroxyl groups and includes Vaiin, Leucine, Isoieucine and Threonine;

aa17b is a neutral, unsubstituted amino acid with 5 to 6 carbon atoms or an acidic amino acid, in particular valine, isoieucine or glutamic acid;

aa18 is a neutral, aliphatic amino acid, substituted or unsubstituted with 2 to 6, preferably 3 to 6 carbon atoms with 0 to 1 hydroxy substituents, in particular alanine, serine or glutamine;

aa19 can be any amino acid, especially an aliphatic or basic amino acid, preferably with 4 to 6 carbon atoms and particularly preferably with 5 to 6 carbon atoms, for example arginine, leucine, Vaiin and glutamic acid;

aa20 is an acidic amino acid or an aliphatic carboxamide-substituted amino acid and includes aspartic acid, asparagine, glutamic acid or glutamine;

aa21 is a neutral, unsubstituted, aliphatic amino acid or a basic amino acid of 3 to 6 carbon atoms and has 0 or 1 hydroxyl or carboxamide group and includes isoieucine, leucine, asparagine, serine, threonine, lysine and arginine;

aa22 is a bond or an acidic amino acid which is aspartic or glutamic acid or a neutral aliphatic amino acid, especially valine;

aa223 is a neutral, aliphatic amino acid with 0 or 1 hydroxyl group, in particular a hydroxyl group and preferably serine;

aa22b igt a neutral, aliphatic amino acid with 4 to 6 carbon atoms, in particular leucine or isoieucine;

aa23 is a bond or a neutral aliphatic amino acid which is substituted or unsubstituted and has 2 to 6 carbon atoms and 0 to 1 hydroxyl substituent, for example glycine, serine, threonine and asparagine;

aa24 is an aliphatic amino acid, in particular a thio-substituted, aliphatic amino acid, preferably methionine, proline or an aromatic amino acid, in particular tyrosine;

aa41 is a neutral, substituted or unsubstituted, aliphatic or acidic amino acid with 4 to 6 carbon atoms, in particular valine, asparagine or aspartic acid;

aa43 is a neutral, aliphatic, acidic or basic amino acid, which can be substituted or unsubstituted with 4 to 6 carbon atoms and includes valine, leucine, isoieucine, arginine, lysine and aspartic acid;

aa44 can be any amino acid, especially other than a basic amino acid, and can be an acidic, neutral or aromatic amino acid and, if not aromatic, have 3 to 6 carbon atoms and especially be aspartic acid, alanine, leucine, tryptophan or threonine: and p Is 0 or 1.

It is of particular interest if pp1 has the following sequence:

pp1'-aa-24-aa-23-aa-22-aa-21-aa-20-aa- '| 9-aa-'18

-aa-17-aa-16-aa-15-aa-i4-aa-13-aa-12-aa-11

aa-10-aa-9-aa-8-aa-7-aa-s-aa-5-aa-4-aa_3-aa-2-aa_1

where pp1 'in combination with the amino acid signs following in this sequence is equivalent to pp1. The above sequence falls within the definition of pp1; pp1 'can be hydrogen or an amino acid sequence. One or more amino acids represented by aa- "(x is any number) can be a bond so that it serves to reduce the number of amino acids in the N-terminal chain. As a result, all or part of it can If a part of the sequence is present, the adjacent amino acids from the sequence are also involved, ie the amino acids in their numerical order without deletions. Normally at least one amino acid is present and usually at least 3 and preferably at least 6 when the remaining upstream amino acids are absent so that pp1 'is connected to aa-1, aa-6 or the like;

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aa-1 can be any amino acid, especially an aliphatic one with 3 to 6 carbon atoms, preferably a neutral or basic one, especially lysine, arginine, proline, asparagine or serine;

aa-2 can be any amino acid, either an aliphatic or aromatic, especially an aliphatic, preferably having 3 to 6 carbon atoms;

aa-3 can be an aliphatic or aromatic, especially an aliphatic, either polar or non-polar having 2 to 6, usually 2 to 5 carbon atoms and generally has 0 or 1 hydroxyl substituents;

aa-4 can be any aliphatic amino acid, neutral or basic, generally with 3 to 6 carbon atoms, preferably with 5 to 6 carbon atoms and e.g. Asparagine, proline or lysine;

aa-5 can be any aliphatic amino acid, especially a neutral one, generally from 3 to 6 carbon atoms, especially valine or isoieucine;

aa-6 is a neutral or acidic amino acid, if it is aliphatic, with 3 to 6, usually with 4 to 6 carbon atoms, for example isoieucine, valine or aspartic acid;

aa-7, aa-8, aa-9, aa-12, aa-14, aa-15, aa-16, aa-17, aa-19, aa-20, aa-21 and aa-23 are aliphatic amino acids with 3 to 6 carbon atoms, either polar or non-polar, in particular polar with 0 or 1 hydroxyl substituents or aromatic;

aa-10 and aa ~ 25 are non-polar, aliphatic amino acids or acidic amino acids with 2 to 6, usually 2 to 3 carbon atoms;

aa-13, aa-22 and aa-24 are polar, aliphatic amino acids or acidic amino acids with 4 to 5 carbon atoms, in particular with a carboxamide substituent;

aa-11 and aa-18 are aliphatic amino acids, either polar or non-polar or acidic amino acids. Of particular interest is an N-terminal fragment with aa-1 to aa-24 or aa-1 to aa-7, in particular aa-1 to aa-6. pp1 'can conveniently be any amino acid sequence that can serve as a fusion protein, particularly to provide an immunogenic peptide for the production of antibodies to the compound.

The primary aspect of the compositions according to the invention are the sequence aa25 to aa40, in particular the sequence from the cysteine in position 28 to the cysteine in position 37 and the loops formed by the cysteines in positions 2 and 10 and the cysteines in positions 15 to 26 will. The compositions according to the invention preferably have a loop which is created by the cysteines in positions 28 and 37 in conjunction with the loop which is formed by the cysteines in positions 15 to 26.

Chimeric polypeptide sequences can be made by combining fragments of different polypeptides, which have a sequence substantially similar to the naturally occurring polypeptide chains, which are associated with naturally occurring growth factors, and poxvirus proteins. The resulting chimeric polypeptides have about 40 to 65 amino acids, usually about 45 to 60 amino acids and especially 49 to 53 amino acids. In any case, the frame structure is maintained by the cysteines by means of the cysteine bridges, which define the loops of the sizes defined above. Accordingly, any fragment of a growth factor can be used which is essentially homologous to VGF (see, for example, FIG. 1) or another growth factor which has the same framework structure as the compositions according to the invention and has a similar physiological activity. This is irrespective of the mammalian source, for example of primates such as humans, rodents such as rats and mice, cattle, birds, pigs etc.

Up to three compounds may be required, depending on the source of the fragments and the length of the polypeptides desired. It is desirable that the connections be at a point between aa-6 and aa1; aa11 and aa1 ^; aa25 and aa29 and aa42 and aa53 can be made. The sequences between about aa-6 and about aa15 are referred to as N-terminal sequences; those between about aa14 and about aa29 are called the central fragment; and those between about aa25 and the end of the peptide (generally aa44 to aa47) are referred to as the C-terminal region. The exact connection point varies depending on the location of the restriction sites etc. In addition, an extremely N-terminal sequence, which contains about aa-24 to aa-7, in particular aa-23 to aa-7, can also be linked to form a chimeric polypeptide.

The fragments generally contain 15 to 50, normally 15 to 45 amino acids, since aa20 is not the twentieth amino acid of the compound, but only the specific sequence that has been specifically defined (see FIG. 1).

Various convenient restriction sites have been incorporated into the synthetic genes used to construct the chimeric polypeptide. If possible, the restriction sites leave the amino acid sequence of the growth factor gene unchanged. In some cases, however, the incorporation of the new restriction site leads to an altered amino acid sequence. It is of particular interest if the coding sequence for VGF for the introduction of a Kpnl restriction site is changed from GDC to GTC by changing the amino acid sequence aa13 to aa15 and for the introduction of an Sphl site in aa23 to aa28 by the sequence GMYCRC is converted to GYACVC. These changes result in convenient sites for association with fragments of the VGF gene with fragments from other growth factors. For example, the gene fragment

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aa26 to aa47 are conveniently linked to a gene fragment from aipha-TGF encoding amino acids corresponding to residues aa ~ 6 to aa2 ^ to obtain a TGF / VGF hybrid polypeptide. Accordingly, the sequence of the chimeric peptide contains aa-6 to aa13 (tN-terminal) and aa14 to aa2s (central fragment) which is derived from alpha-TGF and aa26 to aa47 (C-terminal) derived from VGF as modified above.

The preparation of a plasmid capable of expressing chimeric proteins having amino acid sequences derived from fragments of more than one growth factor or a poxvirus polypeptide with sequence changes necessary to introduce convenient restriction sites are described in detail in the experimental section .

Fusion proteins can also be made when expressing a growth factor or chimeric polypeptide linked to the N-terminal amino acids of a signal sequence or to N-terminal amino acids of highly expressed bacterial bacteriophages or eukaryotic genes. In addition, an enzymatically or chemically foldable site can be provided after the leader sequence to cleave the mature polypeptide from the leader sequence. The preparation of plasmids which are capable of expressing these fusion polypeptides and methods for the cleavage and isolation of the desired polypeptide are described in detail in the experimental part.

Production of growth factors

The main compositions can be made in various ways depending on the size of the composition. In particular below about 80, usually below 60 amino acids, the composition can be produced by means of conventional syntheses. See, for example, Merrifield, Solid Phase Peptide Synthesis "The Peptides Analysis, Synthesis Biology", Special Methods in Peptide Synthesis, Part A, Vol. 2, Gross and Meinhofer, ed. Academic Press, NY, 1980, pages 1-284. See also US-A 4,127,526.

Alternatively, the use of hybrid DNA technology can be used if DNA sequences can be used which encode the desired polypeptide or a precursor thereof. The DNA sequences encoding the growth factor of interest can be synthesized using conventional techniques, for example, as an overlap of individual strands that can be joined together to form the desired coding sequence. The terms can be designated to provide restriction sites, or one or both terms can be blunt-ended for connection to complementary ends of an expression vector. An initial methionine is required for the expression of the sequence. The expression vectors are generally available and have been extensively described in the literature.

Instead of synthesizing a gene of interest, the growth factor can be isolated by various techniques. For example, if the growth factor is a viral growth factor, the mRNA can be isolated from the virus encoding the polypeptide including the growth factor, the mRNA can be transcribed in reverse, the resulting single-stranded (ss) DNA used as a template to create a double-stranded (ds ) DNA and the ds-DNA are isolated. Another technique is to isolate a piece of the viral DNA and conveniently isolate it using a sample containing a region of the most conserved sequences in a growth factor gene to identify the sequences encoding a factor in the viral genome. The sample may be shorter than the entire sequence, but should be at least 10, preferably 14 and most preferably 20 nucleotides in length. Longer oligonucleotides are also useful up to the full length of the growth factor morning. Both DNA and RNA samples can be used.

When used, the samples are typically labeled in a traceable manner (for example with 32p or biotin-labeled nucleotides) and are incubated with a single-stranded DNA or RNA from the organism in which the gene is sought. Hybridization is demonstrated by typically, using nitrocellulose paper with the label after the single-stranded and double-stranded (hybridized) DNA (or DNA / RNA) have been separated. Hybridization techniques which are expedient for use with oligonucleotides are known to the person skilled in the art.

Although samples are normally used with a detectable label that allows easy identification, unlabeled oligonucleotides are also useful, both as precursors to labeled samples or for use in methods that are useful for the direct detection of double-stranded DNA (or DNA / RNA) are. As a result, the term “oligonucleotides” refers to both the labeled and the unlabeled forms.

Once a desired DNA sequence has been obtained, it can be manipulated in a variety of ways to achieve expression; for example, chimeric polypeptides can be produced by combining gene fragments of at least two polypeptides that have sequences that are at least above 30% homologous to naturally occurring growth factors that bind the EGF receptor (“natural ligands”). It is desirable that the three-dimensional structure, in particular the loop structure of the polypeptide, is retained, in particular that

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Part or those parts of the structure which are responsible for the binding to the EGF receptor and for the biological activity of the naturally occurring growth factors which bind to the EGF receptor.

Depending on the source of the fragments and the length of the desired polypeptide, desired restriction sites can be incorporated into the synthetic genes used to construct the chimeric polypeptides described above. Whenever possible, the restriction sites leave the amino acid sequence of the polypeptide unchanged, but in some cases the incorporation of new restriction sites can result in an altered amino acid sequence without changing the activity of the protein.

If the gene is expressed in a host that recognizes the transcriptional and translational, regulatory regions of the wild-type growth factor, its entire genes with its 5 'and 3' regulatory regions of the wild-type can be incorporated into a suitable expression vector. There are numerous expression vectors using mammalian virus replication systems such as Simian virus 40, adenovirus, the bovine papilloma virus, vaccinia virus, insect baculovirus, etc. These replication systems have been developed to provide labels that allow the selection of transvection agents, as well as to provide suitable restriction sites that can be incorporated into the gene.

If a gene needs to be expressed in a host that does not recognize the natural, transcriptional and translational, regulatory areas of the wild type, further manipulations are required. A number of convenient 3'-transcriptional regional regions are known which can be incorporated downstream of the stop codon. The 5 'non-coding regions that are downstream of the gene of interest can be removed by endonuclease restriction, Bal31 resection, or the like.

If, on the other hand, there is a convenient restriction site near the 5 'terminus of the gene of interest, the gene of interest can be restricted and an adapter can be used to connect the gene of interest to the promoter region if the adapter is for the lost one Nucleotides of the gene of interest is required. Numerous strategies can be used to obtain an expression cassette which has a transcriptional regulatory region and a translational start region in the 5'-3 'direction of transcription, which may include regulatory sequences for initiation of regulation; genes of interest under transcriptional and translational control of the start region; and a transcriptional and translational final region.

The following factors, which can influence expression, must be taken into account for the appropriate selection of regulating sequences. For the transcriptional regulation, the amount and the stability of the messenger RNA are important factors which influence the expression of the gene product. The amount of mRNA is determined by the number of copies of the particular gene, the relative efficiency of its promoter, and the factors that regulate the promoter, such as enhancers or repressors. It is assumed that the start takes place in the region which is immediately upstream from the start of the coding sequence.

The promoter in prokaryotic cells contains nucleotide sequences that can impair the efficiency of the transcription. The sequences span 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 essentially the same as corresponding sequences for these regions in bacterial promoters of highly efficient genes. Generally, these regions are about 5 nucleotides and 6 nucleotides in length, respectively, and each sequence can vary in length or in sequence by about one nucleotide. A preferred sequence of the -35 match regulatory sequence comes from the trp promoter, namely TGACA, and for the -10 match regulatory sequence from the lac promoter, namely TATAAT.

Not only the nucleotide sequences, but also the interspaces between matching sequences -35- and -10-regulating regions related to each other are important for maintaining the optimal transcription of the gene of interest. In general, the matching sequences of the -35 and -10 regulatory regions are separated by about 16 to 18 nucleotides, preferably by about 17 nucleotides. Each sequence can vary by approximately 1 nucleotide.

Illustrative, transcriptional regulatory regions or promoters for bacteria include the β-gal promoter, left and right lambda promoters, trp and lac promoters, trp-lac fusion promoters, etc .; Synthetic promoters, the sequences of which are substantially similar to these sequences, can also be used. A preferred promoter for bacteria is a fusion promoter which has the -35 regulatory region of the trp promoter and the -10 regulatory region of the lac promoter. The promoter is particularly preferably one in which the -35-trp match sequence is approximately 17 nucleotides upstream of the -10-lac match sequence. For Hese, glycolytic enzyme promoters such as ADH-I and -II promoters, GPK promoter and PGI promoter, trp promoter, etc .; for SV40 mammalian cells, early and late promoter, the late adenovirus major promoter, 15 promoter or enhancer sequences and the like.

The transcriptional regulatory region may include additional regulatory sequences that allow moderation of the expression time of the gene of interest, e.g. through present or

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Absence of nutrients or expression products in the growth medium, temperature, etc. For example, the expression of the gene of interest can be determined by the temperature of the host cell growth medium by adding a regulating sequence including the bacteriophage lambda PL promoter, the bacteriophage lambda OL operator and the gene CI857 , which encodes the temperature-sensitive Ci receptor in the expression vector, are regulated. This allows regulation of the promoter through interaction between the receptor and the operator at low temperatures, for example at about 30 ° C. Increasing the temperature to about 42 ° C would inactivate the receptor and the gene of interest could be expressed.

