BE1004202A5 - New polypeptides having growth factor activity and nucleic acid sequences encoding polypeptides. - Google Patents

Abstract

New polypeptides capable of binding to a receptor for an epidermal growth factor, in addition to their precursors, which have an analogy with polypeptides encoded by the vaccinia virus. These compounds (hEGF ....) include 6 cysteines which form loops analogous to those which exist in natural growth factors which bind to an epidermal growth factor receptor. These compounds serve as mitogens, additives for culture media and therapeutic agents.

Description

       

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  New polypeptides having growth factor activity and nucleic acid sequences encoding the polypeptides.



  Introduction.



  Technical request.



   The present invention relates to novel polypeptide compositions having growth factor activity, especially hybrid polypeptides, in addition to methods for preparing them using recombinant DNA techniques.



  Background of the invention.



   The polypeptides secreted by mammalian cells are found in significant numbers to have growth factor activity. The sequence of these polypeptides is substantially conserved in a wide variety of mammals. Much of the interest in these compounds is due to their association with oncogenic power. There is also interest in regulating the production of these growth factors and how they act as regulators of cell activity.



   It has also been observed that infection of mammalian cells with viruses induces proliferative growth of infected cells. For the purposes of the invention, particular interest is offered by the viruses of the smallpox group, such as the smallpox virus, the vaccinia virus and the viruses associated with particular diseases such as the Shope fibroma virus, Yaba tumor virus and Molluscum contagiosum virus (MCV).



   It would be interesting to determine whether the viruses which induce cell proliferation during infection produce polypeptides which have a relationship with growth factors by acting

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 as growth factors or as receptors for a growth factor. These compounds could then be used for studies on viral actions, for tests for viruses, as mitogens in nutrient media and for the development of therapeutic agents to treat viral infections. It would also be interesting to develop compounds which could be agonists or antagonists of growth factors for use in vitro in cell cultures, in the examination of mitotic processes and for therapeutic purposes.



  Bibliography.



   Venkatesan et al., J. Virol. (1982) 44: 637-646 describe DNA sequencing of the structural gene encoding vaccinia virus proteins, but there is no indication that any of these proteins has activity growth factor. Cooper et al., Ibib (1981) 37: 284-294, describe the translation of mRNAs encoded in the reverse terminal repeat of the vaccinia virus genome. Proliferative conditions due to viruses of the smallpox family have been described in connection with the Shope fibroma virus (Shope, J. Exp. Med. (1932) 56: 793-822), the 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 the epidermal growth factor (TGM) 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 transforming growth factor (TGF) is indicated by Savage et al., J. Biol. Chem. (1973) 248: 7669-7692. See also Doolittle et al., Nature (1984) 307: 558-560 and the

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 Australian patent application no. 85006288. It has been suggested that the receptor-binding region of the
 EMI3.1
 EGF molecule is found in the loop between the third cysteine residue and the fourth (Komoriya et al., Proc. Natl. Acad. Soi. USA (1983) 81: 1351-1355).



   New descriptions of the vaccinia virus growth factor (VGF) can be found in Brown et al., Nature (1985) 313: 491-492; Reisner, Nature (1985) 313: 801-803 and Blomquist et al., Proc. Natl. Acad. Self. USA (1984) 81: 7263-7367. The growth factor of the natural Shope fibroma virus (SFGF) is described by Chang W. C. et al. in Molecular and Cellular Biol. (1987) 7: 535-540. Shope mentions in J. Exp. Med. (1932) 56: 793-802 that infection of adult rabbits with the Shope fibroma virus induces a rapid proliferation of fibroblasts leading to a benign fibroma. Infection in newborn or adult rabbits without immunity causes rapid proliferation of fibroblasts leading to atypical invasive fibrosarcoma.



  Allison A. C., J. Natl. Cancer Inst. (1966) 36: 869-876: Smith et al., J. Natl. Cancer Inst. (1973) 50: 1529-
 EMI3.2
 1539; Allison A. C., J. Natl. Cancer Inst. (1966) 35: 859-868.



   The growth factor of natural myxomatosis virus (MGF) is described by Upton et al., J. Virol. (1987) 61: 1271-1275. Infection of adult rabbits with the myxomatosis virus causes an invasive myxosarcomatous tumor that proliferates rapidly. Strayer D. S. and Sell S., J. Natl. 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 detected. Porter C. D. and Archard L. C. J.

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  Gen. Virol. (1987) 68: 673-682. Infection with the Molluscum contagiosum virus in humans induces benign skin tumors characterized by rapidly proliferating cells. Brown et al., Sex.



  Transm. Say. (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 with the fibroid virus of Shope, myxomatosis virus and Molluscum contagiosum virus, it is expected to have a related growth factor.



   Mouse EGF (mEGF) (Scott et al., 1983, supra; Gray et al., 1983) supra) and human EGF (hEGT) (Gregory et. Al., Int. J Pept. Protein Res. (1977) 9: 107-118) are known to be homologs of TGFs in humans, mice and rats (Marquardt et al., Proc. Natl. Acad. Sci. USA ( 1983) 80: 4684-4688) and residues 45 to 85 of a polypeptide of 140 residues, which is the vaccinia growth factor (VGF) encoded by the genome of the vaccinia virus (Venkatesan et al., 1982 , supra).



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



   This bibliography is given for reference.



  Overview of the invention
The invention relates to novel polypeptide compositions having an analogy with fragments of viral proteins, which compositions behave as mitogenic agents and can be used in nutritive media, as reagents for detecting receptors for

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 growth factors or the presence of growth factors, and as competitive agents for transforming growth factor and epidermal growth factor. These compositions find a therapeutic application, for example, in aid in the reconstitution of the epithelium and in the healing of burns and wounds. These new compositions can be synthesized.

   The peptides of the invention can be formed in the form of oligopeptides or of fused proteins according to recombination techniques in a wide variety of hosts.



  Description of the drawings.



   Fig. 1 is a comparison of the amino acid sequence of the vaccinia virus protein with known growth factors, where the initial amino acid of VGF is aspartic acid (D) at position 20 from methionine, which position corresponds to aa-24 of the peptides described, the legend for FIG. 1 being the following:
FAMILY OF GROWTH FACTORS MGF Myxomatosis virus growth factor SFGF Shope fibroid virus growth factor VGF Vaccine virus growth factor hTGF human TGF human rTGF TGF rat hEGF EGF human mEGF EGF mouse rEGF EGF of the rat gpEGF EGF of the guinea pig Amphiregulin.



  * The numbers correspond to the amino acid residues in the VGF, as indicated in the brief, and

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Fig. 2 represents the proposed structure of the VGF at maturity, with replacements of asparagine (N), the legend for FIG. 2 being as follows: + Aspartic acid in the SFGF.



  * Possible glycosylation site in SFGF and / or
MGF.



  N The amino acid indicated is replaced by asparagine.



  Specific forms of realization.



   The subject of the invention is novel compositions which can act as agonists or antagonists of epidermal growth factor (EGF) or transforming growth factor (TGF) and which find a wide variety of applications in cell culture, in diagnostic means, in vivo therapy and in combination with other peptides to form hybrid polypeptides serving as immunogens for the production of antibodies. Polynucleotide sequences can be isolated or prepared and can be introduced into an expression vector for the purpose of expressing the compositions of the invention.



   The compositions are analogous to fragments of viral proteins, particularly vaccinia group virus proteins. As shown in Fig. 1, the VGF comprises uncharged and hydrophobic residues near the N-terminal end between residues 5 and 15 and near 11 c-terminal end between residues 100 and 124. These residues can be considered by analogy with glycoproteins integral membranes as a hydrophobic N-terminal signaling sequence which is eliminated by proteolysis during translation or immediately after, and a C-terminal transmembrane sequence which serves to anchor the mature protein in the membrane. Cleavage of the arg-43 viral polypeptide

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 and arg-90 of the mature peptide would lead to the release of a soluble polypeptide.

   The polypeptide W of 140 residues can be considered to give rise first to a membrane-associated protein of approximately 121 residues after elimination of the amino acid sequence 19. After the intracellular treatment, a soluble peptide growth factor d 'about 77 residues is made up. Stroobant P. et al. Cell (1985) 42: 383-393.



   The comparison of the growth factor of the myxomatosis virus (MGF) and the growth factor of the Shope's fibroid virus (SFGF) with the other growth factors of this family (Fig. 1) reveals some notable differences. MGF and SFGF, synthesized as precursor molecules, include a signaling sequence at the N-terminus, but unlike other growth factors, do not include a transmembrane domain at the C-terminus , which indicates that these are secreted molecules. Sequence analysis reveals a significant use of asparagine in MGF and SFGF. In six positions, MGF and MSGF include conserved asparagines where the other growth factors do not have asparagine.

   Among these positions, asparagines replace two highly conserved glycine residues at positions 8 and 31, where the first cysteine is at position 2. These two glycine residues appear in combination with highly conserved tyrosine residues at positions 9 and 32. Due to the highly conserved nature of the amino acids on this face of the molecule, the hypothesis is that the cysteine loops present on this face are involved in the binding to the receptor and in the function of the growth factors of this family.

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   Glycine and asparagine are predicted to have the highest beta turn potential (Chou and Fassman, Ann. Rev. Biochem. (1978) 47: 251-276). Thus, it appears that the conservation of this tower potential is extremely important for the activity of the growth factor. Growth factors other than those of myxomatosis and Shope's fibroma almost exclusively include glycine in the important turn regions of the molecule. In MGF and SFGF, almost all of these glycines have been transformed into asparagines (see Fig. 2). This indicates an important biological function due to the replacement of the highly conserved glycine by the asparagine residue.

   The possibility exists that the replacement of glycine by asparagine in the regions of tower preserved in the structure of the growth factor could lead to an increased functionality of the growth factor, either by accentuation of the binding on the receptor of the EGF or a related receptor, which increases the direct effect of the growth factor in the affected cells, either by increasing the stability of the molecule.



   Two of the new appearances of asparagines in MGF and SFGF offer possible N-linked glycosylation sites. In addition, these sites exist in cysteine loops which tolerate varying numbers of amino acids. Therefore, new appearances of asparagine can offer multiple benefits to the growth factor in addition to increased functionality. One of these could be the stabilization of the protein in vivo by protecting, through glycosylation, degradation or immunogenic sites.



   The polypeptides of the invention are characterized in that they are capable of

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 have at least one loop or circle, and usually three loops or circles, because of cysteines that form bridges, where the physiologically active part of the molecule having activity related to that of a growth factor is about 12 to 65 amino acids, but usually 15 to 60 amino acids.

   Among these three loops, two are from 12 to 15 amino acids (14 to 17 annular amino acids), excluding the cysteine bridge, and more particularly one is 12 or 13 amino acids (14 or 15 annular amino acids ) and the other is 15 amino acids (17 ring amino acids), the proximal loop of the Nterminal end is usually 12 or 13 amino acids and more usually 12 amino acids and the middle loop is 15 amino acids .



   The third loop or C-proximal loop is 8 amino acids (10 annular amino acids) and including the flanking amino acids is of the
 EMI9.1
 following formula:
 EMI9.2
 
 EMI9.3
 or the letter symbols for amino acids have their traditional meaning, C being cysteine, G being glycine, N being asparagine, Y being tyrosine and R being arginine; aa25 is a neutral amino acid which can be aliphatic, in particular of 3 or 4 carbon atoms, or else aromatic, in particular of 9 carbon atoms and comprising 0 or 1 hydroxyl radical,

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 such as alanine, serine, threonine, tyrosine or phenylalanine;

   aa27 is a neutral or basic amino acid, having in particular 3 to 6 carbon atoms, and, when neutral, comprising 0 or 1 carboxamide radical, for example arginine, alanine, valine, leucine, isoleucine, asparagine or glutamine; aa29 is a neutral or basic amino acid, which is aliphatic or aromatic, the aromatic example of which is histidine and the aliphatic example has 2 to 6 and more usually 3 to 6 carbon atoms, and comprises 0 or 1 hydroxyl radicals , for example serine, threonine, leucine, valine, isoleucine or arginine; aa30 is a neutral amino acid, which is aliphatic or aromatic, the aromatic example of which is histine and the aliphatic example has 3 to 6 carbon atoms, and comprises 0 or 1 hydroxyl radical, in particular serine, the isoleucine and valine;

   aa33 denotes a neutral aliphatic amino acid from 2 to 6 and more usually from 3 to 6 carbon atoms comprising 0 or 1 hydroxyl radical, more particularly serine, threonine, valine, leucine or isoleucine, or an acid amino acid, for example glutamic acid or aspartic acid; aa35 is a neutral or acidic amino acid of 3 to 6 carbon atoms, of which an example of neutral is alanine, valine, leucine, isoleucine or serine, and of which an example of acid is acid aspartic or glutamic acid; aa38 is a substituted neutral aliphatic amino acid, the substituent of which is a carboxamide radical and which has 4 or 5 carbon atoms, or

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 EMI11.1
 0 indeed an acidic amino acid, for example asparagine, glutamine, aspartic acid or glutamic acid;

   aa39 is an aromatic amino acid or a neutral aliphatic amino acid of 3 or 4 carbon atoms usually carrying a hydroxyl radical, preferably aromatic, for example phenylalanine, histidine, tyrosine, serine or threonine; aa40 is a substituted or unsubstituted neutral aliphatic amino acid of 3 to 6 carbon atoms, or a basic amino acid, for example alanine, valine, isoleucine, glutamine or arginine;

   m and n represent 0 or 1;
 EMI11.2
 3 4 pp3 and pp4 may be the same or different and are hydrogen atoms indicating the termination of the polypeptide, or are polypeptide chains not exceeding a total of 1000 amino acids and usually not exceeding a total of about 500 amino acids, preferably where at least 90% of the amino acids and more preferably at least about 95% of the amino acids present are in one of the two polypeptide chains; in some cases, the chain may include a single amino acid and a maximum of 100 amino acids, and frequently 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 the natural polypeptide chains associated with the natural growth factors and proteins of the smallpox group viruses, or they may be other than the natural chains or parts thereof associated with the specifically described polypeptide chain by the formula, and usually unrelated.