An example of moderation using growth medium nutrients, regulation of the iac or trp-lac hybrid promoter can be performed using the gene for the Lacl receptor that targets the lac promoter in the region downstream from the -10 -regulating region binds. The Lacl receptor gene can be present on an episome, preferably the Lacl-enhanced mutant, or can be contained in the expression cassette itself. The presence of a significant concentration of the repressor molecule in the growth medium inhibits the promoter function in the absence of an inducer. Accordingly, the addition of IPTG or lactose to the host cell growth medium improves the promoter function. If the bacterial strain is Lac +, lactose can be used as an inducer instead of IPTG. In both eukaryotic and prokaryotic systems, the termination regions can also contain sequences or structures which increase the stability of mRNA types and bring about higher expression. Accordingly, the transcriptional, regulating region can additionally contain regulating sequences which terminate the transcription and which have sequences or structures which inhibit the degradation of the mRNA and consequently increase the stability of the mRNA types and thus bring about a higher expression. Various examples of prokaryotic sequences are known, for example the Trp terminator, the gene 32 (T4) terminator or synthetic terminators which have a sequence similar to that of the gene 32.

In eukaryotic systems, the transcriptional termination, the RNA cleavage and the polyadenylation regions bring about self-maturation for the mRNA transcripts and are therefore necessary for efficient expression. The native 3'-untranslated region may be sufficient, but the polyadenylation signal of, for example, SV40, which in particular has a spitting site, which brings about more efficient expression, could also be used. Alternatively, the 3'-untranslated region expressed by a particular cell type (e.g. Ig in myeloma cells) could be fused to the gene of interest. Such 3 'sequences could also be paired with 5' regulating sequences from the same highly expressed genes and could even include in-frame coding regions to form fusion proteins as described above.

For translational regulation, which is given by the presence of mRNA, the ex-priming by influencing the degree of initiation (ribosome binding to the mRNA), the degree of extension (transport of the ribosome by mRNA), the degree of post-translational changes and the stability of the gene product are influenced. The degree of elongation is likely to be affected by codon use; the use of the codon for side tRNA can reduce the degree of translation. Accordingly, it is preferred to use codons that appear more frequently in genes that are normally expressed by host cells to improve the level of translation.

in prokaryotes there is a match sequence, generally AGGA, called the Shine-Daigarno sequence, downstream of the -35 and -10 regulating regions, which is believed to be involved in ribosomal binding. Optimal binding and initiation of translation can be achieved using a ribosome binding site that is functional in host cells of highly expressed genes. The evidence also indicates the presence of nucleotide sequences around the Shine-Daigarno sequence and sequences within the coding region that can affect ribosome binding, possibly through the formation of structural motifs by which the ribosome recognizes the initiation site. The change in nucleotide sequences of the coding region can therefore be used to achieve optimal binding and initiation of translation. The sequence of the first approximately 7 to 30 codons after the initiation codon ATG can also impair binding and expression. Preferably, the leader sequence and the Shine-Daigarno sequence are obtained from the same gene or if they are obtained from different genes, the codons of the leader sequence can be changed by codon degeneration to approximate a nucleotide sequence of the natural gene which follows the leader sequence, like this in the parallel US Patent Application Serial Number 264,098 dated October 28, 1988, to which express reference is made.

The position of the AGGA sequence with respect to the initiation ATG codon can influence expression. In general, the Shine-Daigarno sequence is about 5 to 9 nucleotides from the initiation codon, although unexpectedly a high level of expression can be achieved when using expression cassettes, where the Shine-Daigarno sequence is preferably about 10 to 13 nucleotides, preferably 11 to 12 nucleotides away from the initiation codon.

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The stability of the mRNA depends on the sensitivity of the mRNA to ribonuclease enzymes. In general, exonuclease digestion is inhibited by the presence of structural motifs at the end of the mRNA, relapse structures, altered nucleotides or specific nucleotides. It is believed that endonuclease digestion occurs at specific identification sites within the mRNA and that stable mRNA does not have such sites. There is also some evidence that mRNAs that have a high degree of translation are also protected against degradation by the presence of ribosomes on the mRNA.

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 a fragment thereof. The stability of the expression product is preferably achieved by providing a synthesis of a fusion product in which the polypeptide of interest is expressed in association with a low sequence. A DNA sequence, which is an N-terminal amino acid sequence of, for example, a highly expressing bacterial or bacteriophage gene, such as the bacteriophage lambda N protein gene, cro gene and ss-galactosidase or a eukaryotic gene, is upstream of and in the reading frame with linked to the gene of interest. The leader sequence normally contains 8 to about 50, preferably 50 to 45 and particularly preferably 18 to 25 N-terminal amino acids.

The expression of the polypeptide of interest as a fused protein with a leader sequence from another gene has several advantages in addition to the stability conferred. For example, the presence of the N-terminal amino acids provides a means of using general purification techniques to purify a number of polypeptides. For example, the N-terminal amino acids of the N protein are particularly antigenic and consequently specific antibodies which act against the N-terminal amino acids of the N protein as agents for the purification of fusion proteins which contain the N-terminus of the N protein. can be used. Furthermore, the N-terminus of the N-protein has a high positive charge, which facilitates the purification of the desired protein by ion exchange chromatography and the like.

The leader sequence can also be a hydrophobic amino acid sequence, which additionally serves as a signal sequence for the secretion. A DNA sequence encoding the signal sequence is linked upstream of and in the reading frame to the gene of interest. Typically, the signal sequence comprises a cleavage site which is recognized by a signal sequence peptide dase. Accordingly, the positioning of the polypeptide of interest immediately after the signal sequence cleavage site allows the polypeptide of interest to be specifically cleaved from the signal sequence and excreted 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 sequence, β-lactamase signal sequence; and toxin signal sequences and mutants thereof. For eukaryotic cells, signal sequences can be the signal sequences of the monkey TGF-ß, P97 gene (human melanoma antigen), K. lactis killer toxin sequence. Alpha mating type factor and other killer toxin signal sequences, or the normal signal sequence associated with the gene of interest, along with mutants of signal sequences.

Other leader sequences that can be used include hydrophilic sequences, such as the N-terminal 41 amino acid residues from amphiregulin, which can cause the function of the polypeptide of interest to change. In addition, a cytotoxic agent such as a toxin A chain fragment, the ricin A chain, the growth-inhibiting peptide of snake venom, or a targeted molecule such as a hormone or an antibody can be covalently coupled to the leader sequence, in most cases a minimal effect on biological activity is exerted on the gene product of interest. As described for other leader sequences, a DNA sequence encoding the leader sequence is linked upstream of and in the reading frame with the gene of interest.

If the leader sequence is not a signal sequence or does not have a convenient natural cleavage site, additional amino acids can be inserted in order to insert an enzymatically or chemically cleavable part for the cleavage of the leader peptide after the purification of the fusion protein in order to subsequently purify the mature one. To enable polypeptide. For example, the introduction of acid labile aspartyl-proline bonds between two segments of the fusion protein facilitates their separation at low pH. This method is not useful for the desired polypeptide if it is acid labile. In a further example, the fusion protein can be cleaved with, for example, cyanogen bromide, which is specific for the carboxy side of the methionine residue. The insertion of a methionine between the leader sequence and the desired polypeptide enables the desired polypeptide to be released. This method is not practical if the desired polypeptide contains methionine residues.

If the leader sequence contains a signal sequence, the genes of interest can be expressed with the secretory leader sequences with or without a leader sequence, provided that the sequence can be retained or cleaved off. In addition, in order to obtain a high proportion of the desired polypeptide as a mature cleaved and refolded peptide which is secreted in the medium, it is desirable to use a promoter, such as the tac promoter, which is used at low temperatures, for example at about 30 ° C , can work. Unexpected

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Higher amounts of the secretion could be obtained at low temperatures. Extremely high levels of expression can prevent all translational changes in the protein, leading to the aggregation and accumulation of uncleaved precursors (i.e. structural protein and secretory leader). Similarly, growth at elevated temperatures, for example at 42 ° C, can also lead to the aggregation and accumulation of unpaired precursor. The production of fused proteins, including the processes for the cleavage and refolding of the polypeptides of interest, are further described in parallel U.S. Patent application Serial Number 264 098.

After each manipulation of DNA in the development of the cassette, the plasmid is cloned and isolated as required, the particular cassette component is analyzed for its sequence to ensure that the own sequence has been obtained. Depending on the nature of the manipulation, the desired sequence can be separated from the plasmid and introduced into another vector or the plasmid can be subjected to a restriction and the expression cassette component can be manipulated appropriately. In some cases, a shuttle vector can be used which is capable of performing replication in different hosts requiring different replication systems. This may require additional labeling agents that work in both hosts. If such labeling agents are required, they can be incorporated into the vector. The plasmid containing the cassette, two replication systems and labeling agents can be transferred from one host to another if necessary. Any useful marker can be used for the selection. It is desirable that it be resistant to neomycin or tetracycline. Although a marker for selection is highly desirable for convenience, other methods of selection for transformed cells have also been described. See, for example, G. Reipin, et al., Current Genetics, 1982, 189-193. The transformed cells can also be subjected to a screening process by means of specific products, for example the synthesis of a desired product can be determined by immunological and enzymatic methods.

The expression cassette can be incorporated into a replication system for episomal maintenance in a convenient cellular host or can be used without a replication system when integrated into the host genome. The DNA can be introduced into the host in a conventional manner, such as by transformation using calcium phosphate-precipitated DNA, transvection by contacting the cells with the virus, microinjection of the DNA into cells, or the like. Once the gene of interest has been incorporated into a convenient host, the host can grow to express the gene of interest.

A variety of host cells can be used. Examples of prokaryotic cells include gram negative organisms such as E. coli, for example JM109, JM101 and JM107; HB101, DH1 or DH5. Gram-positive organisms such as B. subtilis are particularly useful. which have no peripheral space and can directly excrete polypeptides into the growth medium. Eukaryotic host cells can be, for example, yeast cells, insect cells and mammalian cells, for example COS cells, CHO cells, monkey kidney cells and silkworm cells (sf9).

The construction, the gene of interest and flanking regions responsible for regulating expression can be introduced into the expression host by any conventional means, for example by transformation, for example with calcium phosphate-precipitated DNA, by transfection, transduction, conjugation, Microinjection, etc. The host can then be grown in high density in an appropriate nutrient medium. If the promoter is insertable, allowable conditions are used, such as change in temperature, fatigue, or excess of a metabolic product or nutrient, or the like.

For example, if the regulatory sequence contains the bacteriophage lambda PL promoter, the bakteriophagen lambda OL operator and the temperature-sensitive CI857 repressor, the host cells can grow unhindered by the demand for the synthesis of the foreign gene product, which is also toxic to the host organism can be. When the host cells have reached an optimal density, the temperature can be raised to an unacceptable temperature, for example to about 42 ° C., at which moment the Cl repressor becomes inactive, which allows transcription from the PL promoter. Maximum secretion can be obtained using the lac promoter or a trp-lac promoter and inducing with a metabolic inducer such as lactose for a lac + host strain and resulting in lac Ii on the vector. Examples of host cells that can be used in such a system include DH1, DH5 or HB101.

When the product is retained in the host cell, the cells are harvested, lysed and the product isolated and purified, e.g. by extraction, precipitation, chromatography or electrophoresis. If the product is excreted, the nutrient medium can be collected and the product can be isolated in the usual way, for example by affinity chromatography. To produce an active protein, the protein may need to be refolded. If the protein is expressed as a fusion protein with a low sequence, the low sequence can be removed by treatment with, for example, formic acid or cyanogen bromide. The leader sequence is preferably removed after the protein is refolded.

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The recombinant products can optionally be glycosylated with wild-type glycosylation or another glycosylation. In general, glycosylation differs from wild type glycosylation by no more than 50% and usually no more than 20%. The proportion of glycosylation depends partly on the sequence of the particular peptide and also on the producing organism. Accordingly, expression of the product in E. coli cells results in an unglycosylated product and expression of the product in insect cells usually results in a less glycosylated product than expression of the product in mammalian cells.

Use of growth factors

The compositions according to the invention find a large number of applications in vitro and in vivo as agonists or antagonists for growth factors, such as epidermal growth factors (EGF) and transforming growth factors, in particular alpha-TGF. A discussion of growth factors which are used by themselves or in combination with other compositions, in particular polypeptide compositions for regulating the growth of lines and other activities, can be found, for example, in the Handbook of Experimental Pharmacology, Tissue Growth Factors, ed. Baseraga, volume 57, Springer-Verlag, Berlin, 1981, Chapter 3, in particular pages 98-109; and Carpenter, Ann. Rev. Biochem. (1979) 48: 193-216.

The human EGF appears to be identical to the human urogastron and exerts a variety of effects on prenatal and neonatal tissue growth. Such effects are opening the eyes early, healing the wounds, breaking out of the front teeth and accelerated maturation of the lungs. EGF receptors can be found in a variety of adult tissues. EGF can also stimulate the phosphorylation of its own receptor. EGF is also related to improved bone resorption.

The transforming growth factor, especially alpha-TGF, has many analogous activities to those of EGF. TGF binds to the EGF receptor, which leads to phosphorylation of the receptor, improvement of its tyrosine-specific kinase activity and stimulation of cell growth. Cohen, in Biological Response Mediators and Modulators (Ed. August, J.T.), Acade-mic, New York, 1983, pages 7-12; Tarn et al., Nature (1984), 309: 376-378; Ibbotson et al., Science (1983) 221: 1292-1294.

The Myxom and Shope fibroma viruses induce a significant increase in the proliferative potential of cells within the affected area. While vaccinia induces a smaller proliferative event at the site of infection, the Shope fibroma virus induces the main proliferative event, resulting in tumor formation which is benign in adult rabbits but proliferative and invasive in immuno-suppressed adults or in newborns. The myxoma virus induces a rapidly spreading myxosarcoma tumor. Although these proliferative events could be caused by other viral factors, the viral growth factor appears to be heavily involved in the induced cellular proliferation caused by the viral infection.

The compounds according to the invention can be used in particular as medicaments, as agonists for EGF and for wound healing, such as for the epithelization of wounds, such as for burns, eye wounds, surgical cuts and the like. The active ingredient can be present in customary carrier substances, for example Silvaden, in a sufficient proportion for epithelization and / or wound healing, normally in proportions of 0.01 to 10.0 ng / ml, usually 0.075 to 5.0 ng / ml EFG equivalents , preferably 0.5 to 5 pg / ml can be used. The formulation is spread over the wound so that a complete coating of the wound with the formulation is obtained. Treatments can be performed up to four times a day or only every other day or less, depending on the nature of the wound and the response to the treatment, the concentration of the active ingredient, and the like.

The compositions according to the invention can be used as active ingredients in diagnostic tests or for the preparation of reagents, for example for polyclonal or monoclonal antibodies. As active ingredients, they can be used for the detection of analog growth factors or for the detection of antibodies to the growth factors in physiological liquids, such as blood.

Depending on the particular circumstances and the purpose of the reagent, the polypeptide may be labeled or unlabeled. A large number of marking substances have been used which directly or indirectly generate a detectable signal. These labeling substances include radionuclides, enzymes, fluorescent substances, particles, chemiluminescent substances, enzyme substrates or cofactors, enzyme inhibitors, magnetic particles etc. Compare, for example, US Pat. Nos. 3,654,090, 3,817,837, 3,935,074, 3,996,345.4,277,437. 4,374,925 and 4,366,241.

There are a large number of methods for linking the labeling substances with polypeptides, which can include the use of N-terminal amino groups for the functionalization of a pyrazole, while other free amino groups are protected, the pyrazolone then being in contact with various reagents is brought, for example with amino groups, to bind the residue causing the detectable signal. By protecting the arginine amino acids that are involved in, or close to, the third loop, other argins can function.

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be functionalized, for example for the conjugation with amino groups or thio groups in the usual way. Alternatively, the polypeptides can be contacted with the active ingredient, for example an activated carboxylic acid which is substituted at random, the biologically active material being separated from the biologically inactive material as a result of the accidental substitution. Finally, depending on the synthesis method, the polypeptide can be changed in order to obtain the desired functionality as part of the synthesis process.

The compositions according to the invention can also be used for the detection of EGF receptors. The compositions according to the invention can also be used for the detection of the cellular response to EGF and / or TGF by ensuring the competition between these naturally occurring materials and a composition according to the present invention. In this way, changes in the receptor conformation can be detected.

The main compositions can also be used for various therapeutic purposes, including stimulating growth or controlling bone formation. These compounds can be administered intraperitoneally, subcutaneously, intravenously, intraarterially in appropriate physiological carriers or applied directly to the affected area. In addition, the compositions according to the invention can be incorporated into liposomes, where appropriate the use of antibodies for the targeting can be considered. Numerous carriers include phosphate buffered saline, saline, water, or the like. The concentration of the additives can vary widely, depending on the last use and the activity.

Other additives may also be present in the formulations, such as EGf, TGF or other factors, bacteriocides such as antibiotics, bacteriostatics, buffers, etc.

For the production of antibodies, the polypeptides according to the invention in which pp1-4 is hydrogen or short oligopeptide chains (with less than five amino acids) can be linked to antigenic polypeptides or proteins in order to inject into mammalian hosts. The antigenic protein has about 60 amino acids and normally has a molecular weight of no more than 100 ku (kilodalton). Techniques exist for the connection of polypeptides, either at a specific site or at random, using bifunctional reagents such as p-maieimidobenzoic acid, glutaraldehyde, p, p'-benzidine, etc. Common antigenic proteins include bovine serum albumin, keyhole limpet hemocyanin, Tetanus toxoid, etc. The polypeptides according to the invention are linked to the antigenic protein in sufficient numbers to achieve the desired immunogenic response. Typically two or more boost injections are required after the original injection. For antisera, the blood is withdrawn from the immunized host and the immunoglobulin fraction is isolated. For monoclonal antibodies, the spleen is isolated and splenocytes are fused in the usual way with an appropriate fusion partner. The resulting hybridomas are then subjected to screening for antibody binding to the epitopic sites of the polypeptide according to the invention. These antibodies can be used for a variety of purposes, such as diagnostic reagents, therapy, etc. If used as reagents, the antibodies can optionally be labeled as described for the polypeptides.

The invention is illustrated without restriction on the basis of the following examples.

EXPERIMENTAL PART Biological deposits

The following expression plasmids, all of which were transformed into E. coli HB101, were deposited on the date specified with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, U.S.A. and have the following identifications and ATCC accession numbers:

ID

ATCC deposit no. Filing period

PBM11 PBM14

67366

67367

PBM11 / PA / VGF PBM11 / DP / VGFa PBM11 / PA / EGF PBM11 / M5

67417

67418

67419

67436

67437 67547

June 3, 1987 June 3, 1989 June 3, 1989

PBM11 / C2 PBM11 / NDP / EGF

October 23, 1987

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method

The general ionization techniques used are described in Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, CSH, New York. All DNA-modifying enzymes were supplied by commercial manufacturers. They were used in accordance with the manufacturer's instructions. The materials and equipment for DNA purification and separations were used in accordance with the instructions of the suppliers.

Example I

Production of synthetic genes of interest

Synthetic growth factor genes were designed whose host cell codons were optimized for high-level expression. In addition, various convenient restriction sites in the synthetic genes were designed. If possible, the new restriction sites left the amino acid sequence of the growth factor gene unchanged, but in some cases the incorporation of new restriction sites resulted in a changed amino acid sequence. These sites divide the synthetic genes into thirds and result in an N-terminal, a middle and a C-terminal region.

The natural VGF gene product contains an extremely N-terminal region, which has no counterpart in the mature TGF. The VGF fragments that lack this area are said to be garbled. The restriction sites were used for the initial construction of the final gene from partial, synthetic oligonucleotide fragments which extend from one restriction site to the other. The oligonucleotides were synthesized on an Applied Biosystems oligonucleotide synthesizer and were purified on an acrylic amide gel. The oligonucleotides were phosphorylated at the 5 'end using T4 polynucleotide kinase and each oligonucleotide was then cured with its complement.

A. Synthetic TGF oligonucleotides

1. Human N-terminal TGF region

TGF ->

Bs-sHIINcoI

MVVSHFNDCPDSHTQFC 5 'CGCGCCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGC 3' GGTACCAACAAAGAGTGAAATTGCTGACGGGCCTGAGAGTATGAGTCAAAACG

Kpnl F H G T

TTTCATGGTAC 3 'TGF104 AAAGTAC 5' TGF1Û3

2. Modified human mid-TGF range. whereby the humane Seouenz QEDK was changed to QEEK, the Seouenz. which was found in rat TGF

Kpnl sole

CRFLVQEEKPAC 5 'CTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATG 31 TGF101

3 'CATGGACGGCAAAAGACCAAGTCCTTCTTTTTGGCC 5' TGF102

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3. Human C-terminal TGF region vchsgyvgarcehadll c. cgtttgccattctggctacgttggcgcacgttgcgaacacgctgacctgctg

3 • gtacgScggtaagaccgatgcaaccgcgtgcaacgcttgtgcgactggacgac

BamHI

A Ter

GCTTAAG 3 'TGF205

CGAATTCCTAG 5 'TGF206

B. Synthetic VGF oligonucleotides 1. Extreme N-terminal VGF region

HindiII

EDSGNAIETTSPEITNATT 5 * AGCTGACTCTGGTAACGCTATCGAAACTACTTCTCCGGAAATCACTAACGCTACTACT 3 'V 3' CTGAGACCATTGCGATAGCTTTGATGAAGAGGCCTTTAGTGATTGCGATGATGA 5 'V

2. Modified N-terminal VGF region. including the Aso-Pro fission point. with the sequence HGT. which replaces the natural sequence HGD

BamHI

IDPM'DIPAIRLCGPEGDGY 5 'GATCGATCCCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTAC 3' CTAGGGTACCTGTAGGGCCGATAGGCAGACACGCCGGGCCTTCCGCTGCCGATG

Kpnl

C L H G T TGCCTGCATGGTAC 3 'VGF104a ACGGACGTAC 5' VGF103a

3. Modified middle VGF area. where the sequence GYAC replaces the natural sequence GMYC

Kpnl SphI

TCIHARDIDGYAC 5 'CTGCATCCATGCACGTGACATCGACGGCTACGCATG 3' VGFlOla 3 'CATGGACGTAGGTACGTGCACTGTAGCTGCCGATGC 5' VGF102a

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4. C-terminal VGF region at the 5 'end

SphI EcoRI

CRCSHGYTG 51 CCGTTGCTCTCATGGCTACACTGG 3 'VGF1A

3! GTACGGCAACGAGAGTACCGATGTGACCTTAA 5 'VGF2A

5. Modified C-terminal VGF region. 5 'end. where the sequence VCS replaces the natural sequence RCS

SphI EcoRI

VCSHGYTG 5 'CGTTTGCTCTCATGGCTACACTGG 3' VGF1

3 'GTACGCAAACGAGAGTACCGATGTGACCTTAA 5' VGF2

6. C-terminal VGF region. 3'-Fraament. with the ending YQR instead of PNT the derived C-terminus of naturally excreted VGF

EcoRI BamHI

IRCQHVVLVDYQR Ter 5 'AATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCG-TTAAGGATC 3' VGF3 3 'GCAACGGTCGTACAACAAGACCAGGTGATGGTCGCAATTC 5' VGF 4

C. Synthetic EGF oligonucleotides

Three sets of overlapping synthetic oligonucleotides 1 (A, B), 2 (A, B) and 3 (A, B) encoding human EGF were sequenced on an Applied Biosystems oligonucleotide synthesizer and purified on an acrylamide gel. The oiitonucleotides were phosphorylated at the 5 'end using T4-polynucleotodkinase. Each oligonucleotide was cured with its complement.

1.

NcoIEcoRI

MNSDSECPLSHDGY

5 'CATGAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTAC 3' EG FIA

3 'TTAAGACTGAGACTTACGGGCGACAGAGTACTGCCGATGACGGAC 5' EGF2A

2y_

Nsil SphI

CLHDGVCHYIEALDKYAC

5 'TGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATG 3' EGF1B 3 'GTACTGCCGCA.TACGTACATGTAGCTTCGAGACCTGTTCATGC 5' EGF2B

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NCVVGYIGERCQYRDLK 5 'CAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAA

3 'GTACGTTGACGCAACAACCGATGTAGCCGCTTGCAACGGTCATGGCACTGGACTTT

BamHI

W W E L R * '

TGGTGGGAACTGCGTTAAG 3 'EGF3

ACCACCCTTGACGCAATTCCTAG 5 'EGF4

Example il

Description of cloning and expression of plasmids

A. The plasmid pLEBam was used to clone synthetic oligonucleotide fragments because it has the practical BssHI1 and BamHI restriction sites. A plasmid with Ncol and BamHI restriction sites, such as pBM11 or pBM11 / NDP (described below) can be used for cloning the synthetic nucleotide fragments or other DNA sequences of interest.

B. Plasmid oBM11. described in the parallel U.S. Patent application, serial number 264,098 dated October 28, 1988, allows a gene of interest to be cloned at the BamHI restriction site downstream of the DNA sequences encoding the 33 N-terminal amino acids of the bacteriophage lambda N gene. After induction of the lambda PL promoter by inactivation of the temperature-sensitive C1857 repressor at 42 ° C., the foreign gene product is expressed as a C-terminal part of a fusion protein, the N-terminal sequence of which is that of the N gene.

C. Plasmid PBM11M4. described in the parallel U.S. Patent application, serial number 264,098 dated October 28, 1988, is derived from pBM11 and allows a gene of interest in the BamHI restriction site to be cloned directly after the initiating methionine of the gene. The plasmid pBM11 / M4 also contains the Ncol site of the neomycing gene.

D. Plasmid PBM11M5. described in the parallel U.S. Patent application serial number 264,098 dated October 28, 1988 is derived from pBM11, which is an Ncol site. which is present in the neomycin resistance gene has been removed by site-directed mutagenesis. The cloning of a gene of interest in pBM11 / M5 therefore does not require partial digestion of the vector with Nçol.

E. Plasmid pBM11 / NDP. described in the parallel U.S. Patent application, serial number 264,098 dated October 28, 1988, is derived from pBM11 and has DNA sequences which encode an acid-labile Asparagin-Proline dipeptide, which between the sequences which encode the N gene and a gene of interest, is inserted. The plasmid contains an Ncol site and a Çlal site for cloning a gene of interest downstream of the pl promoter.

F. Plasmid PBM11 / PAD. described in the parallel U.S. Patent application, serial number 264,098 dated October 28, 1988, is derived from plasmid PBM11M4 and enables a gene of interest in Hindill. Smal or BamHI can be cloned downstream of a modified alkaline phosphatase signal sequence.

G. Plasmid pBM11 / PAK. described in the parallel U.S. Patent application, serial number 264,098 dated October 28, 1988, is derived from plasmid pBM11M4 and allows a gene of interest in Hindlll. Smal or BamHI can be cloned downstream of an alkaline phosphatase signal sequence.

H. Plasmid oTCPt was designed so that it contains the tac promoter elements and can be used as Cro-SD in order to be able to express the gene of interest according to the alkaline phosphatase signal sequence. An example of the construction of this plasmid is given below in the construction of pTCPt / EGF.

Construction of pTNPt ([trp-35] 217bp [lac-10] [nSD] 8bp [ATG] / alkaline phosphatase signal / linker / trans.term.-NEO)

This plasmid was designed to have tac promoter elements and to use the N gene SD to express a given gene behind the alkaline phosphatase signal sequence. It has a pBR322 background with a neomycin resistance gene. The plasmid pTNPt was constructed as follows:

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(a) Preparation of the 2.8kb EcoRI-BamHI fragment from pBM16t / VGFa. which the Hindlll position is missing

The plasmid pBM16t / VGFa was digested with EcoRI and BamHI and the 2.8 kb fragment was isolated. The 2.8 kb fragment was linked to an EcoRI-BamHI linker and the correct construction was isolated by restriction analysis and designated as intermediate I.

The only Hindlll site in the vicinity of the neomycin resistance gene was removed from intermediate I plasmid by digestion with Hindlll, blunt ends were obtained using a Klenow fragment and these were connected again. This resulted in the intermediate Il-plasmid, which lacked the HindIII site.

The 2.8 kb EcoRI-BamHI fraction of pBM16t / VGFa, which lacked the HindIII site, was isolated by digesting intermediate Ii with EcoRI and BamHI. The resulting fragment was isolated by agarose gel electrophoresis.

(b) Preparation of the 150bp BamHl-Bsml fraction of dBM1 1 / PAK

The plasmid pBM11 / PAK is identical to pBM11 / PAK / EGF, with the exception that it is a connecting region with the HindIII. Contains Smal and BamHI sites downstream of the alkaline phosphatase signal sequence in place of the EGF gene. pBM11 / PAK was digested with Bsml and BamHI and a 150 bp fragment containing the N gene SD, the alkaline phosphatase signal sequence and the connecting region was isolated.

(c) Preparation of the Oligonucleotides TacA + and TacA-

The oligonucleotides TacA + and TacA- were synthesized on an Applied Biosystems oligonucleotide synthesizer and were designed to have an EcoRI overhano at the 5 'end with the trp-35 match sequence provided by lac-10 match sequence through 17 Nucleotides separated, within which an Sstl site was present. The sequence also contained the 5 'end of the lac mRNA, the lac receptor binding site and a Bsml overhano.

TacA + 51AATTACTCCCCATCCCCCTGTTGACAATTAATCATCGAGCTC

GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAG 31

TacA- 51GTGTGAAATTGTTATCCGCTCACAATTCCACACATTATA

CGAGCTCGATGATTAATTGTCAACAGGGGGATGGGGAGT 3 '

(d) Connection and isolation of pTNPt

The 2.8 kb EcoRI-BamHI fragment. the 150bp Bsml-BamHI fragment and the oligonucleotides TacA + and TacA- were linked together using DNA ligase and the DNA was used to transform sufficient JM109 (lacIq). Correct construction was isolated by restriction analysis and DNA sequencing.

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(EcoRI site from pBMll)

I.

GAATTACTCCCCATCC

SstI

trp-35 (17bp) lac-10

CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG)

Bsml

51 lac mRNA-> n mRNA->

TGTGG / AATTGTGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCT

TTGGGGTGTGTGATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGC

TGCCAATGTGCCAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACACACC

-Pvul mSD (8bp) signal Sequenee- -> ACCAAAGCTAACTGAC {ACCA} GAATCCAG ATGAAACAATCTACGATCGCCC

MKQSTIAL

Smal

Hindlll BamEI

TCGCACTTCTCCCACTGCTGTTCACTCCAGTGACAAAAGCTTCCCGGGATCCGTG ALLPLLFTPVTK

(BamHI site from pBMll)

Trans. Term. J

ACTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC

I. The plasmid plac / cro-B aal consists of the operator-promoter region of E. coli lactose (lacV operon, as well as the ribosomal binding sites of lag and ero. The plasmid was constructed as in the parallel US patent application, Serial Number 264 098. The fusion proteins expressed by this vector arise from the N-terminus of the bacteriophage lamb-da-Cro protein, the amino acid sequence which was encoded by the inserted DNA and the C-terminus of ß- The control elements of this vector consist of the operator-promoter region of the E. coli lactose (Jaç) operon, as well as the ribosomal binding sites of lac and ero.

J. Plasmid ptac / cro-β-oal allows a gene of interest to be cloned downstream of the N-terminal 21 amino acids of the bacterial Cro protein. The plasmid was, as in the parallel

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Ieri U.S. Patent application 264 098 constructed. The expression vector ptac / cro-ß-gal is similar to the plac / cro-ß-gal, except that the promoter of the ptac / cro-ß-gal is from the -35 region of the tryptophan operon promoter and the Pribnow Box of the lac operon. This hybrid promoter allows a higher level of expression than plac / cro-ß-gal.

K. The plasmid pRSV allows expression of a gene of interest in eukaryotic cells with the RSV LTR promoter, Gorman et al., Proc. Nati. Acad. Be. USA (1982) 79: 6777-6781.

L. plasmid pAc610. Smith et al., Molec. Cell. Biol. (1983) 12: 2156-2165, allows expression of a foreign gene in insect cells.

M. Plasmids pToxl and dTqx2 allow expression of a gene of interest in yeast cells using the X. lactis-killtoxin sianal sequence (EMBO 6, 229-234 (1987); Biochem., Biophys. Res. Comm. 144: 613-619 ( 1987)). This construction has the URA3 gene for selection in yeast and the AMP gene for selection in bacteria. Transcription is initiated with the CYCI promoter and GAL upstream of the activation sequence (Methods Enzymol., 101, 181-191 (1983) and changes within the 3 'end of the FLP gene (Mol. Cell. Biol., 5, 2770 -2780 (1985) The construction has both the 2 micron origin and the pBR322 origin for replication in yeast and E. coli, respectively. The killer toxin N-terminal region with the signal sequence and the Kex2 cleavage site was located on ligonucleotides introduced and contained the AT-rich region immediately before the initiator codon while maintaining optimal translation initiation and extension signals, and various restriction sites were introduced to allow insertion of genes of interest from both the signal cleavage site and the Kex2 cleavage site.