   The definitions of amino acids are given below.

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 EMI12.1
 
 <tb>
 <tb>



  Neutral <SEP> (Ne)
 <tb> aliphatic <SEP> (Al)
 <tb> no <SEP> substituted <SEP> G, <SEP> A, <SEP> V, <SEP> L, <SEP> I, <SEP> P
 <tb> substituted
 <tb> oxy <SEP> S, <SEP> T
 <tb> thio <SEP> C, <SEP> M
 <tb> amido <SEP> N, <SEP> Q
 <tb> aromatic <SEP> (Ar)
 <tb> no <SEP> substituted <SEP> F
 <tb> substituted <SEP> Y
 <tb> heterocyclic <SEP> H, <SEP> W
 <tb> Loaded <SEP> (at <SEP> pH <SEP> 6.0)
 <tb> basic <SEP> (Ba) <SEP> K, <SEP> R
 <tb> acids <SEP> (Ac) <SEP> D, <SEP> E
 <tb>
 The abbreviations in parentheses refer to specific amino acid groups. By unsubstituted, it is meant that there are no other heterosubstitutants than the carboxyl and amino radicals existing in glycine. Amino acids are natural L amino acids.



   Neutral amino acids can also be described as carrying non-polar or polar (but uncharged) R radicals. The amino acids that meet these definitions are:
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 acid amino acid (S). The immediately flanking amino acids (i.e., amino acids 27 and 38) of the cysteines outside the loop may include

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 at least one charged amino acid and at least one amido-substituted aliphatic amino acid.

   Among the amino acids in the loop (28 to 37), from 4 to 6 and usually 5 amino acids will be neutral aliphatic amino acids and at most 2, but usually 1, will be aromatic amino acids.



   Particular interest is offered by the compounds when the polypeptide has 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 is offered by the polypeptides comprising amino acids 44 to 88 and their fragments of VGF (aal to aa47, see FIG. 1).



   Preferably, aa29 is a substituted or unsubstituted aliphatic amino acid of 3 to 6 carbon atoms comprising 0 or 1 hydroxyl radical, in particular serine or valine, or else an aromatic amino acid, in particular histidine; preferably, aa30 is histidine, serine or an unsubstituted aliphatic amino acid of 5 or 6 carbon atoms, in particular isoleucine; preferably, aa33 is a substituted or unsubstituted aliphatic amino acid comprising 0 or 1 hydroxyl radical and 3 to 6 carbon atoms; preferably, aa35 is an aliphatic amino acid of 3 to 6 carbon atoms; preferably aa38 is glutamine or glutamic acid; preferably, aa39 is aromatic and is more advantageously histidine or phenylalanine; preferably, aa40 is a neutral or basic amino acid.



   A particular interest is offered by the presence of two loops, with the C-proximal loop

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 linked to the second loop, where the amino acids of the second loop can be very varied. The second loop preferably has about 14 to 16 amino acids, excluding the cysteine bridge, and more preferably about 15 amino acids.

   Among the amino acids, 6 to 9 and preferably 7 to 9, but more advantageously approximately 8, are substituted or unsubstituted aliphatic amino acids, the preference being that not more than approximately 3 of the amino acids are substituted and more advantageously that 1 or 2 amino acids are substituted; there will be 2 to 4 aromatic amino acids and more particularly 3 aromatic amino acids, preferably histidine and tyrosine; there will be 2 to 4 acidic amino acids, preferably 3 acidic amino acids and more particularly aspartic acid; and there will be 0 to 2, and more usually 1 basic amino acid, especially arginine.

   It is desirable that the cysteine forming the envisaged loop closest to the cysteine of the other loop is separated therefrom by 0 to 2 amino acids, and more usually by 0 or 1 amino acid, in particular arginine.



   Particular interest for certain applications of the compounds of the invention is offered by the compounds of the following formula:

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 EMI15.1
 or: the symbols aa25 to aa40 of the formula have been described; ppl and pp2 are the same or different and may be hydrogen atoms, indicating the terminal part of the polypeptide shown, or be polypeptides having a total of up to about 1000 amino acids, more usually up to about 500 amino acids, and may comprise a total of barely 1 amino acid, or may be individually or separately polypeptides from 1 to 100 amino acids, more usually from about 1 to 75 amino acids and more particularly from about 5 to 50 amino acids;

   these polypeptides have specific applications in the modification of the sequence described specifically for a predetermined purpose; Ppl and pp2 may be identical to, or be different from, the natural vaccinia virus polypeptide; aal can be any amino acid, more particularly an aliphatic amino acid, a basic amino acid or an acid amino acid, preferably an unsubstituted aliphatic amino acid of 2 to 6 atoms

  <Desc / Clms Page number 16>

 carbon, in particular glycine, leucine, valine, lysine, glutamic acid or aspartic acid; aa3 is a neutral amino acid, in particular from 2 to 4 carbon atoms, and more particularly asparagine, glycine or proline;

   aa4 can be any aliphatic or aromatic amino acid, in particular from 2 to 6 carbon atoms, which can be neutral or acid, and more particularly from 3 to 6 carbon atoms, in particular proline, aspartic acid, histidine , serine or leucine; aa5 is an acidic amino acid or a substituted neutral aliphatic amino acid, in particular glutamic acid or aspartic acid, or else a hydroxy-substituted amino acid of 3 or 4 carbon atoms, more particularly serine; aa6 is an unsubstituted aliphatic amino acid of 2 to 6 and usually of 2 or 3 carbon atoms, or else an aromatic amino acid, in particular glycine, histidine or tyrosine; aa7 is an acidic, basic or neutral amino acid, in particular from 3 to 6 carbon atoms, such as glutamic acid, aspartic acid, lysine or threonine;

   aa8 is an unsubstituted neutral aliphatic amino acid of 2 or 3 carbon atoms, or else an amino acid substituted with a carboxamide radical of 4 or 5 carbon atoms, for example asparagine, glutamine or glycine; aall is a neutral amino acid, either aliphatic or aromatic, more particularly aliphatic, especially having 5 or 6 carbon atoms, more particularly leucine or phenylalanine;

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 aal2 is an aromatic amino acid or a carboxamido-substituted aliphatic amino acid of 4 or 5 carbon atoms, in particular histidine or asparagine; aal2a is a neutral aliphatic amino acid or an acidic amino acid, more particularly glycine, asparagine or aspartic acid;

   aal4 is an acidic amino acid or a substituted or unsubstituted neutral aliphatic amino acid of 4 or 5 carbon atoms comprising 0 or 1 hydroxyl radical, in particular aspartic acid, threonine or valine; aal6 is a bond or a neutral or basic aliphatic amino acid, either substituted or unsubstituted, of 4 to 6 carbon atoms, in particular isoleucine, arginine or methionine;
Arl is an aromatic amino acid which can comprise a carbocycle or a heterocycle, in particular histidine, phenylalanine or tyrosine;

     aal7a is a neutral aliphatic amino acid, either substituted or unsubstituted, from 3 to 6 and usually from 3 to 5 carbon atoms comprising 0 or 1 hydroxyl radical, in particular valine, leucine, isoleucine or threonine: aal7b is an unsubstituted neutral amino acid of 5 or 6 carbon atoms or an acidic amino acid, more particularly valine, isoleucine or glutamic acid; aa18 is a neutral, substituted or unsubstituted aliphatic amino acid of 2 to 6 and preferably of 3 to 6 carbon atoms comprising 0 or 1 hydroxyl radical, in particular alanine, serine or glutamine;

   aa19 can be any amino acid, in particular an acidic aliphatic amino acid or

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 basic, more particularly from 4 to 6 carbon atoms and preferably from 5 to 6 carbon atoms, in particular arginine, leucine, valine or glutamic acid; aa20 is an acidic amino acid or a carboxamido-substituted aliphatic amino acid, in particular aspartic acid, asparagine, glutamic acid or glutamine; aa2l is an unsubstituted neutral aliphatic amino acid or basic amino acid of 3 to 6 carbon atoms having 0 or 1 hydroxyl or carboxamide radical, in particular isoleucine, leucine, asparagine, serine, threonine, lysine or arginine;

   aa22 is a bond or an acidic amino acid, which is aspartic acid or glutamic acid, or a neutral aliphatic amino acid, in particular valine; aa22a is a neutral aliphatic amino acid comprising 0 or 1 hydroxyl radical and in particular a hydroxyl radical, more especially serine; aa22b is an aliphatic or neutral amino acid of 4 to 6 carbon atoms, in particular leucine or isoleucine; aa23 is a bond or a substituted or unsubstituted neutral aliphatic amino acid of 2 to 6 carbon atoms comprising 0 or 1 hydroxyl radical, in particular glycine, serine, threonine or asparagine;

   aa24 is an aliphatic amino acid, in particular a thio-substituted aliphatic amino acid, more particularly methionine or proline, or else an aromatic amino acid, in particular tyrosine; aa41 is a neutral aliphatic amino acid

  <Desc / Clms Page number 19>

 substituted or unsubstituted or acidic from 4 to 6 carbon atoms, in particular valine, asparagine or aspartic acid; aa43 is a neutral, acidic or basic aliphatic acid, substituted or unsubstituted, of 4 to 6 carbon atoms, in particular valine, leucine, isoleucine, arginine, lysine or aspartic acid;

   
 EMI19.1
 aa44 aa44 can be any amino acid, in particular other than a basic amino acid, and can be acid, neutral or aromatic and which, when not aromatic, has 3 to 6 carbon atoms, in particular l aspartic acid, alanine, leucine, tryptophan or threonine, and p represents 0 or 1.



   It is of particular interest that ppl has the following sequence:
 EMI19.2
 where: PP-in combination with the following amino acid symbols in the above sequence is the equivalent of ppl. The above sequence falls under the definition of ppl, ppl can represent a hydrogen atom or an amino acid sequence. One or more of the amino acids, symbolized by aa-x (x being any number) can be a bond, so as to reduce the number of amino acids in the N-terminal chain. Therefore, all or part of the amino acid sequence shown

  <Desc / Clms Page number 20>

 
 EMI20.1
 0 may be present.

   When a fraction of the sequence is present, it is preferable that the sequence comprises contiguous amino acids, i.e. amino acids in numerical order without deletions.



  Usually there will be at least one amino acid, more usually at least three, more usually still at least six, where the remaining upstream amino acids may be absent, so that P can be joined to axa-1, axa-6, and so on; axa-1 can be any amino acid, in particular aliphatic, of approximately 3 to 6 carbon atoms, preferably neutral or basic, more particularly lysine, arginine, proline, asparagine or serine; aa-2 can be any amino acid, either aliphatic or aromatic, in particular aliphatic, more particularly about 3 to 6 carbon atoms;

   aa-3 can be an aliphatic or aromatic, in particular aliphatic, either polar or non-polar amino acid, from 2 to 6 and more usually from 2 to 5 carbon atoms, generally comprising 0 or 1 hydroxyl radical; aa-4 can be any aliphatic amino acid, neutral or basic, generally from 3 to 6 and usually from 5 to 6 carbon atoms, in particular asparagine, proline or lysine; ara-S can be any aliphatic amino acid, in particular neutral, in general from 3 to 6 carbon atoms, in particular valine or isoleucine; aa-6 is a neutral or acidic amino acid, when aliphatic, of approximately 3 to 6 and more usually approximately 4 to 6 carbon atoms, more particularly isoleucine, valine or acid

  <Desc / Clms Page number 21>

 aspartic;

   
 EMI21.1
 aa-7, aa-8, aa-9, aa-12, aa-14, aa-15, aa-16, aa-17, aa-l9, aa "20 aa-21 and aa-23 are amino acids aliphatics of 3 to 6 carbon atoms, either polar or non-polar, in particular polar comprising 0 or 1 hydroxyl radical, or are aromatic amino acids; aa-10 and aa-25 are non-polar aliphatic amino acids or acids amino acids of 2 to 6, and more usually of 2 or 3 carbon atoms; aa ", aa-22 and aa-24 are polar aliphatic amino acids or acid amino acids of 4 or 5 carbon atoms, comprising in particular a carboxamido radical; aa-11 and aa-18 are aliphatic amino acids, either polar or non-polar, or acidic amino acids.