1. Preparation of the 3kb EcoRI Espl fragment from PBR322.

which contains the origin and the AMP resistance gene

The plasmid pLGSD5 (-ATG), a yeast expression vector (Methods Enzymol. 101: 181-191 (1983)) was digested with EcoRI and Espi and the 3rd Okb fragment was isolated.

2. Production of the 2.1 kb EcoRI-Espl fraction of the 2 micronolasmide.

which contains the 3 'end of the FLP gene and the origin

The plasmid pLGSD5 (-ATG) was digested with EcoRI and Espi and the 2.1 kb fragment was isolated.

3. Connection and isolation of intermediate I-plasmid

The 2.1 kb and 3 kb EcoRI and Espl fragments were combined using DNA ligase and the DNA was used to transform HB101 sufficiently. A correct construction was isolated by restriction analysis.

4. Preparation of the 1.8kb EcoRI-BamHl fragment of pLGSD5 (-ATGÌ.

which is the URA3 gene. GAL UAS and the CYCI promoter contained

The plasmid pLGSD5 (-ATG) was digested with EcoRI and BamHI and the 1.8 kb fragment was isolated. The BamHI site is present within a linker that has been inserted 4bp downstream of the CYCl initiator.

5. Preparation of the oligonucleotides pToxl A +. 2A-. 1B +. 2 B-

The oligonucleotides were designed to link the codons for the K. lactis-Kil-lertoxin signal sequence to the BamHI site downstream of the CYCl initiator. To preserve optimal translation initiation, 13bp of an A-T rich sequence was incorporated upstream of the killer toxin initiator codon. This sequence is consistent with the match sequence determined for the yeast initiation codons, a BglII site was placed directly upstream of the A-T region to allow mutagenesis of the initiation region of the signal sequence. A HindIII site was placed 10 nucleotides upstream of the signal cleavage site to enable mutagenesis for upstream sequences and the connection of genes of interest. There was a guarantee on the pToxl. Xhol site present directly downstream of the signal cleavage site, while in pTox2 there is a Nael site at the signal cleavage site for blunt end cloning directly above the signal cleavage site, which was changed from Gly-Leu to Ala-Glys.

The dissolution of the heteroduplex at the cleavage site should lead to the formation of clones which contain the pToxi sequence, which in turn contain the pTox2 sequence. In both constructions, the sequences below encode the rest of the killer toxin precursor sequence down from the Kex2 Lys-Arg cleavage site, at which point different restriction sites (StuI, Sa [l, Acci. Hincll and BamHI) are present to clone the gene of interest facilitate. The Stul site allows blunt end cloning directly after the Arg codon of the Kex2 cleavage site. The oligonucleotides be22

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sit a BamHI-Überhana at the 5'-end and a HindII-Überhana at the 3'-end and enable the cloning of an intermediate vector. The resulting sequence no longer has any of the two positions.

(BamHI) Bglll Hindlll

MNIFYIFLFLLS 1A + 5 'GATCAGATCTAATAATTATAAAATGAATATATTTÎACATATTTTTGTTTTTGCTAAGC 2A- 3' TCTAGATTATTAATATTTTACTTATATAAAATGTATAAAAACAAAAACGATTCGAAG

Signal splitting point Kex2 splitting point

(pToxl) / Aval / Xhol / StuI SalI, Acci BamH

FVQGLEHÏHRRGSLVKRPLSTDP 1B + 5 'TTCGTTCAGGGCCTCGAGCATACTCATAGAAGAGGCTCCTTAGTCAAAAGGCCTTTGTCGACGGATCC 2B- 3 1 AAGTCCGGCCGCTCGTATGAGTATCTTCTCCTAGGTCCTGTGTGTGTGGTGTGTGGGGGGGGTGGTGTGGTGGTGGTGGTG

These oligonucleotides were synthesized on an Applied Biosystems synthesizer and were purified by gel electrophoresis. They were then phosphorylated using T4 kinase and the complementary pairs were melted together, connected and gel purified.

6. Preparation of the EcoRI-HindIII intermediate I vector

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

7. Connection and isolation of pToxl and dTqx2

The 1.8 kb EcoRI-BamHI fragment was linked to the oligonucleotide fragment using DNA ligase and the resulting fragment was gel purified and then linked to the EcoRI HindII II intermediate I vector and the DNA was used to transform sufficient HB101 used. The correct constructions were identified using restriction analysis and DNA sequencing and one clone with the sequence pToxl and one with the sequence pTox2 was isolated. Both clones lacked the BamHI site at the junction of the oligonucleotides and the intermediate I vector.

Example III

Compositions of growth factor genes for expression in prokaryotic cells

A. Preparation of the plasmid pLEBam / TTV

The synthetic chimeric growth factor, which was named TTV or (TGF / TGF / VGF), was assembled in the cloning vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal 2/3 of the gene, with the exception of the sequence QEEK, which was changed from the natural sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream from the natural sequence PNT. The plasmid pLEBam was digested with BssHIl and BamHI. BssHII-BamHI pLEBam was then linked to oligonucleotides TGF101,102,103 and 104 and VGFI, 2,3, and 4 using DNA ligase and the resulting plasmids were used to transform enough HB101. The transformants were selected for ampicillin and by restriction analysis using EcoRI. Ncol and BamHI and subjected to a restriction analysis by Maxa-Gilbert nucleotide sequencing. A correct construction was isolated and designated pLEBam / TTV.

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TGF -Ncol ccatggttgtttctcactttaacgactgcccggactctcatactcagttttgctt mvvshfndcpdshtqfcf

VGF -

Kpnl SphI

tcatggtacctgccgttttctggttcaggaagaaaaaccggcatgcgtttgctct hgtcrflvqeekpacvcs

EcoRI

catggctacactggaattcgttgccagcatgttgttctggtcgactaccagcgt egïtgirc-qhvvlvdyqr

BamHI TAAGGATCC

ter

B. Preparation of plasmid pLEBam / TW

The synthetic chimeric growth factor, referred to as TW or (TGF / VGF / VGF), was assembled in the cloning vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal region of the gene. The middle and the C-terminal region were derived from the mutilated VGF sequence and the end from the sequence YQR upstream of the natural sequence PNT. In addition, the synthetic gene has the modification GYACVC for GMYCRC.

(a) Preparation of 4.3kb Kpnl-Sphl fragment from pLEBam / TTV

The plasmid pLEBam / TTV was digested with Kpnl and Sohl and the 4.3kb Kpnl-Sphl fragment was gel-cleaned. This digestion removes the middle TGF region from the synthetic gene TTV in the cloning plasmid pLEBam.

(b) Connection and isolation of pLEBam / TW

Oligonucleotides VGF101a and 102a were linked to the 4.3 kb Kpnl-Sphl fragment from pLEBam / TTV using DNA ligase and the resulting mixture was used to transform HB101 sufficiently. The transformants were selected for ampicillin and screened by nucleotide sequencing using the Sanger dideoxy method. A correct construction was isolated and designated pLEBam / TW.

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TGF *

Ncol ccatggttgtttctcactttaacgactgcccggactctcatactcagttttgctt mvvshfndcpdshtqfcf

VGF *

Künl SjdIiI

tcatggtacctgcatccatgcacgtgacatcgacggctacgcatgcgtttgctct hgtcihardidgyacvcs

EcoRI

catggctacactggaattcgttgccagcatgttgttctgctcgactaccagcgt hgytgircqhvvlvdy qr

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 33 N-terminal amino acids of the N gene and has the sequence QEEK, which replaces the human sequence QEDK.

(a) Preparation of a 780 bp SohI-PvuI fragment from pBM11 / N / TTV

The plasmid pBM11 / N / TTV was digested with Sohl and Pvul and the 780 bp SphI-PvuI fragment was gel purified. This fragment contained parts of the pBM11 plasmid at the Pvul end and at the Sehl end, the N gene and the N-terminal 2/3 of the human TGF gene.

(b) Preparation of the 5 kb BamHI-Pvul fragment from pBM11 / N / TTV

The plasmid pBM11 / N / TTV was digested with BamHI and Pvul and the 5 kb BamHI-PvuI fragment was gel purified.

(c) Connection and isolation of pBM11 / N / TGF

The oligonucleotides TGF205 and 206, the 780 bp Sohl-Pvul fragment and the 5 kb BamHI-Pvul fragment from pBM11 / N / TTV were combined and used to transform sufficient HB101. The transformants were selected on neomycin and subjected to restriction analysis by restriction analysis using EcoRI and nucleotide sequencing by the Sanger dideoxy method. The correct construction was isolated and designated pBM11 / N / TGF.

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N-gen -

atggat.gcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqvîk

BamHI

aagcagcaaatcccctgttggttggggtaagcgcaaaaccagttcggatccgcat aanpllvgvsakpvrirm tgf -

ggttgtttctcactttaacgactgcccggactctcatactcagttttgctttcat vvshfndcpdshtqfcfh

Kpnl SphI

GGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCCATTCTG GTCRFLVQEEKPACVCHSG

BamHI

GCTAGGTTGGCGCACGTTGCGAACACGCTGACCTGCTGGCTTAAGGATCC YVGARCEHADLLA Ter

B. Modified synthetic TGF-VGF hybrid

1. Production of pBM11 / N / TTV

In this construction, a synthetic modified TTV 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-terminus. This hybrid growth factor contained the amino acid sequence of human TGF in the amino terminal 2/3 of the gene with the exception that the sequence QEEK was present instead of the natural human sequence QEDK. The carboxy terminus was derived from the amino acid sequence of VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

(a) Preparation of the synthetic NcoKstumpfì-BamHl TTV gene

The plasmid pLEBam / TTV was digested with Ncol and the ends blunted by filling the overhangs using the Klenow fragment of DNA polymerase. The DNA was then digested with BamHI and the 170 bp NcoKstumpfi-BamHI TTV fragment was gel purified.

(b) Preparation of the BamHI-digested pBM11 The plasmid pBM11 was digested with BamHI.

(c) Connection and isolation of pBM11 / N / TTV

BamHI digested pBM11, the Ncolfstumpfl-BamHI TTV fragment and the BamHI linker (5'GATCCG3 ') were linked using DNA ligase and the resulting mixture was used to transform HB101 sufficiently. The transformants were selected for neomycin and screened using restriction analysis and nucleotide sequencing using the S'anger dideoxy method. A correct construction was isolated and designated pBM11 / N / TTV.

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N-qen -

atggatgcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqwk

BamHI

aagcagcaaatcccctgttggttggggtaagcgcaaaaccagttcggatccgcat aanpllvgvsakpvrirm tgp -

GGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTTTCAT VVSHFNDCPDSHTQFCFH

Kpnl SphI VGF ■ *

GGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCTCATG GTCRFLVQEEKPACVCSHG

EcoRI

GCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGTTAAG YTGIRCQHVVLVDYQR Ter

BamHI GATCC

Example V

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

A. Modified synthetic VGF

1. Preparation of pBM11 / NDP / VGFA

The N-terminal sequence of the synthetic VGFA gene is a garbled version of the natural VGF sequence and begins with the DIPAIR sequence. In this plasmid, the VGFA fragment 32 amino acids of the lambda-N protein and the dipeptide aspartic acid-proiin is located downstream. To preserve the Konl cloning site, the synthetic sequence was changed to code CLHCGTC instead of the natural VGF sequence CLHGDC and ends with the sequence YQR upstream of the natural sequence PNT. In addition, the VGFA gene encodes the sequence GYACVC, which replaces the natural sequence GMYCRC.

(a) Preparation of the Kpnl-BamHI 89bp C-terminal fragment of the synthetic VGF gene

The plasmid pLEBam / TVV was digested with Kpnl and BamHI and the 80bp KonI-BamHI fragment was gel purified. The fragment contains the C-terminal 2/3 of the synthetic VGF gene, with the Kpnl site at the 5 'end.

(b) Preparation of BamHI-digested dephosphorylated pBM11

The plasmid pBM11 / N / TTV was digested with BamHI and the 5'-phosphates were removed by treatment with intestinal alkaline calf phosphatase. The 5.6 kp BamHI plasmid fragment was gel cleaned.

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(c) Connection and isolation of pBM11 / NDP / VGFA

The oligonucleotides VGF103a, 104a, the 5.6 kb BamHI fragment from pBM11 and the 80bp Kpnl-Bam-HI fragment from pLEBam / TW were combined together using DNA and then used for the sufficient transformation of HB101. The transformants were selected for neomycin and were screened by restriction analysis using Clal and nucleotide sequencing according to the Sanger dideoxy technique. A correct construction was isolated and designated pBM11 / NDP / VGFA. This construction had the sequences GTC and GYACVC instead of the authentic VGF sequences GDC and GMYCRC.

N-qen -►

atggatgcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqwk

Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC AANPLLVGVSAKPVRIDP

Ncol VGF -

CCÄTGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT MDIPAIRLCGPEGDG.YCL

Kpnl SphI

GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT HGTCIHARDIDGYACVCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT HGYTGIRCQHVVLVDYQR

BamHI TAAGGATCC Ter

2. Production of DBM11 / NDPA / GFa

The VGFa N-terminal sequence is a garbled version of the natural VGF sequence and begins with the DIPAIR sequence. In addition, the VGFa sequence contains the modified sequences GTC and GYACRC, instead of the natural VGF sequences GDC and GMYCRC. In this plasmid, the VGFa gene is located downstream of the 32 amino acids of the lambda-N protein and the dipeptide aspartic acid proline. Treatment of the purified fusion protein with formic acid leads to cleavage at the acid-labile aspartic acid-proline-peptide bond, which allows the VGFa protein to be separated from the lambda N-protein amino terminus. The cleavage is of such a nature that the VGFa protein remains with the proline residue at the amino terminus.

(a) Preparation of digested digest. dephosphyrlated pBM11 / DP / VGFA

The plasmid pBM11 / DP / VGFA (10 jig) was digested with 30 units of sol and the 5'-phosphates were removed by treatment with intestinal alkaline calf phosphatase. The 5 kb plasmid fragment was recovered after electrophoresis on an agarose gel.

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(b) Preparation of an EcoRI-Sphl 70bp fragment from pBM11 / DP / VGFA

The plasmid pBM11 / DPA / GFA (10 ng) was digested with 30 units of EcoRI and then with 30 units of SphI. The 70bp fragment was obtained after electrophoresis on an agarose gel.

3. Connection and isolation of dBM1 1 / NDP / VGFa

The 24bp fragment containing oligonucleotides VGF 1A and 2A, the 5bk Sohl fragment and the 70bp EcoRI-Sohl fragment from pBM11 / DP / VGFA were joined together and the mixture was used to make E. coli HB101-capable cells to transform. The transformants were screened by nucleotide sequencing using the Sanger dideoxynucleotide method. A correct clone was isolated and designated pBM11 / NDP / VGFa.

N — gen *

atggatgcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqwk

Clal aagcagcaaatcccctgttggttggggtaagcgcaaaaccagttcggatcgatcc aa npllvgvsakpvridp

Ncol vgp -

ccatggacatcccggctatccgtctgtgcggcccggaaggcgacggctactgcct mdipairlcgpegdgycl

Kpnl SphI

GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCTCT HGTCIHARDIDGYACRCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT HGYTGIRCQHVVLVDYQR

BamHI "TAAGGATCC Ter

B. Modified Synthetic TGF-VGF Hybrids 1. Preparation of pBM11 / NDP / TTV

In this construction, the synthetic modified TTV chimeric gene is expressed as a C-terminal part of a fusion protein with 32 amino acids of the N gene at the N-terminus. An acid-sensitive aspartic acid-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 2/3 of the gene with the exception of the sequence QEEK, which was changed from the natural human TMR sequence QEDK. The carboxy terminus was derived from the amino acid sequence of the VGF and terminated with the sequence YQR upstream of the natural sequence PNT.

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(a) Preparation of the 5bk Ncol pBM11 plasmid fragment

The plasmid pBM11 / NDP / VGFA was digested with Ncol and the 5bk Ncol plasmid fragment was gel purified. This fragment had an Ncol-Uberhana at the aspartic acid-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 the 0.6kb Ncol-BamHI pBM 11 fragment

The plasmid pBM11 / N / TTV was digested with Ncol and BamHI and the 0.6 kb Ncol-BamHI plasmid fragment was gel purified. This fragment had the Ncol overhana in the neomycin resistance gene.

(c) Preparation of the 170bp synthetic TGF / TGF / VGF fragment

The plasmid pLEBam / TTV was digested with Ncol and BamHI and the Ncol-BamHI-170bp fragment containing the synthetic TGF / TGF / VGF gene was gel purified. This fragment had the Ncol overhana at the 5 'end of the gene and the BamHI overhana at the 3' end of the gene.

(d) Connection and isolation of pBM11 / NDP / TTV

The 5bk Ncol and 0.6kb Ncol-BamHI plasmid fragments were linked to the 170bp Ncol-BamHI TTV gene using DNA ligase and the resulting mixture was used to transform the capable HB101. The transformants were selected on neomycin in such a way that only colonies with correctly reconstructed neomycin resistance genes would survive. The transformants were screened using Ncol restriction analysis and nucleotide sequencing using the Sanger dideoxy technique. A correct construction was isolated and designated pBM11 / NDP / TTV.

N-gen -

atggatgcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqwk

Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC AANPLLVGVSAKPVRIDP

Ncol TGF ♦

CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT MVVSHFNDCPDSHTQFCF

Kpnl SphI VGF - *

TCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT HGTCRFLVQEEKPACVCS

EcoRI

CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT HGYTGIR CQHVVLVDYQR

BamHI TAAGGATCC Ter

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2. Production of pBM11 / NDP / VTV

In this construction, the synthetic modified VTV chimeric gene was expressed as a C-terminal part of a fusion protein with the first 32 amino acids of the N gene at the N-terminus. An acid labile aspartic acid proline dipeptide separates the two parts of the fusion. The hybrid growth factor contained the amino acid sequence of the human TGF in the middle region with the amino acid sequence QEEK, which replaces the natural sequence QEDK. The N-terminal and C-terminal regions were derived from the garbled VGF sequence and begin with the DIPAIR sequence and end with the YQR sequence, which is upstream of the natural PNT sequence.

(a) Preparation of a 5bk BamHI-Ncol fragment of pBM11

The plasmid pBM11 / N / TTV was digested with BamHI and Ncol and the Sbk BamHl-NcoI fragment was gel purified. This fragment contains a BamHI overhano at the 3 'end of the sequences which encode the first 32 amino acids of the N gene and an Ncol site in the neomycin resistance gene.

(b) Preparation of a 700 bp Kon I-Ncol fragment from pBM11 / N / TTV

The plasmid pBM11 / N / TTV was digested with Kpnl and Ncol and the 700bp Kpnl-Ncol fragment was gel purified. This fragment is composed of a part of the plasmid pBM11, which contains part of the neomycin resistance gene on the Ncol overhana and the C-terminal VGF region of the synthetic TTV gene on the Koni overhang.

(c) Connection and isolation of pBM11 / NDP / VTV

The oligonucleotides VGF103a and 104a, the 5kb BamHI-Ncol fragment from pBM11 and the 700bp Konl-Ncol fragment from pBM11 / N / TTV were linked together using DNA ligase and then used to transform capable HB101. The transformants were selected for neomycin and were screened by restriction analysis using Clal and nucleotide sequencing by the Sanger dideoxy method.

N-gen -

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA MDAQTRRRERRAEKQAQWK

* "* * * Clal

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC AANPLLVGVSAKPVRIDP

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Ncol VGF ~

CCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT MDIPAIRLCGPEGDGYCL

Kpnl TGF - sole VGF -

GCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT HGTCRFLVQEEKPACVCS

EcoRI

CATGGCTACACTGGAATTCGTTCGCAGCATGTTGTTCTGGTCGACTACCAGCGT HGYTGIRCQHV'VLVDYQR

BamHI TAAGGATCC Ter

3. Production of pBM1 6 / NDP / TVV

In this construction, the synthetic modified TVV chimeric gene was expressed as a C-terminal part of a fusion protein with the first 32 amino acids of the N gene at the N-terminus. An acid labile aspartic acid proline dipeptide separates the two parts of the fusion. The hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal region. The middle and the C-terminal region were derived from the mutilated VGF sequence and end with the sequence YQR. In addition, the synthetic gene has the modification GYACVC instead of GMYCRC.

(a) Preparation of the 4.3 kb Ncol-BglII fragment from pBM11 / NDP / VGFa

The plasmid pBM11 / NDP / VGFa was digested with Ncol and Balli and the 4.3 kb fragment was gel purified. The Ncol overhana is located at the aspartic acid-proline cleavage site, immediately downstream of the first 32 amino acids of the N gene.

(b) Preparation of the 1.2kb BamHI-Bolll fragment from pBM11M5

The plasmid pBM11M5 was digested with BamHI and BglII and the 1.2 kb fragment was gel purified. This fragment differs from the normal pBM11 fragment in such a way that the Ncol site in the neomycin resistance gene has been removed and all resulting vectors which lack this Ncol site are referred to as pBM16.

(c) Preparation of the 170bp Ncol-BamHI TW synthetic gene

The plasmid pLEBam / TW was digested with Ncol and BamHI and the 170bp Ncol-BamHI fraction was gel purified. This synthetic gene fragment had the Ncol site at the 5 'end and the BamHI site at the 3' end.

(d) Connection and isolation of pBM16 / NDP / TW

The 4.3kb Ncol-Bolll fragment from pBM11 / NDP / VGFa and the 1.2kb BamHl-Balll fragment from pBM11M5 and the 170bp Ncol-BamHI TW synthetic gene fragment were joined together using DNA ligase and the resulting mixture was used to transform capable HB101. The transformants were selected for neomycin and screened using restriction analysis and nucleotide sequencing according to the Sanger dideoxy technique. The plasmid designated pBM16 to indicate the loss of the Ncol restriction site in the neomycin resistance gene.

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N-gene -

atggatgcacaaacacgccgccgcgaacgtcgcgcagagaaacaggctcaatgga mdaqtrrrerraekqaqwk

**** Clal aagcagcaaatcccctgttggttggggtaagcgcaaaaccagttcggatcgatc aanpllvgvsakpvridp

Ncol tgf -

ccatggttgtttctcactttaacgactgcccggactctcatactcagttttgctt mvvshfndcpdshtqfcf

Kpnl vgf - SphI

tcatggtacctgcatccatgcacgtgacatcgacggctacgcatgcgtttgctct hgtcihardidgyacvcs

EcoRI

catggctacactggaattcgttgccagcatgttgttctggtcgactaccagcgt hgytgircqhvvlvdyqr

BamHI

taaggatcc

Ter

C. Synthetic EGF

1. Manufacture of dBM1 1 / NDP / EGF

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

(a) Preparation of the 5kb Ncol fragment from pBM11

The plasmid pBM11 / DPA / GFA was digested with Ncol and the 5'-phosphates were removed by treatment with alkaline intestinal calf phosphatase. The 5kb plasmid fragment was gel purified. This fragment had an Ncol overhano at the Asp-Pro site downstream of the sequences encoding the first 32 amino acids of the N gene. The other Ncol site is the neomycin resistance gene.

(b) Preparation of a 0.6 kb Ncol-BamHI fragment from pBM11

The plasmid pBM11 / n / TTV was digested with Ncol and BamHI and the 0.6 kb Ncol-BamHI plasmid fragment was gel purified. This fragment had the Ncol overhang in the neomycin resistance gene.

(c) Connection and isolation of pBM11 / DP / EGF

The three sets of fused EGF oligonucleotides with an Ncol overhang at the 5 'end and a BamHI overhana at the 3' end, the Skb Ncol fragment from pBM11 and the 0.6kb NçoI-BamHI fragment from pBM11 were combined linked using T4 DNA ligase and the resulting mixture was used to transform capable E. coli HB101. The transfor

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Antibodies were selected for neomycin in such a way that only colonies with a correctly reconstructed neomycin resistance gene would survive. The transformants were screened by restriction analysis using EcoRI and BamHI and by DNA sequencing as described above.

N-gen -

ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGCGTCAATGGA

MDAQTRRRERRAEKQAQWK

* * *

AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATCC AANPLLVGVSAKPVRIDP

EGF1 - »■ EcoRI EGF2 ■ *

CATGAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCATGAC MNSDSECPLSHDGYCLHD

EGF 3 -

Nsil SphI

GGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTTGTTG GV CMY IEAL DKYACNCVV G

GCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTA YIGERCQYRDLKWWELR *

BamHI AGGATCC

Example VI

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

A. Manufacture of oBM11 / PAD / EGF

The synthetic oligonucleotides were designed to allow insertion of a DNA encoding a modified phosphatase signal peptide and a junction region with three cloning sites (Hindll, Smal and BamHI) in the pBM11 expression vector downstream of the PL promoter and the ribosomal binding site of the N gene is. The nucleotide sequence was optimized so that it was as similar as possible to the nucleotide sequence of the amino terminus of the lambda-N gene, since the lambda-N gene sequence was developed for efficient ribosome introduction and translation due to its ribosomal binding site. In addition, the second amino acid of the alkaline phosphatase signal sequence, the basic amino acid lysine, was replaced by the acidic amino acid aspartic acid.

1. Production of the 0.17kb EcoRI-BamHI fragment from EGF

The plasmid pBM11 / NDP / EGF (30 j * g) was digested with 30 units of EcoRI and then treated with 4 units of Klenow fragment of DNA polymerase to create blunt ends. The DNA was finally digested with 30 units of BamHI and the 0.17 kb fragment of the EGF gene was recovered on an agarose gel after electrophoresis. The DNA purified in this way had a blunt EcoRI site at the 5 end and a BamHi overhano at the 3 'end.

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2. Preparation of the 0.5kb Pvul-Hindlll fraction of PBM11 / PAD

The plasmid pBM11 / PAD (18 ng) was digested with 30 units of HindIII and then treated with Klenow fragment to blunt the ends. The DNA was then digested with Pvul and the 0.5kb Pvul-HindIII (blunt) fragment was recovered after electrophoresis on an agarose gel.

3. Production of the 5.2kb Pvul-BamHI fragment from pBM1 1 / PAP

The plasmid pBM11 / PAD (18 (xg) was digested with 30 units Pvul and then with 30 units Bam-HI. The 5.2 kb fragment was obtained after electrophoresis on an agarose gel.

4. Connection and isolation of pBM1 1 / PAD / EGF

The 0.17kb EcoRI (blunt) BamHI fraction. the 0.5kb Pvul-Hindlll (blunt) fragment and the 5.2kb Pvul-BamHI fragment were joined together and the resulting mixture was used to transform capable E. coli HB101. The transformants were screened using DNA sequencing as described above. The desired signal sequence / EG F region had the following sequence:

Signal sequence atggatcaatctacaatcgccctcgcacttctcccactgctgttcact mdqstialallpllft egf ccagtgacaaaagctaattctgactctgaatgcccgctgtctcatgac pvtkansdsecplshd

Nsil ggctactgcctgcatgacggcgtatgcatgtacatcgaagctctg gyclhdgvcmyieal

Sole gacaagtacgcatgcaactgcgttgttggctacatcggcgaacgt dkyacncvvgyiger

BamHI

tgccagtaccgtgacctgaaatggtgggaactgcgttaaggatcc cqyrdlkwwelr *

The effectiveness of producing foreign protein in the pBM11 / PAD expression system and the ability to purify functionally active foreign proteins from the fusion product was demonstrated using pBM11 / PAD / EGF as an example. After size exclusion chromatography (TSK-250), 10.3 mg equivalents of the active EGF fusion polypeptide were obtained from 23 g (8 l) of the E. coli. which were able to express the EGF gene. 40% of the EGF activity was derived from the EDF, which was cleaved by the signal sequence.

B. Manufacture of oBM11 / PAD / nVGFa

The synthetic oligonucleotides were designed to link the synthetic VGFa gene with a modified alkaline phosphatase signal sequence to an optimal signal sequence cleavage site by encoding the additional N-terminal residues that occur immediately downstream of the signal sequence cleavage site in natural VGF and above be called the extreme N-terminus. The nVGFa sequence contains the modified sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC and has the sequence YQR upstream of the natural sequence PNT. In this expression system, the signal sequence for the multiple

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of the molecules found at the nVGFa, a fusion protein with nVGFa being formed at the C-terminus.

1. Preparation of the 0.5kb Hindlll-Pvul-digested pBM11 / PAD

The plasmid pBM11 / PAD was digested with HindIII and Pvul and the 0.5 kb fragment was gel purified. The HindIII site is at the C-terminus of the modified alkaline phosphatase signal sequence.

2. Preparation of the 5.2kb-pvul-BamHI dBM1 1 plasmid fraction

The plasmid pBM11 / NDP / VGFa was digested with Pvul and BamHI and the 5.2 kb plasmid fragment was gel purified.

3. Production of the synthetic 170bp Ncolfetumpfeni-BamHI synthetic VGFa gene

The plasmid pBM11 / NDP / VGFa was digested with Ncol and the 5 'overhangs were removed by treatment with S1 nuclease. This resulted in a blunt end at the first codon of the garbled synthetic VGFa gene. The DNA was then digested with BamHI and the 170bp Ncol (blunt egg BamH [fragment was gel purified.

4. Connection and isolation of pBM11 / PAD / nVGFa

The oligonucleotides VGF105 and 106, the 0.5kb Hindlll-Pvul fragment from pBM11 / PAD, the 5.2kb Pvul-BamHl-pBM11 fragment and the 170bp Ncol (blunt) -BamHI synthetic VGFa gene were used together using DNA Ligase linked and the resulting mixture was used to transform a capable HB101. The transformants were selected for neomycin and screened by restriction analysis and nucleotide sequencing using the Sanger dideoxy technique. A correct construction containing the modified alkaline phosphatase signal sequence in the frame with the nVGFa gene was isolated.

Signal sequence. -

ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA MDQSTIALALLPLLFTPVT

nVGF -

CAAAAGCTGACTCTGGTAACGCTATCGAAACTACTTCTCCGGAAATCACTAACGC KADSGNAIETTSPEITNA

TACTACTGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGC TTDIPAIRLCGPEGDGYC

Koni SphI

CTGCATGGTÄCCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCT LHGTCIHARDIDGYACRC-S

ctcatggctacactggÜItcgttgccagcatgttgttctggtcgactaccagcg hgytgircqhvvlvdyqr

BamHI ttaaggatcc Ter

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Example VII

Expression of the polypeptide of interest as a fusion protein with an alkaline phosphatase signal sequence

A. Production of pBM11 / PAK / nVGFa ^ Alkalische Phosphatase- Sianalseauenz / nVGFa with natural VGF terminus and the sequences GTC and GYACRCÌ

The plasmid pBM11 / PAD / nVGF was mutagenized in vitro (Morinaga et al., Biotechnology (1984) 2: 636-643) to change the codons that change the second amino acid in the signal sequence, namely the Asp (D) codon back in that for Lys (K), the rest found in the natural sequence. This mutagenesis also introduced a Pvul site in the signal sequence.

atggatcaatctacaatcgccctcgcacttctcccactgctgttcactccagtgacaaaa mdqstialallpllftpvtk gctgactctggtaacgctatcgaaactacttctccggaaatcactaacgctactact adsgnaiettspeitnatt

Kpnl gacatcccggctatccgtctgtgcggcccggaaggcgacggctactgcctgcatggt dipairlcgpegdgyclhg

SphI

acctgcatccatgcacgtgacatcgacggctacgcatgccgttgctctcatggctacact tcihardidgyacrcshgyt

EcoRI BamHI

ggaattcgttgccagcatgttgttctggtcgactaccagcgttaaggatcc gircqhvvlvdyqr Ter

B. Preparation of pBM11 / PAK / EGF

In this expression cassette, the EGF gene is part of a fusion with the alkaline phosphatase signal sequence.

The plasmid pBM11 / PAD / EGF was mutagenized in vitro to change the codons encoding the second amino acid in the signal sequence to convert the Asp (D) codon to that for Lys (K), the rest, which in the natural sequence is found. A Pvul site also introduces this mutagenesis into the signal sequence.

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Pvul

Signal sequence - *

atgaaacaatctàcgatcgccctcgcacttctcccactgctgttcactccagtga mkqstialallpllftpvt

EGF -

caaaagctaattctgactctgaatgcccgctgtctcatgacggctactgcctgca kansdsecplshdgyclh

Nsil SphI

tgacggcgtatgcatgtacatcgaagctctggacaagtacgcatgcaactgcgtt dgvcmyiealdkyacncv gttggctacatcggcgaacgttgccagtaccgtgacctgaaatggtgggaactgc vgyigercqyrdlkwwelr

BamHI gttaaggatcc if

C. Preparation of TacPak / EGF (alkaline phosphatase sianalsauenz / human EGF

1. Preparation of plasmid fragments

Plasmid p135-1 was derived from plasmid pDR540 (Pharmacia) and contained the Cro gene SD and a BglII site downstream of the lac-SD. pDR540 is an expression vector that contains the trp-lac hybrid promoter. pl35-1 was digested with BglII and BamHI and treated with bacterial, alkaline phosphatase.