   A special interest is offered by a
 EMI21.2
 N-terminal fragment of aa "-up to aa" or from ara-l to aa "and more particularly from aa-l until 1 to aa-6 0 aa". Advantageously, P- can be an unrelated amino acid sequence which serves as a fusion protein, in particular to form an immunogenic peptide in order to produce antibodies against the compound.



   The main aspects of the compositions of the invention are provided by the sequence aa25 to aa40, in particular by the sequence from cysteine at position 28 to cysteine at position 37 and the loops formed by cysteines at positions 2 and 10 and the cysteines at positions 15 and 26. It is desirable that the compositions of the invention comprise the loop created by the cysteines at positions 28 and 37 together with the loop created

  <Desc / Clms Page number 22>

 by cysteines at positions 15 and 26.



   Hybrid polypeptide sequences can be prepared by combining fragments of various polypeptides having a sequence substantially similar to the natural polypeptide chains associated with natural growth factors and natural vaccinia virus proteins. The resulting chimeric polypeptides will have about 40 to 65 amino acids, usually about 45 to 60 amino acids and especially 49 to 54 amino acids.



  In each case, the grid structure of the cysteines is maintained at the cysteine bridges defining the loops of the dimensions described above. Thus, it is possible to use a fragment originating from any growth factor having a sensitive homology with the VGF (see, for example, Fig. 1) or with another growth factor comprising the same grid structure as the compositions of the invention and having a similar physiological activity. This is so independently of the mammal of origin, such as a primate, for example the human being, a rodent, for example the rat or the mouse, a bovine, a bird, a pig, etc.



   Up to three junctions may be required depending on the origin of the fragments and the length of the desired polypeptide. It is desirable that the junctions be made at some point between aa-6 and aal; aal1 and aal5; aa25 and aa29; and aa42 and aa53. The sequence between approximately aa-6 and approximately aal5 is called N-terminal sequence and that between approximately aal4 and approximately aa29 is called central fragment, while that between approximately aa25 and the end of the peptide (generally aa43 to aa47) is called domain C-terminal.



  The exact junction point varies with the location of the restriction sites, and so on. In addition, a

  <Desc / Clms Page number 23>

 extreme N-terminal sequence comprising about aa-24 to aa-7, and more usually aa-23 to aa-7 can also be linked to the hybrid polypeptide.



   The fragments will generally be about 15 to 50 and usually 15 to 45 amino acids since aa20 does not denote the twentieth amino acid of the compound but only that of the specific sequence which has been specifically defined (see Fig. 1).



   Various convenient restriction sites are provided in the synthetic genes used to construct the hybrid polypeptides. Where possible, the restriction sites leave the amino acid sequence of the growth factor gene unaltered. However, in some cases, the incorporation of the new restriction sites gives an amino acid sequence which is modified. A particularly interesting case is that where the sequence coding for VGF is modified so as to introduce a KpnI restriction site by changing the amino acid sequence from aal3 to aal5 from GDC to GTC and introducing a SphI site from aa23 to aa28 by changing the sequence from GMYCRC to GYACVC. These modifications provide convenient sites for assembling fragments of the VGF gene with fragments of other growth factors.

   For example, the fragment of
 EMI23.1
 % 47 can gene aa26 to aa47 can be advantageously assembled with a gene fragment of -TGF which codes for the 6% amino acids corresponding to residues aa-6 to aa25 in order to obtain a hybrid polypeptide TGF / VGF. Thus, the sequence of the hybrid peptide will comprise aa-6 up to aal3 (N-terminal part) and aal4 up to aa25 (central fragment) originating from-TGF, as well as aa26 up to aa47 (C-terminal domain) from VGF, modified as above.



   Preparation of capable plasmids

  <Desc / Clms Page number 24>

 to express the hybrid proteins having the amino acid sequences derived from fragments of more than one vaccinia virus growth factor or polypeptide with sequence modifications when necessary to introduce an appropriate restriction site is described in detail in the experimental part.



   It is also possible to prepare fusion proteins where a growth factor or else a hybrid polypeptide is expressed united to the N-terminal amino acids of a signaling sequence or else to the N-terminal amino acids of a bacteria gene, highly expressed bacteriophage or eukaryote. In addition, an enzymatic or chemical cleavage site can be provided after the signal sequence to allow cleavage separation of the mature polypeptide from the signal sequence. The preparation of plasmids capable of expressing these fusion polypeptides and the methods for cleavage and isolation of the desired polypeptide are described in detail in the experimental part.



  Preparation of growth factors.



   The compositions of the invention can be prepared in various ways depending on the size they have. In particular, below about 80 and more especially below about 60 amino acids, the compositions can be prepared by synthesis according to traditional methods, 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 eds., Academic Press, NY, 1980, pp. 1 to
 EMI24.1
 284. See also U.S. Patent No. 4,127,526.



  Alternatively, hybrid DNA technology can be applied, whereby it is possible to use DNA sequences which encode the

  <Desc / Clms Page number 25>

 
 EMI25.1
 0 desired polypeptide or a precursor thereof. The DNA sequences coding for the growth factor of interest can be synthesized according to traditional techniques, such as the superposition of single strands which can be ligated to define the desired coding sequence. The ends can be designed to provide restriction sites or one or both ends can be made non-cohesive for ligation with the complementary ends of an expression vector. An initial methionine is provided for expression of the sequence.

   Expression vectors are generally available and have been described in detail in the literature.



   Instead of synthesizing the gene of interest, it is possible to isolate the growth factor using different techniques. For example, when the growth factor is a viral growth factor, it is possible to isolate from the virus the mRNA which codes for the polypeptide comprising the growth factor,
 EMI25.2
 subject the mRNA to reverse transcription, use the resulting single strand DNA (ss) as a template to prepare double strand DNA (ds) and isolate the DNA gene (ds). Another technique is to isolate a fragment of viral DNA and using a suitably degenerate probe that includes a region of the best conserved sequences in a growth factor gene, to identify the sequences encoding a factor in the viral genome.

   The probe can be considerably shorter than the complete sequence, but must be at least 10, preferably at least 14 and more preferably at least 20 nucleotides in length. Longer oligonucleotides are useful too, up to full length
 EMI25.3
 of the growth factor gene. Probes so much ge DNA

  <Desc / Clms Page number 26>

 that RNA can be used.



   For use, the probes are typically labeled in a detectable manner (for example with 32p or biotinylated nucleotides) and are incubated with RNA or single stranded DNA from the organism in which a gene is being sought. Hybridization is detected by means of the marker after the DNA (or DNA / RNA) single strand and double strand (hybridized) have been separated, typically on nitrocellulose paper.



  Hybridization techniques suitable for oligonucleotides are well known to those skilled in the art.



   Although probes with a detectable marker that allow easy identification are normally used, unlabeled oligonucleotides are also useful, both as precursors to labeled probes and for use in methods which provide direct protection of DNA ( or DNA / RNA) double strand.



  Consequently, the term "oligonucleotide" designates both marked and unlabeled forms.



   When the desired DNA sequence has been obtained, it can be manipulated in various ways for expression; for example, hybrid polypeptide sequences can be prepared by combining gene fragments from at least two polypeptides having sequences that are at least more than 30% homological with the natural growth factors that bind to the receptor. EGF ("natural ligands").

   It is highly desirable that the three-dimensional structure, especially the loop structure, of the polypeptide be preserved, and this is particularly so of the part or parts of the structure which may be responsible for binding to the EGF receptor and for the biological activity of natural growth factors which bind to

  <Desc / Clms Page number 27>

 the EGF receptor.



   Depending on the origin of the fragments and the length of the desired polypeptide, suitable restriction sites can be incorporated into the synthetic genes used to construct the hybrid polypeptides as described above. Where possible, the restriction site (s) leave the amino acid sequence of the polypeptide unaltered.



  However, in some cases, the incorporation of one or more new restriction sites can lead to an amino acid sequence which is altered without affecting the activity of the protein.



   When the gene is to be expressed in a host which recognizes wild type growth factor translation and transcription regulatory regions, the complete gene with its wild type 5 'and 3' regulatory regions can be introduced into a vector. appropriate expression. There are different expression vectors using the replication systems of mammalian viruses, such as simian virus 40, adenovirus, bovine papilloma virus, vaccinia virus, insect baculovirus, etc. These replication systems have been developed to provide markers that allow the selection of transfectants, as well as to provide convenient sites of restriction in which the gene can be inserted.



   When the gene is to be expressed in a host that does not recognize natural wild type translation and transcription regulatory regions, further manipulation is necessary. Various regulatory regions of transcription 3 ′ are known which can be inserted downstream of the termination codons. The 5 ′ non-coding region upstream of the gene of interest can be eliminated by restriction

  <Desc / Clms Page number 28>

 with an endonuclease, resection by Bal31 and so on.

   Alternatively, when a convenient restriction site exists near the 5 ′ end of the gene of interest, the latter may be subject to restriction and an adapter may be used to assemble the gene of interest with the promotion region, in which case the adapter replaces the lost nucleotides of the gene of interest. Different strategies can be used to constitute an expression cassette which, in the 5'-3 'transcription direction, comprises a transcription regulatory region and a translation initiation region, which can also include regulatory sequences allowing the induction of regulation, the gene of interest under the control of transcription and translation of the initiation region; and a transcription and translation termination region.



   The choice of appropriate regulatory sequences should take into account the following factors which influence expression. In terms of regulating transcription, the quantity and stability of messenger RNA are important factors that influence the expression of gene products. The amount of mRNA is determined by the number of copies of the particular gene, the relative effectiveness of its promoter and by factors that regulate the promoter, such as enhances or repressors. The initiation is believed to be in a region just upstream from the start of the coding sequence.



   The promoter in prokaryotic cells includes nucleotide sequences which can influence transcription efficiency. The sequences include regulatory regions at about -35 and -10 nucleotides from the start of the DNA chain. Effective promoters include those

  <Desc / Clms Page number 29>

 wherein the nucleotide sequence of regulatory regions -35 and -10 is substantially the same as the consensus sequences for these regions in bacterial promoters from highly efficient genes. In general, these regions are about 5 nucleotides and 6 nucleotides in length, respectively, and each sequence can vary by about one nucleotide in length and / or in sequence.

   A preferred sequence for the consensus regulatory sequence-35 extends from the trp promoter, namely TGACA, and for the consensus regulatory sequence-10 extends from the lac promoter, namely TATAAT.



   Not only the nucleotide sequences, but also the distance of the consensus sequences of the regulatory regions -35 and -10, from one another, are important for obtaining an optimal transcription of the gene of interest. In general, the consensus sequences of regulatory regions -35 and -10 are separated by about 16 to 18 nucleotides and preferably about 17 nucleotides.



  Each sequence can vary by approximately 1 nucleotide.



   Examples of transcription regulatory regions or promoters are in particular, for bacteria, the p-gal promoter, the left and right lambda promoters, the trp and lac promoters, the trp-lac fusion promoter, etc. ; synthetic promoters which have sequences substantially similar to these sequences can also find their application. A preferred promoter for bacteria is a fusion promoter comprising the regulatory region -35 from the trp promoter and the regulatory region -10 from the lac promoter.

   Very advantageously, the promoter is a promoter in which the consensus sequence trp-35 is located at

  <Desc / Clms Page number 30>

 approximately 17 nucleotides upstream of the lac-10 consensus sequence. For yeasts, they are promoters of glycolytic enzymes, such as the ADH-I and-II promoters, the GPK promoter and the PGI promoter, in addition to the trp promoter, etc. ; for mammalian cells, these are the early and late SV40 promoters, the major late adenovirus promoters, the I5 promoter or the enhancer sequences and so on.



   The transcriptional regulatory region can also comprise regulatory sequences which make it possible to modulate the expression time of the gene of interest, for example under the effect of the presence or absence of nutrients or expression products in the growth medium. , temperature, etc. For example, the expression of the gene of interest can be regulated by the temperature of the growth medium of the host cells by using a regulatory sequence comprising the PL promoter of the bacteriophage lambda,
 EMI30.1
 the OL operator the OL operator of the bacteriophage lambda and the CI857 gene which codes for the temperature-sensitive CI repressor in the expression vector.

   This allows regulation of the promoter by an interaction between the repressor and the operator at low temperatures, for example at around 30oC. Raising the temperature to around 42 C would inactivate the repressor and allow expression of the gene of interest.



   As an example of the modulation by means of nutrients in the growth medium, the regulation of the lac promoter or of the hybrid trp-lac promoter can be done by means of the gene for the LacI repressor, which binds to the region of the lac promoter at the of the regulatory region-10. The LacI repressor gene may be present on an episome, preferably the LacI mutant under enhancer effect, or may be understood

  <Desc / Clms Page number 31>

 in the expression cassette itself. The presence of a significant concentration of the repressor molecule in the growth medium inhibits the function of the promoter in the absence of inducers. Thus, the addition of IPTG or lactose to the growth medium of the host cells enhances the function of the promoter.

   When the bacterial strain is Lac +, lactose can be used as an inducer instead of IPTG. In both eukaryotic and prokaryotic systems, the termination regions may also contain sequences or structures which increase the stability of the mRNA and allow higher expression. Thus, the transcriptional regulatory region can additionally comprise regulatory sequences which terminate transcription and which constitute sequences or structures which inhibit the degradation of mRNA and thus increase the stability of mRNA and allow higher expression. Various examples of prokaryotic sequences are known, for example the Trp terminator, the 32 (T4) gene terminator or the synthetic terminators which are similar in sequence to the 32 gene.



   In eukaryotic systems, the end of transcription, RNA cleavage and polyadenylation regions ensure appropriate maturation of the
 EMI31.1
 mRNAs transcribed and are necessary for efficient expression. The 3'native untranslated region may suffice, but the polyadenylation signal from, for example, SV40, in particular including a splicing site which provides more efficient expression, could also be used. Alternatively, the 3 'untranslated region from a gene strongly expressed in a particular type of cell (eg, Ig in myeloma cells) could be fused with the gene of interest.

   Such 3 ′ sequences could

  <Desc / Clms Page number 32>

 be paired with 5 'regulatory sequences from the same high expression gene and could even include coding regions in a reading frame to produce fusion proteins as described below.



   In terms of translation regulation, given the presence of mRNA, the expression can be regulated by influencing the speed of initiation (binding of the ribosome to the mRNA), the speed of elongation (translocation of the ribosome to the mRNA), the speed of modifications after translation and the stability of the gene product. The speed of elongation is probably influenced by the use of a codon and the use of codons for rare tRNAs can lower the speed of translation. It is therefore preferable to use codons which frequently appear in the genes normally expressed by the host cell in order to increase the speed of translation.



   In prokaryotes, downstream of regulatory regions -35 and -10, there is a consensus nucleotide sequence, generally AGGA, known as the Shine-Dalgarno sequence, which is believed to be involved in the fixation of the ribosome. The optimum ribosome binding and start of translation can be achieved by using a functional ribosome binding site in the host cell from a highly expressed gene. Various indications reveal the presence of nucleotide sequences surrounding the Shine-Dalgarno sequence and of sequences in the coding region which can influence the binding of the ribosome, perhaps by the formation of structural motifs by means of which the ribosome recognizes the initiation site.



  A modification of the nucleotide sequences of the coding region can therefore be used to ensure optimum fixation and translation initiation. The

  <Desc / Clms Page number 33>

 sequence of the first some 7 to 30 codons after the ATG initiation codon can also influence fixation and expression. Preferably, the signal sequence and the Shine-Dalgarno sequence are obtained from the same gene or, when they have been obtained from different genes, the codons of the signal sequence can be modified by degeneration of codons to closer to the
 EMI33.1
 p nucleotide sequence of the natural gene which succeeds the signal sequence, as described in patent application PCT / US88 / 03972 of October 28, 1988.



   The position of the AGGA sequence relative to the ATG initiation codons can influence expression. Generally, the ShineDalgarno sequence is located approximately 5 to 9 nucleotides away from the initiation codon, but unexpectedly, high levels of expression can be obtained using expression cassettes in which the sequence of Shine-Dalgarno is located approximately 10 to 13 nucleotides and preferably 11 or 12 nucleotides away from the initiation codon.



   The stability of mRNA is governed by the sensitivity of mRNA to ribonucleases. In general, digestion with exonucleases is inhibited by the presence of structural patterns at the ends of the mRNA, by palindromic structures, by modified nucleotides or by specific nucleotides. Endonuclease digestion is believed to occur at specific recognition sites within the mRNA, and stable mRNA would be free from such sites. There are also indications that mRNAs undergoing high translation rates are also protected against degradation by the presence of ribosomes on mRNA.



   The stability of the expression product can be

  <Desc / Clms Page number 34>

 acquired by performing the synthesis of a fusion protein in which the desired polypeptide is expressed together with a second polypeptide or fragment thereof. Preferably, the stability of the expression product is ensured by carrying out the synthesis of a fusion protein in which the polypeptide of interest is expressed united to a signal sequence. A DNA sequence encoding or an N-terminal amino acid sequence originating, for example, from a highly expressed bacterium or bacteriophage gene, such as the lambda bacteriophage N protein gene, the cro or P- gene galoctosidase or a eukaryotic gene is assembled upstream of the gene of interest and in a reading frame with this gene.

   The signal sequence usually includes about 8 to about 50, preferably about 15 to about 45, and more preferably 18 to 25 N-terminal amino acids.



   Expression of the polypeptide of interest as a protein fused to a signal sequence from another gene offers various advantages in addition to ensuring stability. For example, the presence of N-terminal amino acids provides a means for applying general purification techniques to the purification of any of a variety of polypeptides.



  For example, the N-terminal amino acids of the N protein are particularly antigenic and, therefore, specific antibodies directed against the N-terminal amino acids of the N protein can be used as a means for purifying fusion proteins containing the N-terminus of protein N. In addition, the N-terminus of protein N has a strongly positive charge which facilitates purification of the desired protein by ion exchange chromatography or the like.

  <Desc / Clms Page number 35>

 
 EMI35.1
 



  0 The signal sequence can also be a sequence of hydrophobic amino acids which can also serve as a signaling sequence for secretion. A DNA sequence coding for the signaling sequence is assembled upstream of the gene of interest and in a reading frame with it. Typically, the signaling sequence includes a cleavage site which is recognized by a peptidase from the signaling sequence. Thus, the positioning of the polypeptide of interest, directly after the cleavage site of the signaling sequence, allows this polypeptide of interest to be specifically separated from the signaling sequence by cleavage and secreted in the state of mature polypeptide.

   Examples of hydrophobic amino acid sequences are in particular the signaling sequence for bacterial alkaline phosphatase, the OMP-A-B-C- signaling sequences
 EMI35.2
 Crazy ; the LPP signaling sequence, the j-lactamase signaling sequence and the signaling sequences of toxins and their mutants. In the case of eukaryotic cells, the signaling sequences can be the signaling sequence of monkey TGF-, of the gene P97 (human melanoma antigen), the sequence of the killer toxin of K. lactis, the factor of the conjugation type a : and other killer toxin signaling sequences or the normal signaling sequence associated with the gene of interest in conjunction with the mutants of the signaling sequences.



   Other signal sequences which can be used are in particular hydrophilic sequences, for example the 41 N-terminal amino acid residues of amphiregulin which can ensure the modification of the function of the polypeptide of interest. In addition, a cytotoxic agent, such as a fragment of chain A of

  <Desc / Clms Page number 36>

 toxin, the castor chain, the snake venom growth arrest peptide or a targeting molecule, such as a hormone or antibody, can be covalently linked to the signal sequence with, most often, minimal effect on the biological activity of the genetic product of interest. As has been described for the other signal sequences, a DNA sequence coding for the signal sequence is linked upstream of the gene of interest and in reading frame with it.



   When the signal sequence is not a signaling sequence or does not contain an appropriate natural cleavage site, additional amino acids can be inserted to provide an enzymatic or chemical cleavage site intended for cleavage of the signal peptide, after the purification of the fusion protein, to allow subsequent purification of the mature polypeptide. For example, the introduction of labile aspartyl-proline bonds in an acid medium between the two segments of the fusion protein facilitates their separation at low pH values. This process is not suitable if the desired polypeptide is labile in an acid medium. Also, for example, the fusion protein can be cut, using, for example, cyanogen bromide, which is specific for the carboxyl side of methionine residues.

   The positioning of a methionine between the signal sequence and the desired polypeptide allows the release of the desired polypeptide. This method is not suitable when the desired polypeptide contains methionine residues.



   When the signal sequence includes a signaling sequence, genes of interest with secretory signal sequences can be expressed with or without the signal sequence under conditions where the sequence can be retained or cut. In addition, for

  <Desc / Clms Page number 37>

 to obtain a high proportion of the desired polypeptide in the form of a mature cut and folded peptide secreted in the medium, it is preferable to use a promoter, such as the tac promoter, which can act at a lower temperature, for example around 30 ° C. Unexpectedly, higher levels of secretion can be obtained at lower temperatures.

   Extremely high expression levels can prevent complete changes in protein translation from taking place, leading to accumulation and aggregation of uncut precursor (i.e., protein of structure and secretory signal sequence ). Similarly, growth at elevated temperatures, for example 42OC, also tends to induce aggregation and accumulation of uncut precursor. The preparation of fused proteins and in particular the methods of cutting and folding the polypeptide of interest are described in more detail in the above-mentioned E.U.A. patent application No. 264,098.



   After each manipulation of DNA in the development of the cassette, the plasmid must be cloned and isolated and, as necessary, the particular compound of the cassette must be analyzed for its sequence in order to verify that the appropriate sequence has been obtained. Depending on the nature of the manipulation, the desired sequence can be excised from the plasmid and introduced into a different vector, or the plasmid can be restricted and the expression cassette component can be manipulated, as appropriate . In some cases, use is made of a shuttle vector, this vector being capable of replication in different hosts which require different replication systems.

   This may or may not require markers

  <Desc / Clms Page number 38>

 which are functional in both hosts. When such markers are required, they can be included in the vector. The plasmid containing the cassette, two replication systems and the marker (s) can be transferred from one host to another, as required. Any useful marker can be chosen for selection. Resistance to neomycin or tetracycline is interesting.



  However, although a marker for selection is highly desirable for convenience, other techniques for screening for transformed cells have been described. See, for example, G. Reipin, et al., Current Genetics, 1982, 189-193. Transformed cells can also be sorted using the specific products they form, for example the synthesis of the desired product can be detected by immunological or enzymatic methods.



   The expression cassette can be included in a replication system for maintaining the episome in an appropriate cellular host or can be provided without a replication system, in which it can integrate into the genome of the host. DNA can be introduced into the host according to known techniques, such as transformation, using DNA precipitated by calcium phosphate, transfection by contact of the cells with the virus, microinjection of the DNA into the cells, And so on. When the gene of interest has been introduced into the appropriate host, the latter can be cultured for the expression of the gene of interest.



   Various host cells can be used. Examples of prokaryotic host cells are in particular Gram-negative organisms, such as E. Coli, for example JM109, JM101 and JM107, in addition to HB101, DH1 and DH5. Particular organizations

  <Desc / Clms Page number 39>

 suitable are Gram-positive organisms such as B. subtilis which have no periplasmic space and can secrete the polypeptides directly into the growth medium. The eukaryotic host cells can be yeast cells, insect cells or mammalian cells, for example COS cells, CHO cells, monkey kidney cells and silkworm cells (sf9).



   The construct containing the gene of interest and the flanking regions ensuring expression regulation can be introduced into the expression host by any suitable means, for example transformation, by means, for example, of DNA precipitated by the calcium phosphate, transfection, transduction, conjugation, micro-injection, etc. The host can then be grown to a high density in an appropriate nutrient medium. When the promoter is capable of induction, permissive conditions will then be used, for example a temperature change, an exhaustion or an excess of a metabolic product or of a nutrient, and so on.



   For example, when the regulatory sequence comprises the PL promoter of bacteriophage A, the operator OL of bacteriophage A and the temperature-sensitive repressor CI857, the host cells can be cultured at the permissive temperature, generally around 30 ° C., temperature at which transcription from the PL promoter is suppressed and host cells can be cultured unimpeded by requests for synthesis for the foreign gene product, which may additionally be toxic to the host organism.

   When the host cells have reached an optimal density, the temperature can be raised to a non-permissive value, for example

  <Desc / Clms Page number 40>

 about 42 OC, when the CI repressor becomes inactive, which allows transcription from the PLI promoter Maximum secretion can be obtained using the lac promoter or a trp-lac promoter, and induction with a metabolic inducer such as lactose for a lac + host strain with contribution of lac Iq on a vector. Examples of host cells, which could be used with such a system, include DH1, DH5 and HBlOl.