The plasmid pBM11 / PAK / EGF was digested with Pvull and BamHI and the 230 bp fragment encoding part of the alkaline phosphatase signal sequence and the human EGF were isolated.

2. Preparation of the TacPakl and TacPak2 ligonucleotides

The synthetic oligonucleotides TacPakl and TacPak2 were designed with an overhang compatible with the Bloll site of pl35-1 and a Pvull overhang, compatible with the Pvull site in alkaline phosphatase / EGF PvuIl / BamHI fraction. The oligonucleotides were synthesized on an Applied Biosystems oligonucleotide synthesizer.

Belli Pvul

Bql

TacPakl 5 'GATCTATGAAACAATCTACGAT 3' TacPak2 3 'ATACTTTGTTAGATGC 5'

3. Connection and isolation of the TacPak / EGF clone

The Balll-BamHI digested pl35-1, the 230bp PAK / EGF fragment and the oligonucleotides TacPakl and TacPak2 were linked using DNA ligase, transformed into a capable HB101 and a correct construction was isolated by "DNA sequencing.

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(Hindlll site of pDR540)

i

AAGCTTACTCCC

trp-35 (I6bp) lac-10

CATCCCCCTG [TTGACA] ATTAATCAÎCGGCTCG (TATAATG)

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

Pvul croSD (llbp) BglII {AGGA} GGTTCAGATCT

Signal sequence

ATGAAACAATCTACGATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA MKQSTIALALLPLLFTPVT

EGF ->

CAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCA • KANSDSECPLSHDGYCLH

Ns i I SphI

TGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTT DGVCMYIEALDKYACNCV

GTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC VGYIGERCQYRDLKWWELR

BamHI

GTTAAGGATCC *

Example VII

Expression of a polypeptide of interest as a fusion protein with an alkaline phosphatase signal sequence using an expression cassette containing a transcriptional termination region

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A. Preparation of pTCPt / EGF iïtrp-35116bDHac-1 OïïlacSDH 1 bp lATGI / alkaline phosphatase-Si-na / human EGF / trans. term.NEO)

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

1. Production of the 420bp Hindlllfstumpfenl-BamHI fragment from TacPak / EGF

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

2. Preparation of the 2.8 kb EcoRlfetumpfenl-BamHI fragment from pBM1 6t / 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 was isolated. This DNA fragment contained the pBR322 origin, the neomycin resistance gene without its Ncol site and the Gen32-like transcription terminator downstream of the BamHI site.

3. Connection and isolation of pTCPt / EGF

The 2.8 kb EcoRI (blunt) BamHl fragment from pBM16t / NDP / VGFa was linked to the 420bp HindIII (blunt) BamHl fragment from TacPak / EGF and the resulting DNA was used to transform capable JM109 (lacIq). A correct construction has been isolated by its resistance to neomycin and by DNA sequencing.

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CHindiII site of pDR540)

I.

AAGCTTACTCCC

trp-35 (16bp) lac-10

CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG {TATAATG}

mRNA 51 lacl binding site lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC {AGGA} AACAGGATCACTA

Pvul croSD (llbp) BglII signal sequences2: ->

{AGGA} GGTTCAGATCT ATGAAACAATCTACGATCGCCCTCGCACTTCTCC

MKQSTIALALLP

EGF ->

CACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGTC LLFTPVTKANSDSECPLS

Nsil

TCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGAC HDGYCLHDGVCMYIEALD

SphI

AAGTACGCATGCAACTGCGTTGTTGGCTACATCGGC KYACNCVVGYIG

BamHI

GAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGA ERCQYRDLKWWELR *

Trans. Term. CTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATC

B. Preparation of nTCPt / nVGFa iïtm-35l16bpriac-10ïïlacSD1 rcroSDlHhorATGl / alkaline phosphatase sianal / N-terminal VGFa with the sequence GTC and GYACRCVtrans. term. -NEO

This plasmid has the tac promotor elements and uses cro-SD to express the modified VGF gene with the N-terminal extension downstream of the alkaline phosphatase signal sequence. The plasmid has a pBR322 background with a neomycin resistance gene.

1. Preparation of the 350hn-Pvul-BamHI fragment from pBM1 1 / PAK / nVGFa

The plasmid pBM11 / PAK / nVGFa was digested with Pvul and BamHI and the 350bp fragment was isolated by gel electrophoresis. This fragment contains most of the alkaline phosphatase signal sequence and the nVGFa gene.

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2. Preparation of the 2.8kb Pvul-BamHI fragment from pTCPt / EGF

The plasmid pTCPt / EGF was digested with Pvul and BamHI and the 2.8 kb fragment was isolated by gel electrophoresis.

Connection and isolation of pTCPt / nVGFa

The 2.8kb fragment and the 350bp fragment were joined using DNA ligase and the DNA was used to transform capable JMI09 (laclq). Correct construction was isolated using restriction analysis.

3. Connection and isolation of pTCPt / nVGFa

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

(Hindlll site of pDR540)

AAGCTTACTCCC

trp-35 (16bp) lac-10

CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG)

mRNA 51 lacl binding site lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC {AGGA} AACAGGATCACTA

Pvul croSD (llbp) Bgll.I Signal SequenS ->

{AGGA} GGTTCAGATCT ATGAAACAATCTACGATCGCCCTCGCACTTCTCCC

MKQSTIALALLP

nVGF ->

ACTGCTGTTCACTCCAGTGACAAAAGCTGACTCTGGTAACGCTATCGAAACTACT LLFTPVTKADSGNAIETT

TCTCCGGAAATCACTAACGCTACTACTGACATCCCGGCTATCCGTCTGTGCGGCC SPEITNATTDIPAIRLCGP

Kpnl

CGGAAGGCGACGGCTACTGCCTGCATGGTACCTGCATCCATGCACGTGACATCGA EGDGYCLHGTCIHARDID

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SphI EcoRI

CGGCTACGCATGCCGTTGCTCTCATGGCTACACTGGAATTCGTTGCCAGCATGTT GYACRCSHGYTGIRCQHV

BamHI Trans.

GTTCTGGTCGACTACCAGCGTTAAGGATCCGTGACTAATTGGGGACCCTAGAGGT

VLVDYQR *

Term.

CCCCTTTTTTATTTTAAAACGATC

C. Preparation of pTNPt / EGF trtrp-35H7bpnac-10ïïnSD18bp TATGI / alkaline phosphatase-Si-anal / human EGF / trans. term.-NEO

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

1. Preparation of 2.8kb Pvul-BamHI-pTNPt

The plasmid pTNPt was digested with Pvul and BamHI and the 1.8 kb fragment was isolated by gel electrophoresis.

2. Production of the 300bo Pvul-BamHI fraction from oBMU / PAK / EGF

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

3. Connection and isolation of pTNPt / EGF

The 2.8 kb fragment and the 300 bp fragment were linked using DNA ligase and the DNA was converted into capable JM109 (lacIq). Correct construction was isolated by restriction analysis and DNA sequencing.

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CEcoRI site of pBMll)

l

GAATTACTCCCCATCC

SstI

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

CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG) TGTGG / AATTG

Bsml mRNA-> n mRNA->

TGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCTTTGGGGTGTGT

GATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGCTGCCAATGTGC CAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACACACCACCAAAGCTAA

Pvul nSD (8bp) signal sequence ->

CTGAC {AGGA} GAATCCAG ATGAAACAATCTACGATCGCCCTCGCACTTCTC

MKQSTIALALL

EGF ->

CCACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGT PLLFTPVTKANSDSECPLS

Nsil

CTCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGA HDGYCLHDGVCMYIEALD

SphI

CAAGTACGCATGCAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCAGTACCGT KYACNCVVGYIGERCQYR

BamHI BamHI

GACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGACTAATTGGGGA

DLKWWELR *

(BamHI site of pBMll) Trans. Term. 1

CCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC

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Example IX

Composition of the growth factor gene for expression in eukaryotic cells

A. Preparation of the plasmid pRSV / VGF

The transfection of this plasmid in ovary lines of a Chinese hamster led to the production of the natural VGF.

fai Production of HindllKstumpfi-BolllKstumpfl-pRSV

The plasmid pRSV was digested with HindIII and BglII and the 4kb fragment was gel purified. The protruding ends were blunted with the Klenow fragment of DNA polymerase and then the 5'-phosphates were removed with alkaline intestinal calf phosphatase.

(b) Production of the Ddel fragment. which contains the VGF gene

A subcloned genomic fragment of vaccinia DNA was digested with Ddel and the protruding ends were blunted using the Klenow fragment and a 550bp Ddel fragment was gel purified.

(ci connection and isolation of pRSV / VGF

The 4 kb Hindllltstumpfì-BglIKstumpfì fragment from pRSV and the 550 bp Ddel (stump) fragment of vaccinia DNA were joined together using DNA ligase and the transformants were selected with ampicillin and screened by restriction analysis. A correct construction was isolated and designated pRSV / VGF.

(d) Transfection of CHO cells by pRSV / VGF

The plasmid pRSV / VGF was co-transfected with a plasmid pRSV with calcium phosphate precipitation in Chinese hamster cells. The transfectants were screened by Southern dot blot hybridization and a positive clone named pRSV / VGF52 was isolated.

B. Preparation of plasmid pAcA / GF

An additional expression system that was used to express the recombinant VGF protein is an insect system. Compare Maeda et ai. and Carboneil et al., supra. In such a system, the Autographa Californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Soodoptera fruoiperda cells. The envelope gene can be cloned into non-essential regions (e.g. the polyhedrin 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 leads to inactivation of the polyhedrin gene and production of an incomplete recombinant virus (i.e. the virus lacks the protein envelope which is encoded by the polyhedrin gene). These recombinant viruses are then used to infect Soodoptera fruoiperda cells. in which the inserted gene is expressed.

The VGF gene of interest was placed under the control of an appropriate promoter for an insect cell system. The plasmid pAc610 contains the polyhedrin gene, which has an ampiciliin-resistant marker. Polylinkers are inserted into this gene 50 bases downstream of the transcription start site of the polyhedrin gene and 7 bases before the first ATG. The VGF gene is cloned at a suitable polylinker site so that it is under the control of the polyhedrin promoter. The ATG methion start codon and the translational termination codons are those of the VGF gene. The transcriptional start sites and the polyadenylation signals are those of the polyhedrin gene.

(ai Production of Smal-BamHl-pAc610

The plasmid pAc610 was digested with Smal and BamHI.

(b) Production of the HindllKstumpfi-Bolll fragment of the VGF gene

The plasmid pRSVA / GF was digested with HindIII and the protruding ends were blunted using the Klenow fragment. The DNA was then digested with BglII and the 560 bp fragment was gel purified.

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(c) Cleaning and isolation of dAc / VGF

The Smal-BamHI fraction of pAc610 and the 560 bp fragment of pRSV / VGF containing the VGF gene were linked using DNA ligase and the transformants were screened by restriction analysis. A correct construction was isolated and designated pAc / VGF.

(dì transfection of Sf9 cells with pAcA / GF

The plasmid pAcA / GF was co-transfected with wild-type AcNPV baculovirus DNA in Sf9 insect cells (Spodoptera fruoiperdai). The transfectants were screened for the inclusion of negative phenotypes in plaque tests. A negative plaque inclusion was isolated and after 5 Rounds of consecutive piaque cleaning made a higher titer of the recombinant virus stock.

C. Preparation of pAc / SFGF

Baculovirus expression from natural Shope fibroma virus growth factor.

(a) Preparation of BamHI-digested pAc610

The plasmid pAc610 was digested with BamHI and Smal. The Smal site of this plasmid is located downstream of the baculovirus polyhedrin promoter.

(b) Production of a 430 bp Hincll-Sau3a fraction of a genomic Shope fribromavirus DNA

The 3.7kb BglII fragment of the 19kb BamHl-C fragment of the genomic Shope fibromavirus DNA was cloned in BamHI-digested SP64 plasmid and with SP64 / 3.7. designated. The plasmid SP64 / 3.7. was digested with Hincll and the Ikb-Hincll fragment was gel cleaned. The Ikb fragment was then digested with Sau3a and the 430bp Hincll-Sau3a fragment was gel purified.

(c) Connection and isolation of pAc / SFGF

The BamHI-Smal-digested pAc610 became the 430bp HincllSau3a fragment. which contains the SFGF gene, and the resulting mixture was transformed into suitable E. coli HB101. The transformants were screened by restriction analysis and the correct construction was isolated and the pAc / SFGF was designated.

Example X

Preparation of polypeptides A. Festo rabbit synthesis of VGF and TGF

1. Synthetic VGF fraction

The sequence, which corresponds to the mutilated VGF and begins with the DI PAIR sequence and ends with the LVDY sequence, was assembled on an Applied Biosystems Model 430 A peptide synthesizer, using the standard method. Treatment of the peptide obtained with liquid HF under customary “low-high” conditions gave the crude deblocked peptide. The peptide was subjected to chromatofocusing on PBE94 (Pharmacia). The partially purified peptide was eluted at pH 6.5. The brief treatment with 0.2 N sodium hydroxide removed the cysteine protecting groups (ethyl carbamoyl) and the peptide was oxidized in the presence of oxidizing and reducing glutathione. Purification by gel filtration and HPLC gave highly purified VGF with the following sequence:

DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSHGYTGIRCQHWLVDY

2. Synthetic human and rat TGF-alpha

Human and rat TGF-alpha were chemically synthesized essentially as above for the VGF and were provided by Penninsula Labs.

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B. Isolation of recombinant polypeptides produced in procarvontic cells

1. Growth factors which were produced in vectors based on pBM, using the PL promoter and the ts-CI reoressor

E. coli B (HB101), which contained the pBM11 / NDP / growth factor plasmids, were grown in Luria broth at 30 ° C. The culture density was measured at 550 nm and when the density reached an absorbance of 0.7 to 0.9, the synthesis of the growth factor fusion protein was initiated by increasing the temperature to 42 ° C. The culture was incubated at this temperature for 5 to 20 hours and then the bacteria were isolated by centrifugation and cooled to -70 ° C until used.

For the isolation of the recombinant protein, the cells were thawed in buffer solution which contained 0.05 M NaHaPCU, pH 7.2, 0.5 M NaCl, 0.01 M EDTA. 150 ml of buffer was used to prepare 50 g of bacteria. The cells were disrupted by sonication on ice for 15 minutes using a 1/4 inch probe, with 50% pulses at 60 watts of force. After disruption of the cells, the insoluble protein was centrifuged at 12,000 revolutions / min. collected in a GSA rotor for 90 min. The beads containing the insoluble protein were then resuspended in 50 ml of 6 M guanidine hydrochloride. The insoluble material was removed by centrifugation at 25,000 rpm for 2 hours. collected on a Beck-man type 30 ultracentrifuge rotor. The supernatant was collected and stored at -20 ° C until further use.

The fusion protein was purified on either Sephacryl S300 or Fractogel HW-55 columns equilibrated with 1 M guanidine hydrochloride. Fractions containing the fusion protein were identified as those fractions which determined a polypeptide having a molecular weight corresponding to the molecular weight of the polypeptide encoded by the synthetic gene by determination on a 15% polyacrylamide urea gel.

To produce the active form of the recombinant growth factor, the protein was refolded by containing it in 50 mM Tris-HGI buffer, pH 8.7, which contained 1 M guanidine hydrochloride, 1.24 mM reduced glutathione and 0.25 mM oxidized glutathione , incubated at 4 ° C for 3 to 10 days. The biological activity of the growth factor was checked by a competitive receptor binding test as described above (see Example I.C). After a maximum level of activity was obtained, the protein was freeze-dried against distilled water and to dryness.

If the leader sequence was to be removed, the protein was cleaved either by resuspension in 70% formic acid and incubation at 40 ° C for 3 days or by overnight incubation at room temperature in a 100-fold molar excess of cyanogen bromide. The cleaved product was dialyzed against distilled water and freeze-dried to dryness.

For further purification, the recombinant growth factor was resuspended in 40% acetonitrile, 0.1% TFA and purified by HPLC using a BioRad TSK-250 column. The fractions containing the growth factor were pooled and subjected to further purification using reverse phase HPLC, either Waters jiBondapk C-18 or Rainin Dynamax C-8. The eluent consisted of a linear gradient of 20 to 40% acetonitrile, which contained 0.1% TFA. The fractions containing the receptor binding activity were pooled, freeze-dried and stored at -20 ° C until used.