   When the product is retained in the host cell, the cells are harvested and subjected to lysis, then the product is isolated and purified by extraction, precipitation, chromatography, electrophoresis, etc. When the product is secreted, the nutrient medium can be collected and the product can be isolated according to traditional techniques, for example affinity chromatography. To obtain an active protein, it may be necessary to allow the protein to fold. If the protein is expressed as a fusion protein with the signal sequence, the latter can be removed by treatment with, for example, formic acid or cyanogen bromide. The signal sequence is preferably eliminated after refolding of the protein.



   The recombinant product may or may not be glycosylated, the glycosylation being wild type or other. As a general rule, glycosylation will not differ from wild-type glycosylation by more than about 50% and usually by more than about 20%. The extent of glycosylation depends in part on the sequence of the particular peptide, as well as on the organism in which it is produced. Thus, expression of the product in E. coli leads to an unglycosylated product, and expression of the product in insect cells leads, in general, to a

  <Desc / Clms Page number 41>

 glycosylation less important than the expression of the product in mammalian cells.



  Use of growth factors.



   The compositions of the invention have a wide variety of in vitro and in vivo applications as agonists or antagonists of growth factors, such as epidermal growth factor (EGF), and transforming growth factor, in particular 1'ct- TGF. A discussion of growth factors used as such or in conjunction with other compositions, particularly polypeptide compositions, for regulating cell growth and for other activities can be found, for example, in Handbook of Experimental Pharmacology, Tissue Growth Factors, publisher Baseraga, volume 57, Springer-Verlag, Berlin 1981, chapter 3, in particular on pages 98 to 109, and in Carpenter, Ann.



  Rev. Biochem. (1979) 48: 193-216.



   Human EGF appears identical to human urogastrone and has various effects on the growth of prenatal and neonatal tissue. These effects include early opening of the eyes, wound healing, eruption of the incisors and accelerated maturation of the lungs. EGF receptors are found in various adult tissues. EGF has been shown to stimulate the phosphorylation of its own receptor. EGF is also found to be associated with increased bone resorption.



   The transforming growth factor, in particular a-TGF, has many activities similar to that of EGF. TGF binds to the EGF receptor and leads to phosphorylation of the receptor, an increase in its tyrosine-specific kinase activity and a stimulation of cell growth. See Cohen in: Biological

  <Desc / Clms Page number 42>

 Response Mediators and Modulators (editor:
 EMI42.1
 a August, J. T.), Academic, New York, 1983, pages 7 to 12; Tarn et al., Nature (1984) 309: 376-378, Ibbotson et al. ; Science (1983) 221: 1292-1924.



   The myxomatosis and Shope fibroma viruses induce a significant increase in the proliferative potential of cells in the affected region. Vaccinia virus induces minor proliferation at the site of infection, but Shope's fibroid virus induces major proliferation leading to tumor formation which is benign in adult rabbits but which is proliferative and invasive in newborns and adults whose immunity has been terminated. The myxomatosis virus induces a rapidly spreading myxosarcomatous tumor. Although these proliferative events may be due to other viral factors, viral growth factors appear to be highly involved in induced cell proliferation caused by viral infection.



   The compounds of the invention have a
 EMI42.2
 particular application as drugs as EGF agonists and for wound healing, such as the formation of an epithelium on wounds such as burns, eye wounds, surgical incisions, and so on. The active ingredient can be used in a suitable excipient, for example Silvadene, in an amount sufficient to promote the formation of an epithelium and / or the healing of wounds, generally in amounts
 EMI42.3
 from about 0.01 to 10.0 zig per ml, usually from about 0.075 to 5.0 g per ml, and preferably from 0.05 to 5 µg per ml of EGF equivalents. The pharmaceutical composition is spread over the wound to ensure complete coverage.

   The frequency of treatment may

  <Desc / Clms Page number 43>

 be high, for example four times a day, or low, for example every other day if not less, depending on the nature of the wound, its response to treatment, the concentration of the active ingredient, and so on.



   The compositions of the invention can be used as reagents for diagnosis or for the preparation of reagents such as polyclonal or monoclonal antibodies. As reagents, they can be used for the detection of analogous growth factors or for the detection of antibodies against growth factors in physiological fluids, for example blood.



   Depending on the particular protocol and the application envisaged for the reagent, the polypeptide may or may not be labeled. A wide variety of markers have already been used to give a detectable signal directly or indirectly. These markers are in particular radionuclides, enzymes, fluorescent agents, particles, chemiluminescent agents, substrates or cofactors for enzymes, enzyme inhibitors, magnetic particles, etc. See, for example, U.S. Pat. Nos. 3,654,090, 3,817,837.3 935,074.3 996,345, 4,277,437.4 374,925 and 4,366,241.



   To assemble the markers and the polypeptides, there are many methods which can consist in using the amino radical Nterminal to fix a function by forming a pyrazolone, while the other free amino radicals are protected, the pyrazolone being able then to be brought into contact with various reagents, for example amino radicals, to fix the fragment which generates the detectable signal. By protecting the arginines associated with or close to the third loop,

  <Desc / Clms Page number 44>

 it is possible to functionalize the other arginines with a view to conjugation with amino radicals or thio radicals, according to known techniques.

   Alternatively, the polypeptide can be contacted with an active agent, for example an activated carboxylic acid, and randomly substituted, in which case, the biologically active material can be separated from the biologically inactivated material as a result of random substitution. Finally, according to the synthesis process, the polypeptide can be modified to provide the desired functionality, as part of the synthesis technique.



   The compositions of the invention can be used for monitoring EGF receptors.



  These compositions can also be used to monitor the cellular response to EGF and / or TGF by establishing competition between these factors of natural origin and a composition in accordance with the invention. In this way, changes in the conformation of the receptor can be monitored.



   The compositions of the invention can also be used for various therapeutic purposes, in particular stimulation of growth or regulation of bone formation. These compounds can be administered in suitable physiological excipients by the intraperitoneal, subcutaneous, intravenous or intraarterial route, or by application at the desired site. In addition, the compositions of the invention can be introduced into liposomes, which may or may not involve the use of antibodies for orientation to the site. Various excipients include saline phosphate buffered saline, physiological saline, water, and so on. The concentration of the active ingredient varies greatly with its

  <Desc / Clms Page number 45>

 end use and its activity.



   The pharmaceutical compositions can contain other constituents, such as EGF, TGF or other growth factors, bacteriocides, for example antibiotics, bacteriostats, buffers, etc.



   To prepare antibodies, the polypeptides of the invention where PP- "represent hydrogen atoms or short oligopeptide chains (less than five amino acids) can be combined with polypeptides or antigenic proteins for injection into host mammals. The antigenic protein must contain at least about 60 amino acids and usually does not exceed 100 kilodaltons (kDal). There are many techniques for assembling polypeptides, either at a specific site or at random, at using bifunctional reagents, such as p-maleimidobenzoic acid, glutaraldehyde, p, p'-benzidine, etc. Common antigenic proteins include albumin from beef serum, crack hemocyanin, tetanus toxoid , etc.

   The polypeptides of the invention are joined to the antigenic protein in a sufficient number to induce the desired immunogenic response.



  Usually two or more booster shots are needed after the first injection. For antisera, blood is taken from the immune host and the fraction containing the immunoglobulins is isolated.



  For monoclonal antibodies, the spleen is removed and the splenocytes are fused with an appropriate fusion partner according to standard techniques.



  The resulting hybridomas are then sorted, the criterion being the attachment of the antibodies to the epitopic sites of the polypeptide of the invention. These antibodies can be used for a variety of purposes, such as

  <Desc / Clms Page number 46>

 example as diagnostic reagents, therapeutic agents, etc. Antibodies used as reagents can be labeled or unlabeled, as described for the polypeptides.



   The examples below are given by way of illustration and not by way of limitation.



  Experimental part.



  Contents.



  EXAMPLE I. Preparation of interesting synthetic genes.



   A. Synthetic oligonucleotides of TGF.



   B. Synthetic VGF oligonucleotides.



   C. Synthetic EGF oligonucleotides.



  EXAMPLE II Description of the cloning and expression of plasmids.



   A. The expression plasmid pLEBAm.



   B. The expression plasmid pBM11.



   C. The expression plasmid pBM11M4.



   D. The expression plasmid pBM11M5.



   E. The expression plasmid pBMIl / NDP.
 EMI46.1
 



  F. The expression plasmid pMBMi / PAD.



  G. The expression plasmid pBMIl / PAK.



  H. The expression plasmid pTCPt.



  I. The plac / cro-gal expression plasmid.



  J. The ptac / cro-gal expression plasmid.



  K. The expression plasmid pRSV.



  L. The expression plasmid pAc610.



  M. The expression plasmids pToxl and pTox2.



   N. TakPak.



  EXAMPLE III.- Assembly of growth factor genes for expression in prokaryotic cells.
 EMI46.2
 



  A. Preparation of the plasmid pLEBam / TVV.



  B. Preparation of the plasmid pLEBam / TVV with the VGF and its fragments.

  <Desc / Clms Page number 47>

 



  EXAMPLE IV Expression of the polypeptides of interest in the state of fusion protein with protein N.



   A. The modified synthetic TGF.



   B. The modified synthetic TGF-VGF hybrid.



  EXAMPLE V. Preparation of the polypeptide of interest in the state of fusion protein with protein N and a cleavage site.



   A. The modified synthetic VGF.



   B. The modified synthetic TGF-VGF hybrids.



   C. Synthetic EGF.



  EXAMPLE VI Preparation of the polypeptide of interest in the state of a fusion protein with the signaling sequence of the modified alkaline phosphatase.



   A. Preparation of pBMIl / PAD / EGF.



   B. Preparation of pBMll / PAD / nVGFa.



  EXAMPLE VII Expression of the polypeptide of interest in the state of a fusion protein with an alkaline phosphatase signaling sequence.



   A. Preparation of pBM11 / PAK / nVGFa (alkaline phosphatase signaling sequence / nVGF / a with N-terminus of natural VGF and GTC and GYACRC sequences).



   B. Preparation of pBMIl / PAK / EGF.



   C. Preparation of TacPak / EGF (alkaline phosphatase / human EGF signaling sequence).

  <Desc / Clms Page number 48>

 



  EXAMPLE VIII Expression of a polypeptide of interest in the state of a fusion protein with the alkaline phosphatase signaling sequence, using an expression cassette containing a transcription termination region.
 EMI48.1
 



  A. Preparation of pTCPt / EGF ([trp-35 2 16pub C lac-10 LiacSD J llpb ATG / alkaline phosphatase signaling sequence / human EGF / trans. Term.-NEO).



  B. Preparation of pTCPt / nVGFa trp-35 3 16pb C lac-10 E lacSD croSDj llpb ATG 3 / alkaline phosphatase signaling sequences / VGFa-n-terminal with GTC and GYACRC sequences) / trans. term.-NEO).
 EMI48.2
 



  C. Preparation of pTNPt / EGF (C trp-35 J l7pb [Yac-10 nSD J 8 pb [TG / alkaline phosphatase signaling sequence / human EGF / trans. Term.-NEO).



  EXAMPLE IX Assembly of growth factor genes for expression in eukaryotic cells.



   A. Preparation of the plasmid PRSV / VGF.



   B. Preparation of the plasmid pAc / VGF.



   C. Preparation of the plasmid pAc / SFGF.



  EXAMPLE X Preparation of polypeptides.



   A. Solid phase synthesis of BGF and TGF.



   B. Isolation of the recombinant polypeptides prepared in prokaryotic cells.



   C. Isolation of VGF and SFGF produced by expression of natural genes in eukaryotes.



   D. Natural EGF.

  <Desc / Clms Page number 49>

 



  EXAMPLE XI.- Activity dosages.



   A. Mitogenic dosage.



   B. Determination of colony growth stimulation on soft agar.



   C. Assay of inhibition of binding to the EGF receptor.



   D. Radioimmunoassay.



   E. Healing.



  EXAMPLE XII Biological activity of the recombinant growth factors prepared in prokaryotic cells.



   A. Attachment to the EGF receptor.



   B. Mitogenic activity.



   C. Healing.



  EXAMPLE XIII Biological activity of the VGF prepared in eukaryotic cells.



   A. VGF prepared in monkey kidney cells (BSC-1).



   B. VGF prepared in CHO cells.



   C. The VGF prepared in the silkworm.



   D. Immunological comparison of VGF and TGF.



  Biological deposits.