(a) TGF and modified TGF (i) N / TGF

The recombinant, modified, human TGF was prepared from plasmid pBM11 / N / TGF and contained 33 amino acids of the N gene at the N-terminus and the sequence change QEEK instead of the natural human sequence QEDK.

(b) Modified and garbled VGF

(i) PAD / nVGFa

Recombinant modified VGF was prepared from plasmid pBM11 / PAD / nVGFa, which contained the extreme N-terminal sequence of VGF and the modified sequences 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 was mutilated in the YQR sequence at the C-terminus.

(ii) NDP / VGFa

The recombinant modified VGF was derived from the plasmid pBM11 / NDP / VGFa, which was used in the

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DIPAIR sequence begins and ends at the YKQR sequence in the VGF. It has the modified sequences GTC and GYACRC instead of the natural VGF sequences GDC and GMYCRC. The VGFa fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N-terminus and the acid-labile dipeptide aspartic acid proline.

(iii) VGFa

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

(iv) NDP / VGFA

The recombinant modified VGF was made from plasmid pBM11 / NDP / VGFA, which begins with the DIPAIR sequence and ends with the YKQR sequence in VGF, and has the modified sequences GTC and GYACVC instead of the natural VGF sequences GDC and GMYCRC. The VGFA fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N-terminus and the acid-labile dipeptide aspartic acid proline.

(c) Chimeric TGF / VGF hybrid

(i) N / TTV (TGF / TGF / VGF)

Recombinant modified TTV was made from pBM11 / N / TTV and contained the amino acid sequence of the human TGF in the amino terminal 2/3 of the gene with the exception of the sequence QEEK, which was changed from the natural human sequence QEDK. The carboxy terminus was changed 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 fusion protein with 33 amino acids of the N gene at the N-terminus.

(ii) NDP / TTV

Recombinant TTV was produced from plasmid pBM11 / N / TTV and modified as described in (a), except that the TTV fragment is expressed as a fusion protein with 32 amino acids of the N gene at the N-terminus and the acid-labile dipeptide aspartic acid proline has been.

(iii) NDP / VTV

The recombinant modified VTV was prepared from plasmid pBM11 / NDP / VTV and contained the amino acid sequence of the human TGF, the middle region with the amino acid sequence QEEK replacing the natural sequence QEDK. The N-terminal and C-terminal regions were derived from the garbled VGF sequence and begin with the DIPAIR sequence and end with the YQR sequence. The VTV fragment was expressed on a fusion protein with 32 amino acids of the N gene at the N-terminus and in the acid-labile dipeptide aspartic acid proline.

(iv) NDP / TVV

Recombined modified VTV was made from plasmid pBM11 / NDP / TVV and contained the amino acid sequence of human TGF in the N-terminal region of the gene. The middle and the C-terminal region were derived from the mutilated VGF sequences and change with the YQR sequence. In addition, the synthetic gene has the change GYACVC instead of GMYCRC. The TW fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N-terminus and the acid-labile dipeptide aspartic acid proline.

2. Growth factors made in vectors containing the tac or lac promoter

Bacterial hosts containing expression cassettes containing the tac or lac promoter were grown at 30 to 37 ° C to an optical density of A600 = 0.2 to 0.8, either in LB broth or in a chemically defined medium such as M9 medium, which was provided with thiamine and glucose. An appropriate antibiotic was added to the growth medium to select the hosts for the expression cassette. The bacterial cultures were induced at a concentration of 100 to 1000 mM IPTG and grown at 30 ° C for 16 to 24 hours. For the expression cassettes lacking the lacl gene of the bacterial host, the bacterial hosts carried an F factor with the lacql gene, such as MJ109, XL1, JM103, etc. For the expression cassettes that carried the lacl gene, examples of bacterial hosts HB101, DH1, DH5 etc. If the bacterial host contains a lac operon (lac +), the expression cassette can be induced with 1% lactose

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will. After the induction period, the growth factors can be isolated either from the medium or from cell beads.

(a) PAK / EGF

Human EGF made from the TAcPak / EGF and pTcCPt / EGF expression cassettes was isolated from the medium in an active form, removing the alkaline signal sequence. Approximately 85% of the active EGF was found in the medium, with the rest associated with the cell beads. This expression cassette gave 4 mg / l of the active EGF. The cells were removed from the medium by centrifugation and the medium was filtered through an Amicon SY30, 30,000 Mr cut-off spiral filter and then passed through a Q-Sepharose column and the high purity human EGF was 0 in 20 mM NaPÜ4 pH 7 , up to 0.5 M NaCI gradient eluted. Alternatively, the growth factors can be isolated from the cell beads by osmotic shock or sonication and purified by essentially the same method as the method described above.

(b) PAK / nVGFa

The recombinant nVGFa was produced from the expression cassette pTCPt / nVGFa. The nVGFa was isolated from the cell beads by sonication and contained approximately 40% of the total bacterial protein.

C. Isolation of the VGF and the SFGF. produced by eucavontic expression of the natural gene

1. Production of VGF from monkey kidney cells

The natural VGF was produced by infecting monkey kidney cells with the vaccinia virus. Ceropithecus monkey kidneys (BSC-1) cell monolayers were coated with 15 plaque-forming units (pfu) per cell of purified VV (strain WR, grown in Hela cells) and by sucrose density gradient sedimentation (Moss, B. , (1981) in Gene Amplification and Analysis, ed. Chirickjian, JG and Papis, TS (Elsevier / North-Holland, NY), volume 2, pages 253-266)). The infected cells were incubated at 37 ° C with approximately 1 ml Eagle's basal medium with 2% fetal calf serum per 2x106 cells. Mock-infected cells were treated in an identical manner. The cell culture supernatants were clarified by centrifugation and low speed and freeze-dried. The residue was then again suspended in 1M acetic acid and dialyzed intensively against 0.2M acetic acid. The insoluble material was removed by centrifugation and the supernatant was freeze-dried and resuspended in 1/100 of the original volume of 1M acetic acid and stored at 4 ° C.

2. Production of VGF in CHO cells

The natural VGF was also produced by transfection of the plasmid pRSV / VGF, which contained the VGF gene fragment in Chinese hamster ovary cells. The transferred cells were expanded and the medium and cell beads were tested for the presence of VGF by immunoprecipitation using an antiserum against an N-terminal VGF peptide.

3. Production of VGF in silkworms using the baculovirus promoter

(a) Transformation

The host cell for AcNPV is Spodoptera fruoiperda (sf9). To produce a recombinant virus stock, the VGF-containing plasmid was mixed with AcNPVDNA and transfected into sf9 cells using the calcium phosphate technique. The recombinant viruses were isolated from the medium and the plaque was purified on sf9 cells. The recombinant plaques were identified by hybridization using radiolabeled VGF DNA as a sample.

(b) Expression

Once a recombinant virus is identified, it is spread to sf9 cells. The recombinant virus stock is then used to infect sf cells, the cells are lysed and the supernatants screened for the production of the VGF protein, the protein being purified as described above for the vaccinia virus-infected mammalian cells.

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The predicted sequence of the VGF produced in the insect cells is:

DSGNAIETTSPEITNATTDIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSHGYTGIR CQHVVLMYQRSENPNTTTSYIPSPGIMLVLVGIIIITCCLLSVYRFTRRTKLPIQDMWP

A potential glycosylation site is present on the asparagine at position 15 of the N-terminal end of the polypeptide.

This 121-residue sequence starts with the N-terminal sequence which was determined directly for the purified VGF from W-infected monkey cells (i.e. at residue 20 of the open VGF reading frame) and continues to the last rest of the open reading frame. The signal peptide (residues 1 to 19 of the open reading frame) is therefore missing, but includes the transmembrane sequence (YIPSPGIMLVLVGIIIITCCLLSVY).

The predicted molecular weight of the 121 residual peptide is 13,304, excluding carbohydrate. The apparent molecular weight of the baculovirus-produced VGF is 17,000, which is significantly smaller than that obtained from VV-infected cells, suggesting that the two forms are likely to be developed differently. The baculovirus-produced VGF may lack an additional part of the N-terminal sequence or part of the C-terminal sequence or both, or could differ in the type and extent of the glycosylation.

4. Preparation of SFGF in silkworms using the baculovirus promoter

(a) Transfection of sf9 cells with pAc / SFGF

The plasmid pAc / SFGF was co-transfected with AcNPV DNA of wild-type baculovirus in sf9 insect cells (Soodoptera fruoioerdai. The transfectants were screened for the exclusion of the negative Phe-notype in plaque tests. An exclusion-negative plate was isolated and after A high titer of a recombinant virus stock was produced after 5 rounds of successive plate cleaning, which according to Southern analysis contained the SFGF gene.

D. Natural EGF

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

Example XI

Activity tests

A. Mitooen test

The mitogenesis tests were carried out as follows: diploid, human fibroblasts from cell cultures of newborn foreskin were made to a density of 3x104 cells / hole (96-hole plates, Nunclon, Roskilde, Denmark) and were changed in Eagle medium according to Dulbecco (GIBCO) / 10% newborn calf serum grown. The cultures were then placed in medium containing 0.2% newborn calf serum and 2 days later the EGF (10 ng / ml) or the growth factor to be tested (10 ng equivalents of EGF / ml) was added. After 8 hours, the cultures were labeled with 5- [125l] -lod-2'-deoxyuridine (Amersham, 10 p.Ci/mi, 5 Ci / mg; 1 Ci = 37 GBq) and the amount of the TCA insoluble in the Material incorporated isotopes were determined as described (Twardzik et al., Proc. Nati. Acad. Sei. USA (1985) 182: 5300-5304).

B. Soft Aoar Colonial Growth Stimulation Test

A 0.5 ml base layer of 0.5% agar (Agar Noble; Difco Laboratories, Detroit, Michigan) in growth medium was applied to 24-well Costar tissue culture plates. 1/2 ml 0.3% agar in growth medium containing 1 to 1.5.104 cells / ml NRK cells or other cell lines of interest and various concentrations of the tested factor were at 37 ° C in a humid atmosphere with 5% CO2 incubated in air and after 7 days 0.5 ml of 0.3% agar in growth medium with the same content of the factor to be determined was added. The colonies were numbered, detached and removed. The number of colonies with more than 6 cells was counted.

C. EGF Recipe Inhibition Inhibition Test

The radioreceptor test was carried out as follows: the binding of 125I-labeled EGF (1251-EGF) to its receptor on monolayers of A431 cells was modified by the method of Cohen and Carpenter, Proc. Nati. Acad. Be. USA (1975) 71: 1317-1321). The cell

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lines (1x103 per hole) were fixed on 24-hole plates (Linbro, Flow Laboratories) with 10% formalin in phosphate-buffered saline before the test. The formalin-fixed cells are not stripped from the plates as easily as is the case with unfixed cells, and the replicate values were therefore constant. Under these test conditions, 125 | _eGF (1x1 010 cpm / nmoi) saturates the binding test at 3nM; the tests were carried out at 10% of the saturation value. The growth factor concentrations were expressed as ng equivalents of EGF / ml, i.e. the amount required to produce an inhibition of 125l EGF binding equivalent to that produced by a known amount of EGF.

D. Radioimmunoassav

Each 50 pJ reaction contained the following: 20 mM sodium phosphate at pH 7.4, 200 mM NaCl, 40 mM di-thiothreitol, 0.1% bovine serum albumin, 0.1% NaN3, 125sl-labeled peptide (2x10 * cpm) accordingly the 17 carboxy-terminal residues of alpha-TGF (Linsley et al., Proc. Nati. Acad. Sei. USA (1985) 82: 356-369), antiserum with a final dilution of 1: 5000 and other additions as indicated. The reaction was initiated by the addition of antiserum and was continued at 23 ° C for 90 min. An equal volume of 10% formaiin-fixed S. aureus (Pansorbin, Calbiochem) was then added and the incubation was continued at 23 ° C. for a further 30 min. The immunoadsorbent was removed by sedimentation and the amount of bound 125 I-labeled peptide was measured. The amount of peptide bound was measured for non-specific binding in the absence of the antibody (less than 5% of the total) and expressed as a percentage of the maximum binding.

E. Wound healing

1. Middle dermal, thermal injuries

Mid-dermal thermal injuries were made to the dorsal thorax of anesthetized Yorkshire pigs (30 pounds), with the back shaved and hair removed with commercial hair removal cream. A brass template (3x3 cm, 147 gm) was equilibrated in a water bath at 70 ° C and brought into firm contact with the skin for exactly 10 seconds. The resulting bubble was then removed. 5 middle dermal burns were placed on each side of the spine, each approximately 2.5 cm apart. The burns were treated twice daily with approximately 3 ml of carrier cream (Siivadene) alone or with growth factor, or the wounds remained untreated. After 9 or 10 days of treatment with natural VGF, the pigs were anesthetized and the scab was removed from the burns. Biopsies of each burn in the re-epithelized areas were taken.

2. Mid-term donor transplant injury

A 5 month old, 20.5 kg small pig was anesthetized with 20 mg / kg ketamine and 2 mg / kg rompum. The dorsal thorax was shaved, rubbed with betadine, and washed carefully with saline. A number of six 5x5 donor sites were performed on each side of the dorsal thorax with a Padgett dermatome at 60/1000 inches by taking two strips at 30/1000 inches. The topical therapy contains 1 ml saline in 20 g Siivadene, evenly distributed over six wounds on the left side. The right side was treated with 1 ml of the test growth factor in 20 g of Silvaden evenly distributed between the six wounds. All wounds were given a burn dressing, Chux, Ace-Wickel and Gerkin. The animals were anesthetized as described above on the days after surgery 1, 2, 3, 4, 7, 8, 9, 10 and 11. The bandages were removed and the wounds were rubbed with betadine and rinsed carefully with saline. The appropriate agent was applied and the wounds were healed as described above.

Example XII

Biological activity of recombinant growth factors produced in prokaryotic cells A. EGF receptor binduno

This test determines a molecule's ability to bind the EGF receptor, as measured by its ability to inhibit EGF binding to its receptor. All growth factors and chimeric growth factors, whether modified, mutilated, isolated, were found to be active in the EGF receptor binding test. A summary of these results is shown below:

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table

EGF receptor binding of recombinant growth factors

peptide

Expression cassette

EGF purity

Binding to receptor

N / TGF modified, mutilated fusion pBM11 / N / TGF

95%

Yes

PAD / nVGFa modified, mutilated fusion pBM11 / PAD / nVGFa

95%

Yes

NDP / VGFa modified, mutilated fusion pBM11 / NDP / VGFa

95%

Yes

N / TTV modified, garbled, chimeric fusion pBMl 1 / NDP / VGFa

95%

Yes

NDP / TTV modified, garbled, chimeric fusion pBM11 / N / TTV '

95%

Yes

NDP / VTV modified, garbled, chimeric fusion pBM11 / NDP / TTV

95%

Yes

NDP / TW modified, garbled, chimeric fusion pBM11 / NDP / VTV

95%

Yes

PAK / EG F

pBM11 / PAK / EGF

95%

Yes

EGF

pBM11 / PAK / EGF

95%

Yes

PAD / EGF

pBM11 / PAD / EGF

95%

Yes

EGF

pBM11 / PAD / EGF

95%

Yes

PAK / EGF

TacPak / EGF ■

95%

Yes

EGF

TacPak / EGF

95%

Yes

PAK / EGF

pTCPt / EGF

95%

Yes

EGF

pTCPt / EGF

95%

Yes

A comparison of the binding inhibition curves for natural mouse EGF and bacterially expressed, recombined, chimeric growth factor N / TTV (polypeptide fusion of 32 N-terminal amino acids of the lambda N gene and the modified and mutilated TGF / VGF hybrid ) led to the realization that there were no differences in the binding activities.

B. Mitogenic activity

The activity of various purified growth factors was tested and the activity was comparable in all cases to the effect caused by the EGF. The tested compounds are shown below:

table

Mitogenic activity of growth factors

Peptides Mitogenic Activity * •

TTV modified, garbled, chimeric Yes

N / TTV modified, garbled, chimeric fusion Yes

* as measured by 3H thymidine or 125l IdU incorporation.

C. Wound Healing 1. Middermal Injuries

The effect of natural or synthetic TGF, EGF and VGF as well as recombinant growth factors on middle dermal injuries was tested as described in example VEI. The percentage of the original burn area that healed was determined by computer aided telemetry and the percentage of wound re-epithelization was determined. About 15% of the untreated wounds were re-epithelized. Treatment with Silvaden alone or Sil-vaden with EGF resulted in approximately 50% re-epithelization, while treatment with synthetic TGF or natural VGF resulted in approximately 90% re-epithelization. The optimal concentration to promote re-epithelization of EGF was 1 to 10 ng / ml, while the synthetic TGF and the natural VGF showed a maximum response at 0.1 ng / ml.