   The following expression plasmids, all transformed into E. Coli HB101, were deposited on the date indicated in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 and their identification and ATCT designation are as follows:

  <Desc / Clms Page number 50>

 
 EMI50.1
 
 <tb>
 <tb> Identification <SEP> Designation <SEP> ATCC <SEP> Date <SEP> from <SEP> deposit
 <tb> PBMll <SEP> 67366
 <tb> PBM14 <SEP> 67367
 <tb> PBMl1 / PA / VGF <SEP> 67417 <SEP> 3 <SEP> June <SEP> 1987
 <tb> PBMll / DP / VGFa <SEP> 67418 <SEP> 3 <SEP> June <SEP> 1987
 <tb> PBM11 / PA / EGF <SEP> 67419 <SEP> 3 <SEP> June <SEP> 1987
 <tb> PBM1l / M5 <SEP> 67436
 <tb> PBM11 / C2 <SEP> 67437
 <tb> PBMl1 / NDP / EGF <SEP> 67547 <SEP> 23 <SEP> October <SEP> 1987
 <tb>
 Processes.



   The general cloning techniques described by Maniatis et al., 1982 were applied in "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory, CSH, New York. All DNA modifying enzymes were purchased from commercial suppliers. They were used in accordance with the manufacturer's instructions. Agents and devices for DNA purification and separation were used according to the supplier's instructions.



  EXAMPLE I. Preparation of interesting synthetic genes.



   Synthetic growth factor genes have been developed using codons of host cells optimized for high levels of expression.



  In addition, various convenient restriction sites have been arranged in the synthetic genes. Where possible, the new restriction sites have left the growth factor gene amino acid sequence unchanged, but in some cases the incorporation of the new restriction site has resulted in a modified amino acid sequence. These sites first divide the synthetic genes into thirds giving an N-terminal domain, a median domain and a C-terminal domain.

  <Desc / Clms Page number 51>

 



   The natural VGF gene product contains an extreme N-terminal domain which has no counterpart in mature TGF. Fragments of VGF free from this domain are called truncated. The restriction sites were used for the initial constructions of the final genes using partial synthetic oligonucleotide fragments extending from one restriction site to another. The oligonucleotides were synthesized on an apparatus for the synthesis of Applied Biosystems oligonucleotides and were purified on an acrylamide gel.



  The oligonucleotides were phosphorized at the 5 ′ end using T4 polynucleotide kinase and each oligonucleotide was then renatured with its complement.



  A. Synthetic oligonucleotides of TGF.



  1. The N-terminal domain of human TGF:
TGF->
BssHIINcoI M V V S H F N D C P D
 EMI51.1
 5'CGCGCCATGGTTGTTTCTCACTTTAACGACTGCCCGGA 3'GGTACCAACAAAGAGTGAAATTGCTGACGGGCCT KpnI S H T Q F C F H G T CTCTCATACTCAGTTTTGCTTTCATGGTAC 3'TGF104 GAGAGTATAAGTAA10

  <Desc / Clms Page number 52>

 2. The median domain of human TGF modified with the human sequence QEDK modified in QEEK, the sequence observed in rat TGF:
KpnI SphI
C R F L V Q E E K P A C
 EMI52.1
 5'CTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATG 3'TGF101 3'CATGGACGGCAAAAGACCAAGTCCTTCTTTTTGGCC 5'TGF102 3.

   The C-terminal domain of human TGF:
SphI VCHSGYVGARC
 EMI52.2
 5'CGTTTGCCATTCTGGCTACGTTGGCGCACGTTG 3'GTACGCAAACGGTAAGACCGATGCAACCGCGTGCAAC
BamHI
E H A D L L A Ter
CGAACACGCTGACCTGCTGGCTTAAG 3'TGF205
GCTTGTGCGACTGGACGACCGAATTCCTAG 5'TGF206 B. Synthetic oligonucleotides of VGF: 1. The N-terminal domain of VGF:
HindIII
E D S G N A I E T T 5'AGCTGACTCTGGTAACGCTATCGAAACTAC 3'CTGAGACCATTGCGATAGCTTTGATG
S P E I T N A T T
TTCTCCGGAAATCACTAACGCTACTACT 3'V
AAGAGGCCTTTAGTGATTGCGATGATGA 5'V

  <Desc / Clms Page number 53>

 2.

   The N-terminal domain of the modified VGF comprising a
 EMI53.1
 0 Asp-Pro cleavage site, with the HGT sequence replacing the natural HGD sequence:
BamHI
I D P M D I P A I R L C
5'GATCGATCCCATGGACATCCCGGCTATCCGTCTGT 3'CTAGGGTACCTGTAGGGCCGATAGGCAGACA KpnI
 EMI53.2
 GPEGDGYCLHGT GCGGCCCGGAAGGCGACGGCTACTGCCTGCATGGTAC 3'VGF104a CGCCGGGCCTTCCGCTGCCGATGACGGACGTAC 5'VGF103a 3. The central region of VGF including modified GYAC sequence replacing the natural sequence GMYC: KpnI SphI TCIHARDIDGYAC 5'CTGCATCCATGCACGTGACATCGACGGCTACGCATG 3'VGFl0la 3'CATGGACGTAGGTACGTGCACTGTAGCTGCCGATGC 5'VGF102a 4. The C-terminal domain of VGF, 5 'end: SphI EcoRI
C R C S H G Y T G
 EMI53.3
 5'CCGTTGCTCTCATGGCTACACTGG 3'VGF1A 3'GTACGGCAACGAGAGTACCGATGTGACCTTAA 5'VGF2A 5.

   The C-terminal domain of the modified VGF, end S ', with the VCS sequence replacing the natural TCS sequence:
SphI EcoRI
V C S H G Y T G
 EMI53.4
 5'CGTTTGCTCTCATGGCTACACTGG 3'VGF1 3'GTACGCAAACGAGAGTACCGATGTGACCTTAA 5'VGF2

  <Desc / Clms Page number 54>

 6. The C-terminal domain of the VGF, 3 ′ fragment, ending at YQR instead of PNT, the C-terminal domain deduced from the natural secreted VGF:
EcoRI I R C Q H V V L
5'AATTCGTTGCCAGCATGTTGTTCT 3'GCAACGGTCGTACAACAAGA
BamHI
V D Y Q R Ter
GGTCGACTACCAGCGTTAAGGATC 3'VGF3
CCAGCTGATGGTCGCAATTC 5'VGF4 C.

   EGF synthetic oligonucleotides:
Three sets of overlapping synthetic oligonucleotides 1 (A, B), 2 (A, B) and 3 (A, B) encoding human EGF were synthesized on an apparatus for the synthesis of Applied Biosystems oligonucleotides and were purified on an acrylamide gel. The oligonucleotides were phosphorylated at the 5 ′ end using T4 polynucleotide kinases. Each oligonucleotide was renatured with its complement.



  1.



   NcoIEcoRI
M N S D S E C P
5'CATGAATTCTGACTCTGAATGCCC 3'TTAAGACTGAGACTTACGGG
L S H D G Y
GCTGTCTCATGACGGCTAC 3'EGF1A
CGACAGAGTACTGCCGATGACGGAC 5'EGF2A

  <Desc / Clms Page number 55>

   2. NsiI
C L H D G V C M Y
5'TGCCTGCATGACGGCGTATGCATGTAC 3'GTACTGCCGCATACGTACATG
SphI
 EMI55.1
 I E A L D K Y A C ATCGAAGCTCTGGACAAGTACGCATG 3'EGF1B TAGCTTCGAGACCTGTTCATGC 5'EGF2B 3.



   SphI
N C V V G Y I G E R C Q 5'CAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCA
3'GTACGTTGACGCAACAACCGATGTAGCCGCTTGCAACGGT
BamHI YRDLKWWELR *
GTACCGTGACCTGAAATGGTGGGAACTGCGTTAAG 3'EGF3
CATGGCACTGGACTTTACCACCCTTGACGCAATTCCTAG 5'EGF4 EXAMPLE II.Description of the cloning and expression of plasmids.



   A. Plasmid pLEBam was used to clone synthetic oligonucleotide fragments due to its convenient BssHII and BamHI restriction sites. A plasmid with NcoI and BamHI restriction sites such as pBM11 or pBM11 / NDP (described below) can be used to clone the synthetic nucleotide fragments and other DNA sequences of interest.



   B. The plasmid pBM11, described in EUA patent application No. 264,098 of October 28, 1988, allows the cloning of a gene of interest downstream of the DNA sequences coding for the 33 N-terminal amino acids of the N gene bacteriophage lambda at a restriction site

  <Desc / Clms Page number 56>

 BamHI. During the induction of the lambda PL promoter by inactivation of the temperature-sensitive repressor C1857 at 42 C, the foreign gene product is expressed as the C-terminal part of a fusion protein whose N-terminal sequence is that of the gene NOT.



   C. The plasmid pBMllM4, described in E.U.A. patent application no. 264,098 of October 28, 1988, is derived from pBMll and makes it possible to clone a gene of interest at a BamHI restriction site directly after the
 EMI56.1
 initiation methionine of the N gene. The plasmid of m e e pBM11 / M4 also contains an NcoI site in the neomycin gene.
 EMI56.2
 



  D. The plasmid pBMUMS, described in E.U.A. patent application no. 264,098 of October 28, 1988, is derived from pBM11 in which an NcoI site present in the neomycin resistance gene has been deleted by site-oriented mutagenesis. The cloning of a gene of interest in pBMIl / MS therefore does not require partial digestion of the vector with Ncol.



   E. The plasmid pBM11 / NDP, described in the
 EMI56.3
 E.U.A. patent application No. 264,098 of October 28, 1988, is derived from pBMl1 and includes DNA sequences coding for an aspartic acid-proline dipeptide labile in acid medium inserted between the sequences coding for the N gene and a gene of interest. The plasmid contains an Ncol site and a ClaI site for the cloning of a gene of interest downstream of the PL promoter.



   F. The plasmid pBMll / PAD, described in EUA patent application No. 264,098 of October 28, 1988, is derived from the plasmid pBMllM4 and makes it possible to clone a gene of interest at a HindIII, SmaI or BamHI site downstream of a sequence of modified alkaline phosphatase signaling.



  * G. The plasmid pBMll / PAK, described in the

  <Desc / Clms Page number 57>

 E.U.A. patent application no. 264,098 of October 28, 1988, derived from the plasmid pBMllM4 and makes it possible to clone a gene of interest at a HindIII, SmaI or BamHI site downstream of an alkaline phosphatase signaling sequence.



   H. The plasmid pTCPT is designed to have the elements of the tac promoter and uses the cro SD to express the gene of interest behind the alkaline phosphatase signaling sequence. An example of the construction of this plasmid is given below by the construction of pTCPt / EGF.



  Construction of pTNPt (1 trp-35 117bpl lac-10 H nSD 1 8bpl ATG 1 / alkaline phosphatase signaling sequences / linker segment / term. Trans.-NEO).



   This plasmid is designed to possess the elements of the tac promoter and uses the SD gene N to express a given gene behind the alkaline phosphatase signaling sequence. It includes a background structure of pBR322 with the neomycin resistance gene. The plasmid pTNPt was constructed as follows: (a) Preparation of the 2.8 kb EcoRI-BamHI fragment of pBM16t / VGFa free from the HindIII site.



   Plasmid pBM16t / VGFa was put to digest with EcoRI and BamHI and the 2.8 kb fragment was isolated. The 2.8 kb fragment was ligated with an EcoRI-BamHI linker segment and by restriction analysis, a correct constructed product called intermediate I was isolated.



   The unique HindIII site close to the neomycin resistance gene of the intermediate plasmid I was deleted by digestion with HindIII, creating blunt ends with a Klenow fragment and re-ligation. This resulted in the intermediate plasmid II free from the HindIII site.

  <Desc / Clms Page number 58>

 



   The 2.8 kb EcoRI-BamHI fragment of pBM16t / VGFa free from the HindIII site was isolated by intermediate II digestion with EcoRI and BamHI. The resulting 2.8 kb fragment was isolated by agarose gel electrophoresis.



  (b) Preparation of the BamHI-BsmI fragment of 150 bp of pBM11 / PAk.



   The plasmid pBMll / PAK is identical to pBMll / PAK / EGF, except that it contains a linker region with HindIII, SmaI and BamHI sites downstream of the alkaline phosphatase signaling sequence instead of the l gene 'EGF. PBM11 / PAK was digested with BsmI and BamHI and the 150 bp fragment containing the SD gene N, the alkaline phosphatase signaling sequence and the linker region was isolated.



  (c) Preparation of the TacA + and TacA- oligonucleotides.



   The oligonucleotides TacA + and TacA-were synthesized on an apparatus for the synthesis of Applied Biosystems oligonucleotides and arranged to include an EcoRI overhanging at the end. 5 ′, with the consensus sequence trp-35 separated from the consensus sequence lac-10 by 17 nucleotides among which is an SstI site.



  The sequence also contains the 5 ′ end of the lac mRNA, the lac repressor binding site and a BsmI overhang.



   TacA + 5'AATTACTCCCCATCCCCCTGTTGACAATTAATCATCGAGCTC GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAG 3 '
TacA-5'GTGTGAAATTGTTATCCGCTCACAATTCCACACATTATA
CGAGCTCGATGATTAATTGTCAACAGGGGGATGGGGAGT 3 '

  <Desc / Clms Page number 59>

 (d) Ligation and isolation of pTNPt.



   The 2.8 kb EcoRIBamHI fragment was ligated, the 150 bp BsmI-BamHI fragment and the oligonucleotides TacA + and TacA-with DNA ligase and DNA was used to transform competent JM109 (lacIq) . By restriction analysis and
 EMI59.1
 S. DNA sequencing, a correct construct was isolated.



   (EcoRI site of pBMll)
 EMI59.2
 I GAATTACTCCCCATCC Sst I trp-35 (17pb) lac-10 CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG) BsmI 5'lac mRNA-> n mRNA-> TGTGG / AATTGTGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCT TTGGGGTGTGTGATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTAGC TGCCAATGTGCCAATCGCGGGGGGTTTTCGTTCAGGACTACAACTGCCACACACC PvuI mSD (8bp) Seq. by signa ACCAAAGCTAACTGAC {AGGA} GAATCCAG ATGAAACAATCTACGATCGCCC
M K Q S T I A L
SmaI
HindIII BamHI TCGCACTTCTCCCACTGCTGTTCACTCCAGTGACAAAAGCTTCCCGGGATCCGTG
A L L P L L F T P V T K

  <Desc / Clms Page number 60>

   (BamHI site of pBMll) Term. trans. 1 ACTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC
I.

   The plasmid plac / cro- consists of the
 EMI60.1
 0 eron lactose (lac) operator-promoter region of the lactose operon (lac) of E. coli, as well as the ribosomal binding sites of lac and cro. The plasmid was constructed as described in EUA patent application No. 264,098 of October 28, 1988. The fusion proteins expressed by this vector consist of the Nterminal part of the Cro protein of the bacteriophage, the amino acid sequence encoded by l DNA and the C-terminal domain of P-galactosidase. The control elements of this vector consist of the operator-promoter region of the lactose (lac) operaton of E. Coli, and the lac and cro ribosomal binding sites.



   K. The plasmid ptac / cro-6-gal makes it possible to clone a gene of interest downstream of the 21 N-terminal amino acids of the bacterial Cro protein. The plasmid was constructed as described in EUA patent application No. 624,098 of October 28, 1988. The expression vector ptac / cro-P-gal is similar to plac / cro-û-gai, with the exception that the ptac / cro-f3-gal promoter consists of region-35 of the promoter of the tryptophan operon and in the Pribnow box of the lac operon. This hybrid promoter allows a higher degree of expression than plac / cro-P-gal.



   K. The plasmid pRSV allows the expression of a gene of interest in eukaryotic cells with the
 EMI60.2
 promoter RSV LTR, Gorman et al., Proc. Natl. Acad.



  Sci. USA (1982) 79: 6777-6781.



  L. Plasmid pAc610, Smith et al., Molec.

  <Desc / Clms Page number 61>

 



  Cell. Biol. (1983) 12: 2156-2165, allows the expression of a foreign gene in the cells of an insect.



   M. Plasmids pToxl and pTox2 allow the expression of a gene of interest in yeast cells using the killer toxin signaling sequence of K. lactis (EMBO 6, 229-234 (1987); Biochem, Biophys. Res. Comm. 144,613-619 (1987)). This constructed product includes the URA3 gene for selection in yeast and the AMP gene for selection in bacteria. Transcription begins with the CYC1 promoter and the "upstream activation sequence" GAL (Methods Enzymol., 101, 181-191 (1983)) and ends in the 3 'end of the FLP gene (Mol. Cell.



  Biol., 5, 2770-2780 (1985)). The product constructed includes both the origin of the 2 micron plasmid and the origin of pBR322 for replication in yeast and E. coli, respectively. The Nterminal region of killer toxin, with the signaling sequence and the Kex2 cleavage site, was introduced onto the oligonucleotides and limited in the region rich in A-T immediately before the initiator codon to conserve translation initiation signals and optimal elongation. Various restriction sites have been arranged to allow the insertion of genes of interest downstream of the signaling cleavage site and of the Kex2 cleavage site.



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



   The plasmid pLGSD5 (-ATG), which is an expression vector in yeasts (Methods Enzymol. 101: 181-191 (1983)), was put to digest with EcoRI and EspI and the fragment of 3 was isolated, 0 kb.

  <Desc / Clms Page number 62>

 



  2. Preparation of the 2.1 kb EcoRI-EspI fragment of the 2 micron plasmid containing the 3 ′ end of the FLP gene and the origin.



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



  3. Ligation and isolation of the intermediate plasmid I.



   The EcoRI-EspI fragments of 2, 1 kb and 3 kb were ligated together using DNA ligase and the DNA was used to transform competent HB101. By restriction analysis, a correct constructed product was isolated.



  4. Preparation of the 1.8 kb EcoRI-BamHI fragment of
 EMI62.1
 pLGSD5 (-ATG) containing the URA3 gene, the upstream activation sequence GAL and the promoter CYC1.



   Plasmid pLGSD5 (-ATG) was digested with EcoRI and BamHI and the 1.8 kb fragment was isolated. The BamHI site is present in a linker segment inserted at 4 base pairs upstream of the initiator CYC1.



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



   Oligonucleotides were developed to unite the codons for the killer toxin signaling sequence of K. lactis at the BamHI site downstream of the initiator CYC1. To maintain optimal translation initiation, 13 base pairs of the rich A-T sequence were included upstream of the killer toxin initiator codon. This sequence conforms to the consensus sequence determined for the initiation codons of the yeast codons. A BG1I site was placed directly upstream of the A-T region to allow mutagenesis of the initiation region and of the signaling sequence. A HindIII site was placed 10 nucleotides upstream of the signal cleavage site to allow mutagenesis of the sequences in

  <Desc / Clms Page number 63>

 downstream and ligation of an interesting gene.

   In pToxl, an AvaI site, XhoI was located directly downstream of the signal cutoff site, while in pTox2, a NaeI site was located at the signal cutoff site for blunt-end cloning directly beyond the signal cut which was changed from Gly-Leu to AlaGlys. The resolution of the heteroduplex at the cleavage site should lead to the formation of clones containing the pToxl sequence and of clones containing the pTox2 sequence. In the two products constructed, the subsequent sequences code for the rest of the sequence
 EMI63.1
 0 killer toxin precursor downstream to the Kex2 Lys-Arg cleavage site, point at which various restriction sites (StuI, SalI, AccI, HincII and BamHI) have been arranged to facilitate the cloning of a gene of interest .

   The StuI site allows blunt-end cloning directly after the Arg codon of the Kex2 cleavage site. The oligonucleotides have a BamHI overhang at the 5 ′ end and a HindIII overhang at the 3 ′ end to allow cloning into the intermediate vector I. The resulting sequence no longer has either of the two sites.



   (BamHI) BglII
 EMI63.2
 M 1A + 5'GATCAGATCTAATAATTATAAAAT 2A-3'TCTAGATTATTAATATTTTA
HindIII
N I F Y I F L F L L S GAATATATTTTACATATTTTTGTTTTTGCTAAGC CTTATATAAAATGTATAAAAACAAAAACGATTCGAAG

  <Desc / Clms Page number 64>

 
Sign break site.



   (pToxl) / AvaI, XhoI
F V Q G L E H T H R R
1B + 5'TTCGTTCAGGGCCTCGAGCATACTCATAGAAG
2B-3'AAGTCCGGCCGCTCGTATGAGTATCTTC
A G (pTox2) NaeI
Cut-off site Kex2 / StuI SalI, AccI BamHl
G S L V K R P L S T D P
AGGCTCCTTAGTCAAAAGGCCTTTGTCGACGGATCC
TCCGAGGAATCAGTTTTCCGGAAACAGCTGCCTAGGTCG
These oligonucleotides were synthesized on an apparatus for the synthesis of Applied Biosystems oligonucleotides and were purified by gel electrophoresis. Then they were phosphorylated with T4 kinase and the complementary pairs were renatured together, ligated and gel purified.



  6. Preparation of the intermediate vector 1 EcoRI-HindIII.



   Intermediate vector 1 was digested with EcoRI and HindIII, then treated with alkaline calf phosphatase. This treatment eliminated a small EcoRI-HindIII fragment and left a HindIII site remaining upstream of the FLP3 'sequence.

  <Desc / Clms Page number 65>

 



  7. Ligation and isolation of pToxl and pTox2.



   The 1.8 kb EcoRIBamHI fragment was ligated with the oligonucleotide fragment using DNA ligase and the resulting fragment was gel-purified, then ligated with EcoRI-HindIII intermediate vector 1 and DNA was used to transform competent HB101. The correct constructs were identified by restriction analysis and DNA sequencing and one clone comprising the sequence pTox1 and one comprising the sequence pTox2 was isolated. The two clones were free from the BamHI site at the junction of the oligonucleotides and of intermediate vector 1.



  EXAMPLE III.- Assembly of growth factor genes for expression in prokaryotic cells.



  A. Preparation of the plasmid pLEBam / TTV.



   The synthetic hybrid growth factor called 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 two-third amino-terminal of the gene, except for the sequence QEEK which was modified from the natural human sequence QEDK.



  The carboxyl terminus originating from the amino acid sequence of VGF and was terminated by the sequence YQR upstream of the natural sequence PNT. Plasmid pLEBam was allowed to digest with BssHII and BamHI. The BssHII-BamHI fragment of pLEBam was then ligated with oligonucleotides TGF101, 102,103 and 104 and VGF1, 2,3 and 4 using DNA ligase and the resulting plasmids were used to transform competent HB101s. Transformants were selected on ampicillin and sorted by restriction analysis using EcoRI, NcoI and

  <Desc / Clms Page number 66>

 BamHI, as well as by nucleotide sequencing according to the Maxam-Gilbert protocol. We isolated a correct constructed product denoted pLEBam / TTV.
 EMI66.1
 



  TGF NcoI CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT M V V S H F N D C P D S H T Q F C F VGF
KpnI SphI
TCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT
H G T C R F L V Q E E K P A C V C S
EcoRI
CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT
H G Y T G I R C Q H V V L V D Y Q R
BamHI
TAAGGATCC
Ter B. Preparation of the plasmid pLEBam / TVV.



   The synthetic hybrid growth factor noted TVV (or TGF / BGF / VGF) was assembled in the cloning vector pLEBam. This hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain of the gene. The middle domain and the C-terminal domain derive from the truncated VGF sequence and terminate with the YQR sequence upstream from the natural PNT sequence. In addition, the synthetic gene includes the modification GYACVC replacing GMYCRC.



  (a) Preparation of a 4.3 kb KpnI-SphI fragment of pLEBam / TTV.



   Plasmid pLEBam / TTV was digested with KpnI and SphI and the 4.3 kb KpnI-Sph-I fragment was gel purified. This digestion eliminates the area

  <Desc / Clms Page number 67>

 median of the TGF outside the synthetic TTV gene in the cloning plasmid pLEBam.



  (b) Ligation and isolation of pLEBam / TTV.



   The oligonucleotides VGF1010a and 102a were ligated with the 4.3 kb KpnISphI fragment of pLEBam / TTV using DNA ligase and the resulting mixture was used to transform competent HB101s. The transformants were selected on ampicillin and sorted by nucleotide sequencing according to the Sanger-dideoxy method. We isolated a correct constructed product denoted pLEBam / TTV.
 EMI67.1
 



  TGF NcoI CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT
M V V S H F N D C P D S H T Q F C F
VGF
KpnI SphI TCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT HGTCIHARDIDGYACVCS
EcoRI CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGCTCGACTACCAGCGT H G Y T G I R C Q H V V L V D Y Q R
BamHI TAAGGATCC Ter EXAMPLE IV Expression of the polypeptide of interest as a fusion protein with protein N.



  A. The modified synthetic TGF.



  1. Preparation of pBMll / N / TGF.



   Human modified TGF was expressed in this system as part of a 33 acid fusion

  <Desc / Clms Page number 68>

 N-terminal amines of the N gene; it includes the QEEK sequence replacing the human QEDK sequence.



  (a) Preparation of a 780 bp SphI-PvuI fragment of PBM11 / N / TTV.



   Plasmid pBM11 / N / TTV was digested with SphI and PvuI and the 780 bp SphI-PvuI fragment was gel purified. This fragment contains a part of the plasmid of pBM11 at the PvuI end and at the SphI end, the N gene and the two-thirds N-terminals of the human TGF gene.



  (b) Preparation of the 5 kb BamHI-PvuI fragment of pBM11 / N / TTV.