Experiments similar to those described above were performed to determine the effect on wound healing of TGF, a modified, garbled form of VGF (VGFa), or a modified, garbled, chimeric fusion of TGF and VGF (TTV), all of which by the recombinant technology was made in bacteria. These recombinant growth factors and

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Hybrid growth factors accelerated wound healing to the same extent as synthetic TGF or natural VGF with an optimal concentration of 0.1 ng / ml.

2. Middle dermal donor transplant injuries

The modified, mutilated VGFa was tested for its ability to accelerate wound healing in the donor translocation model. The treatment schedule was the same as described above (see Example I) using 1 ml VGFa, 5 ng / ml in 20 g Silvaden. Photographs were taken every day. A summary is given below:

table

Effect of recombinant VGFa in wound healing

Treatment * POD wound condition ** 7

POD 8

POD 9

POD 10

Saline solution very open open

mostly healed with sth. healed

VGFa modified, open;

predominantly healed healed healed mutilated obviously. Epithelization

* Silvaden as carrier ** POD = postoperative day

The modified, mutilated VGFa accelerated the healing of the wound compared to the control with the carrier material. The photographs (not available here) of POD 8 showed significant differences between saline and VGFa.

Example XIII

Biological activity of VGF produced in eukaryotic cells A. VGF. produced in monkey kidney cells (BSC-1 Ì

1. The medium derived from BSC-1 cells 24 hours after infection with VV was tested for the presence of material containing 125I-EGF for binding to EGF receptor-rich human epidermoid carcinoma cells (A431) can compete. The VV-infected cells released an effective activity that competed with the EGF, the activity being referred to as VGF. The control medium volume from mock-infected BSC-1 control cultures contained minimal activity in competition with the EGF.

Evidence of VGF production at the earliest tested time, 2 hours after infection, showed increased levels of VGF in the culture medium. Maximum amounts of this activity were found in the culture supernatant after 12 hours, with only a slight increase after 24 hours.

The extent of VGF production turned out to be a function of the multiplicity of the virus infection, as shown in the following table:

table

Effectiveness of multiplicity of infection on VGF release

Virus multiplicity pfu per cell

Released VGF ng eq. of EGF per ml

0

-

10th

2.7

20th

4.5

40

6.1

80

10.1

The cultures of the BSC-1 cells were infused in the pfu-pro-ratio as indicated and the supernatants (approximately 1 ml / 2x106 cells) were harvested 24 hours after infection. Samples were acidified, lyophilized and tested by double radioreceptor assays for EGF as described (Delarco and Todaro, Proc. Nati. Acad. Sei. USA (1978) 75: 401-405). nq ae. = Nanoqram equivalents

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2. Partial cleaning of the VGF

The activity that competes with EGF and was found in W-infected BSC-1 cells was partially purified from the acid-extracted culture supernatants 24 hours after infection as described above. The acid solubilized polypeptides (10.5 mg) from the supernatants were applied to a Biogel P10 column equilibrated with 1M acetic acid and samples from each fraction were tested for activity competing with EGF. The active main peak (fraction 42) was eluted somewhat after the Mr 29,000 carbon anhydrase marker with an apparent Mr of 25,000. Molecular weight was confirmed using tandem-linked Bio-Sil TSK 250 HPLC size columns, with all activity as the major peak in the region being eluted with the Mr 25,000 protein marker. One microgram of the partially purified VGF was equivalent to 90ng EGF in the radioreceptor competition test.

3. Further cleaning by VGF

The pooled fractions of the gel filtration column (25-35) were concentrated by vacuum centrifugation, resuspended in 0.05% trifluoroacetic acid (TFA), clarified and injected into 3.9 mm x 20 cm jxBondapak Ci8 columns (Waters, Milford, MA). The peptides were treated with a linear 20 to 60% acetonitrile gradient in 0.05% TFA at a flow rate of 1.0 ml / min. eluted at 22 ° C. Aliquots of each fraction were tested for EGF competitive activity by a radioreceptor test. The peptides that corresponded to the peak activity were collected and diluted with 0.05% TFA and again injected into a jiBondapak column and isolated using isocratic conditions. The acetonitrile concentration was about 22 to 25%.

A comparison of the proportion of VGF produced by BSC-1 cells infected with 15pfuVV per cell with that of alpha-TGF produced by retrovirus-transformed cells is shown in the following table:

table

Comparison of the biological activity of VGF with alpha-TGF and EGF

Growth factor 1

EGF receptor binding 2 ng eq. to EGF / ml / medium

Introduction d. DNA synthesis [125l] IdU incorporated. (cpm / vessel)

Stimulation d. anchorage-independent. Cell growth4

Soft agar colonies / plate none

-

1,779

<20

VGF

2.3

3,760

108

TGF-alpha

0.2

NT

294

EGF

-

8,482

346

NT = not tested

1VGF (90 ng equivalents (eq) to EGF / ng protein) were purified by gel filtration after elution from a Ci8 iiBondapak column (Waters Associates). Alpha-TGF were purified from Snyder-Theiien Felin sarcoma virus-transformed Fisher rat embryo cells, according to Marquardt et ai., Proc. Nati. Acad. Be. USA (1983) 80: 4684-4688; the EGF was purified from submaximal glands of mice (Cohen et al., J. Biol. Chem. (1980) 255: 41834-41842).

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

3The mitogenesis tests were carried out as described; the calm cultures of dipioid, human fibroblasts were treated with 10 ng purified EGF / ml or with the same number of EGF equivalents VGF / ml. The values for the [125l] iodo-deoxyuridine ([125l] ldU) represent an average of three determinations.

4 The number of soft agar colonies represents an average number of colonies which contain a minimum of 20 NRK cells / six random low-energy fields 10 days after the start (1.5 × 104 cells / ml) with purified EGF (5 ng / ml) or contain the same number of EGF equivalents VGF / ml and 2.0 ng TGF-β / ml purified from human platelets, according to Assoian et al., J. Biol. Chem. (1983) 258: 7155-7160. The plates with NRK cells treated with TGF-ß alone above did not form colonies.

B. VGF produced in CHO cells

The biological activity of VGF produced in CHO cells was determined using the EGF bin

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manure tests evaluated, the culture medium being used without further purification. The growth factor bound to the EGF receptor.

C. VGF made in silkworms

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

table

Percent epithelialization of wounds after growth factor treatment

Pig 1 Of course. VGF ng / mi

(9 days after combustion)

right side

Silvaden

Untreated

0.1

0.1

15%

0%

70%

h-EGF (ig / ml

65%

Left side

Silvaden

Untreated

0.1

0.1

0.1

75%

55%

60%

70%

30%

Pig 2

VGF * jig / ml

(10 days after combustion)

right side

Silvaden

Untreated

0.1

0.5

1.0

50%

0%

95%

95%

60%

Left side

Silvaden

Untreated

0.1

0.5

1.0

50%

0%

60%

75%

40%

Pig 3

Rat TGF ng / ml

(9 days after combustion)

right side

Silvaden

Untreated

0.1

1.0

10th

25%

5%

90%

85%

65%

* VGF: Natural VGF made from Vaecinia virus-infected cells

D. Immunological comparison of VGF and TGF

In the radioimmunoassay described above, a 50% shift in the antigen from the antibody to the carboxy-terminal 17 amino acids of the rat TGF-alpha molecule was found at an antigen concentration of approximately 0.2 to 0.3 ng equivalents of EGF, the 125i-labeled alpha-TGF competes with alpha-TGF. When VGF was tested at equivalent concentrations, no competition was found. In a competitive immunoassay for native EGF, VGF preparations, a minimal shift (less than 10%) of the 125 | -EGF from a polyclonal antibody to the native EGF was found even when tested with 50 ng EGF equivalents / ml.

It is evident from the above results that the VGF is a potent epithelializing agent which satisfactorily withstands comparison with other growth factors which have been previously tested and which have mitogenic activity. The polypeptides according to the invention can be used with various host organisms for the induction of an immunogenic response, an improved cellular proliferation, for wound healing or the like. Epithelialization and vascularization have been observed in wounds, as well as rapid restoration of wound strength, i.e. a resistance to separation or tearing. The host organisms include mammals such as rodents, pets, primates or humans.

According to the invention, the new compounds are widely used in various fields, for example as diagnostics, for in vivo and in vitro effects on cells, as mitogens, additives in nutrient media, as agonists and antagonists against EGF and TGF, as immunogens, as therapeutics, for improving wound healing and the like.

Furthermore, by providing small oligopeptides with binding activity, the oligopeptide can be used alone or in combination with other polypeptides to fuse with other polypeptides to change the properties of the other polypeptides, thereby binding the polypeptide to cells with growth factor receptors . As a result, numerous polypeptides can be reversibly bound to cells which have growth factor receptors, the properties of the cells being influenced in a predetermined manner.

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Claims (33)

Claims 1. polypeptide, characterized by a) its ability to bind to an EGF receptor, b) its substantially the same loop structure as naturally occurring growth factor capable of binding to the EGF receptor as a natural ligand; and c) at least 30% homology with the natural EGF receptor binder, the polypeptide having three regions, each one Has an amino acid sequence which is at least 30% homologous to the fragments of the same or different natural ligands and has the same relative position as these fragments of the natural ligand, the ranges being defined as follows: first region of at least 11 N-terminal amino acids, which begin at amino acid aa-6 to aa1 and end at amino acid aa11 to aa15; second region of at least 11 central fragment amino acids, which begin at amino acids aa11 to aais and end at amino acids aa25 to aa £ 9; third region of at least 14 amino-C-terminal amino acids, which begin at amino acid aa2® to aa29 and end at aa40 to aa53, with the proviso that if the polypeptide has an amino acid sequence that is identical to the natural ligand, a fourth region of at least 6 extremely N-terminal amino acids, which begin at aa-7 to aa-i and at amino acid aa- «to aa -7 ends, is present and has a sequence that is different from the extreme N-terminal amino acids of the natural ligand. 2. Polypeptide according to claim 1, characterized in that it has a fourth region of at least 6 extreme N-terminal amino acids, which begin at aa-7 to aa-1 and end at aa-45 to aa-7. 3. Polypeptide according to claim 1 or 2, characterized in that it comprises the following amino acid sequence or a fragment thereof of at least 37 amino acids, which contains at least amino acids 1 to 37: aa -24 -20 -15 -10 -5 -1 dsgnaiettspeitnattdipair 1 5 10 15 20 lcgpegdgyclhxìgdcihxSard 25 30 35 40 idgx3mycrcshgytgircqhvv 45 50 53 lvdyqrsenpnt wherein X1 is a bond or an amino acid; X2 represents a bond or 1-2 amino acids; X3 represents a bond or 1-4 amino acids; Xi, X2 and X3 can be the same or different. 4. A polypeptide according to claim 3, wherein at least one amino acid of said aa14 is threonine, aa24 is tyrosine, aa25 is alanine and aa27 is valine. 5. Polypeptide according to claim 3, characterized in that the amino acid sequence contains aa-6 to aa47. 6. Polypeptide according to claim 5, characterized in that aa14 is threonine, aa24 is tyrosine and aa25 is alanine. 7. The polypeptide according to claim 6, wherein aa-6 to aa13 of the amino acid sequence is replaced by the following sequence: aa -6 -11 5 10 13 vv-shfndcpdshtqfcfhg. 8. Polypeptide according to claim 6, characterized in that aa14 to aa27 of the amino acid sequence is replaced by the following sequence: 56 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 681 081 A5 aa 14 20 25 27 TCRFLVQEDKPACV. 9. Polypeptide according to claim 8, characterized in that aa22 is replaced by glutamic acid. 10. DNA sequence, characterized in that it encodes a polypeptide according to one of claims 1 or 2. 11. DNA sequence according to claim 10, characterized in that the EGF ligand is VGF, TGF-a, MGF, EGF, SFGF or amphiregulin. 12. DNA sequence according to claim 10, characterized in that the DNA sequence encodes a leader sequence, where an amino acid sequence which can be selectively cleaved is optionally inserted in the carboxy terminus of this leader sequence which is terminated with the amino terminus first region or if the fourth region is present, is connected to the amino terminus of the fourth region. 13. DNA sequence according to claim 12, characterized in that the leader sequence is an N-terminal fragment of a lambda-N protein of a lambda-Cro protein or of amphiregulin. 14. DNA sequence according to claim 12, characterized in that the leader sequence is a signal sequence. 15. DNA sequence according to claim 14, characterized in that the signal sequence is an alkaline phosphatase signal sequence, a monkey TGF-p signal sequence or a K-lactis killer toxin signal sequence. 16. DNA sequence according to one of claims 10 to 15, characterized in that at least one of said regions has at least 30% homology to a fragment of VGF, TGF-a, EGF or SFGF. 17. DNA sequence according to claim 10, characterized in that it has at least one change in the sequence which encodes a natural ligand and leads to the following changes in the amino acid sequence: HGD to HGT; GMYC to GYAC; RCS to VCS; CLHGDC to CLHGTC; and GMYCRC to GYACVC. 18. DNA sequence according to claim 10, characterized in that it comprises a polypeptide and a leader sequence with 8 to 45 N-terminal amino acids of a bacteriophage lambda N protein or Cro protein, amphiregulin, TR Gen32 or a bacterial, alkaline Phosphatase signal sequence, which is connected to the amino terminus of the polypeptide, optionally encoded via an amino acid sequence which can be cleaved selectively. 19. DNA sequence according to claim 18, characterized in that it encodes a polypeptide with a leader sequence of 32 of the 33 N-terminal amino acids. 20. DNA sequence according to claim 19, characterized in that the encoded polypeptide is TGF-a, VGF, VGFA, VGFa or EGF. 21. DNA sequence according to claim 18, characterized in that it encodes a polypeptide, wherein the leader sequence is an alkaline phosphatase signal sequence. 22. DNA sequence according to claim 21, characterized in that the encoded polypeptide is VGFa, NVGFa or EGF. 23. DNA sequence according to claim 19, characterized in that it encodes the polypeptide with the following amino acid sequence: MVVSHFNDCPDSHTQFCFHG TCRFLVQEEKPACVCSHGYT GIRCQHVVLVDYQR. 24. DNA sequence according to claim 19, characterized in that it encodes the polypeptide with the following amino acid sequence: MDIPAIRLCGPEGDGYCLHG TCRFLVQEEKPACVCSHGYT GIRCQHVVLVDYQR. 25. DNA sequence according to claim 19, characterized in that it encodes the polypeptide with the following amino acid sequence: MVVSHFNDCPDSHTQFCFHGT CIHARDIDGYACVCSHGYTGI RCQHVVLVDYQR. 57 5 10th 15 20th 25th 30th 35 40 45 50 55 CH 681 081 A5 26. DNA sequence according to claim 18, characterized in that it encodes a polypeptide with a leader sequence comprising about 21 N-terminal amino acids of Cro protein. 27. DNA sequence according to claim 18, characterized in that it encodes a polypeptide with a leader sequence which comprises about 8 terminal amino acids of the T4 gene 32. 28. DNA sequence according to claim 27, characterized in that the encoded polypeptide has the following amino acid sequence: VVSHFNDCPDSHTQFCFHGT CRFLVQEEKPACVCSHGYTG IRCQHVVLVDYQR. 29. DNA sequence according to claim 10, characterized in that it encodes a polypeptide according to one of claims 3-9. 30. Expression cassette, characterized in that it contains, in the direction of transcription, a functional, transcriptional and translational initiation control region in a host cell, and a DNA sequence according to claim 10. 31. Host cell, characterized in that it contains an expression cassette according to claim 30. 32. A method for producing a polypeptide according to claim 1 or 2, characterized in that a host cell according to claim 31 is grown in a suitable culture medium, the polypeptide being expressed and isolated. 33. Plasmid for the production of a host cell according to claim 31, characterized in that the plasmid comprises the functional regions of pBM11 / NDP / EGF; pBM11 / PAD / EGF; pBM11 / PAK / EGF; pBM11 / N / TGF; pBM11 / NDP / VGFA; pBM11 / NDP / VGFa; pBM11 / PAD / nVGFa; pBM11 / PAK / nVGFa; pRSV / VGF; pAc / SFGF; pAc / VGF; Tac / Pak / EGF; pTCPt / EGF; pTCPt / nVGFa; pBM11 / N / TTV; pBM11 / NDP / TTV; pBM11 / NDP / VTV or pBM16 / NDP / TVV contains. 58