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



  (c) Ligation and isolation of pBMIl / N / TGF.



   The oligonucleotides TGF 206 and 206, the SphI-PvuI fragment of 780 bp and the BamHI-PvuI fragment of 5 kb of pBM11 / N / TTV were ligated and the product was used to transform competent HB101. The transformants were selected on neomycin and sorted by restriction analysis using EcoRI and by
 EMI68.1
 0 nucleotide sequencing according to the Sanger-dideoxy process.

   A correct constructed product denoted pBMIl / N / TGF was isolated. N gene ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K

  <Desc / Clms Page number 69>

 
BamHI AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCCGCAT
A A N P L L V G V S A K P V R I R M
TGF- GGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTTTCAT
V V S H F N D C P D S H T Q F C F H KpnI SphI GGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCCATTCTG G T C R F L V Q E E K P A C V C H S G
BamHI GCTAGGTTGGCGCACGTTGCGAACACGCTGACCTGCTGGCTTAAGGATCC
Y V G A R C E H A D L L A Ter B. The modified synthetic TGF-VGF hybrid.



  1. Preparation of pBMll / NiTTV.



   In this constructed product, a synthetic modified TTV hybrid gene was expressed as the C-terminal part of a fusion protein comprising the first 33 amino acids of the N gene at the N-terminal end. This hybrid growth factor contained the amino acid sequence of human TGF in the amino-two-thirds of the gene, with the exception that the QEEK sequence was modified from the natural human QEDK sequence. The carboxyl terminus was derived from the amino acid sequence of VGF and terminated by the sequence YQR upstream of the natural sequence PNT.



  (a) Preparation of the synthetic TTV NcoI (franc) BamHI gene
Plasmid pLEBam / TTV was digested with NcoI and the ends blunted by completing the overhangs with the Klenow fragment of DNA polymerase. The DNA was then digested with BamHI and the Ncol fragment (franc) - gel purified -

  <Desc / Clms Page number 70>

 BamI of 170 bp of TTV.



  (b) Preparation of pBM11 digested with BamHI.



   Plasmid pBM11 was put to digest with BamHI.



  (c) Ligation and isolation of pBMll / N / TTV.



   BamHI digested pBM11, the NcoI (franc) -BamI fragment from TTV and the BamHI linker segments (5'GATCCG3 ') were ligated together using DNA ligase and the resulting mixture was used to transform competent HB101 . The transformants were selected on neomycin and sorted by restriction analysis and nucleotide sequencing according to the Sanger-dideoxy method.

   A correct constructed product pBMll / N / TTV was isolated. N-p gene ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA MDAQTRRRERRAEKQAQWK
BamHI AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCCGCAT
A A N P L L V G V S A K P V R I R M
TGFGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTTTCAT
V V S H F N D C P D S H T Q F C F H
KpnI SphI VGFGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCTCATG G T C R F L V Q E E K P A C V C S H G
EcoRI GCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGTTAAG
Y T G I R C Q H V V L V D Y Q R Ter BamHI GATCC

  <Desc / Clms Page number 71>

 EXAMPLE 5 Preparation of the polypeptide of interest in the state of fusion protein with protein N and a cleavage site.



  A. The modified synthetic VGF.



  1. Preparation of pBMIl / NDP / VGFA.



   The N-terminal sequence of the synthetic VGFA gene is a truncated version of the natural VGF sequence and begins with the DIPAIR sequence.



  In this plasmid, the VGFA fragment is located downstream of 32 amino acids of the N protein of lambda and of the dipeptide aspartic acid-proline. In order to preserve the KpnI cloning site, the synthetic sequence was modified so as to encode CLHCGTC instead of the CLHGDC sequence of the natural VGF and it ends with the YQR sequence upstream of the natural PNT sequence. In addition, the VGFA gene codes for the sequence GYACVC which replaces the natural sequence GMYCRC. at. Preparation of an 80 bp C-terminal KpnI-BamHI fragment of the synthetic VGF gene.



   Plasmid pLEBam / TVV was digested with KpnI and BamHI and the 80 bp KpnI-BamHI fragment was gel purified. This fragment contains two-thirds Cterminals of the synthetic VGF gene with the KpnI site at the 5 'end. b. Preparation of pBMll digested with BamHI and dephosphorylated.



   Plasmid pBMIl / N / TTV was digested with BamHI and the 5'-phosphates were removed by treatment with intestinal alkaline phosphatase from calves. The 5.6 kb BamHI fragment of the plasmid was gel purified.

  <Desc / Clms Page number 72>

 vs. Ligation and isolation of pBMll / NDP / VGFA.



   The oligonucleotides VGF 103a and 104a, the 5.6 kb BamHI fragment of pBM11 and the 80 bp Kpnl-BamHI fragment of pLEBam / TVV were ligated together using DNA ligase, and the product was used for transform competent HB101s. The transformants were selected on neomycin and sorted by restriction analysis using ClaI and by nucleotide sequencing according to the Sanger-dideoxy method. A correct construct was noted pBM11 / NDP / VGFA. This constructed product includes the GTC and GYACVC sequences instead of the GDC and GMYCRC sequences of authentic VGF. N gene.



  ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K ****
Clal
 EMI72.1
 AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC A A N P L L V G V S A K P V R I D P Nco I VGF CCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT + M D I P A I R L C G P E G D G Y C L Kpn I Sph GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT
H G T C I H A R D I D G Y A C V C S
EcoRI CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT H G Y T G I R C Q H V V L V D Y Q R

  <Desc / Clms Page number 73>

 
BamHI
TAAGGATCC
Ter 2. Preparation of pBMIl / NDP / VGFa.



   The N-terminal sequence of VGFa is a truncated version of the sequence of natural VGF and begins with the sequence DIPAIR. In addition, the VGFa sequence contains the modified GTC and GYACRC sequences instead of the GDC and GMYCRC sequences of natural VGF. In this plasmid, the VGFa gene is located downstream of 32 amino acids from the N protein of lambda and the dipeptide aspartic acid-proline. By treating the purified fusion protein with formic acid, the aspartic acid-proline peptide bond, which is labile in acid medium, is cut, which allows the VGFa protein to be separated from the amino terminus of the protein. N of lambda. The cleavage is such that the VGFa protein remains with the proline residue at the amino terminus. at. Preparation of pBMIl / DP / VGFA digested with SphI and dephosphorylated.



   Plasmid pBM11 / DP / VGFA (10iLg) was digested with 30 units of SphI and the 5'phosphates were removed by treatment with calf intestinal alkaline phosphatase. The 5 kb plasmid fragment was collected after electrophoresis on an agarose gel. b. Preparation of a 70 bp EcoRI-SphI fragment of pBM11 / DP / VGFA.



   Plasmid pBMIl / DP / VGFA (10 g) was put to digest with 30 units of EcoRI, then 30 units of SphI.

  <Desc / Clms Page number 74>

 After electrophoresis on an agarose gel, the 70 bp fragment was collected.



  3. Ligation and isolation of pBMll / NDP / VGFa.



   The 24 bp fragment containing the oligonucleotides VGF 1A and 2A, the
 EMI74.1
 5 bb SphI fragment and the 70 bp EcoRI-SphI fragment of pBM11 / OP / VGFA and the mixture was used to transform E. coli HB101 cells. competent coli.



  The transformants were sorted by nucleotide sequencing according to the Sanger-dideoxy method. A correct clone noted pBM11 / NDP / VGFa was isolated. N gene ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K * * * *
Clal AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATCC
A A N P L L V G V S A K P V R I D P NcoI VGFCCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT
M D I P A I R L C G P E G D G Y C L KpnI SphI GCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCTCT HGTCIHARDIDGYACRCS
EcoRI CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT H G Y T G I R C H V V L V D Y Q R
BamHI TAAGGATCC Ter B. The modified synthetic TGF-VGF hybrids.



  1. Preparation of pBMll / NDP / TTV.

  <Desc / Clms Page number 75>

 



   In this constructed product, the synthetic modified TTV hybrid gene is expressed as a terminal part of a fusion protein comprising the first 32 amino acids of the N gene at the N end. An aspartic acid-proline dipeptide labile in acid medium separates the two parts of the fusion product. The hybrid growth factor contains the amino acid sequence of human TGF in the aminoterminal two-thirds of the gene, with the exception that the QEEK sequence is modified compared to the QEDK sequence of natural human TMR. The carboxyl terminus originated from the amino acid sequence of VGF and was terminated by the sequence YQR 11 upstream from the natural PNT sequence. at. Preparation of the 5 kb NcoI fragment of the plasmid pBM11.



   Plasmid pBMIl / NDP / VGFA was put to digest
 EMI75.1
 with Ncol and the 5 kb col plasmid fragment was gel purified. This fragment includes an NcoI overhang at the aspartic acid-proline cleavage site downstream of the sequences coding for the first 32 amino acids of the N gene. The other NcoI site is in the neomycin resistance gene. b. Preparation of the 0.6 kb NcoI-BamHI fragment of pBM11.



   Plasmid pBM11 / N / TTV was digested with NcoI and BamHI and the 0.6 kb NcoI-BamI fragment of the plasmid was gel purified. This fragment includes the overhang of NcoI in the neomycin resistance gene. vs. Preparation of the 170 bp synthetic TGF / TGF / VGF fragment.



   Plasmid pLEBam / TTV was digested with NcoI and BamHI and the 170 bp NcoIBamHI fragment containing the synthetic gene was gel purified.

  <Desc / Clms Page number 76>

 TGF / TGF / VGF. This fragment includes the Scot overhang at the 5 'end of the gene and the BamHI overhang at the 3' end of the gene. d. Ligation and isolation of pBMIl / NDP / TTV.



   The 5 kb NcoI and 0.6 kb NcoI-BamHI plasmid fragments were ligated together with the 170 bp NcoI-BamHI fragment of the TTV gene using DNA ligase and the resulting mixture was used to transform competent HB101s. The transformants were selected on neomycin, so that only colonies comprising correctly reconstructed neomycin resistance genes can survive.



  The transformants were sorted by restriction analysis with NcoI and by nucleotide sequencing according to the Sanger-dideoxy method. A correct constructed product denoted pBMIl / NDP / TTV was isolated. N gene ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K
ClaI AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC
A A N P L L V G V S A K P V R I D P NcoI TGFCCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT
 EMI76.1
 M V V S H F N D C P D S H T Q F C F KpnI SphI VGFATCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT H G T C R F L V Q E E K P A C V C S

  <Desc / Clms Page number 77>

 
EcoRI CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT H G Y T G I R C Q H V V L V D Y Q R
BamHI TAAGGATCC Ter 2. Preparation of pBMIl / NDP / VTV.



   In this constructed product, the synthetic modified VTV hybrid gene was expressed as a terminal part of a fusion protein comprising the first 32 amino acids of the N gene at the N end. An aspartic acid-proline dipeptide labile in acid medium separates the two parts of the fusion product. This hybrid growth factor contained the amino acid sequence of human TGF in the middle domain with the acid sequence. QEEK amines replacing the
 EMI77.1
 01 natural sequence QEDK. The N-terminal domain and the C-terminal domain originated from a truncated VGF sequence and began with the DIPAIR sequence and ended with the YQR sequence which is upstream from the natural PNT sequence. at. Preparation of a 5 kb BamHI-NcoI fragment of pBM11.



   Plasmid pBM11 / N / TTV was digested with BamHI and NcoI and the 5 kb BamHI-NcoI fragment was gel purified. This fragment contains a BamHI overhang at the 3 ′ end of the sequences coding for the first 32 amino acids of the N gene and an NcoI site in the neomycin resistance gene. b. Preparation of a 700 bp KpnI-NcoI fragment of pBM11 / N / TTV.



   Plasmid pBM11 / N / TTV was digested with Kpnl and NcoI and the fragment was gel purified

  <Desc / Clms Page number 78>

 700 bp KpnI-NcoI. This fragment is made up of a part of the plasmid pBM11 containing a part of the neomycin resistance gene overhanging NcoI, as well as the C-terminal domain of VGF of the synthetic gene TTV overhanging KpnI. vs. Ligation and isolation of pBMll / NDP / VTV.



   The oligonucleotides VGF 103a and 104a, the BamHINcoI fragment of 5 kb of pBM11 and the fragment KpnI-NcoI of 700 bp of pBMll / N / TTV were ligated together using DNA ligase and the product was then used for transform competent HB101s. The transformants were selected on neomycin and they were sorted by restriction analysis using ClaI and by nucleotide sequencing according to the Sanger-dideoxy method.
 EMI78.1
 N gene ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K ****
Clal AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC
A A N P L L V G V S A K P V R I D P NcoI VGFCCATGGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGCCT
M D I P A I R L C G P E G D G Y C L KpnI TGF + SphI VGF.



  GCATGGTACCTGCCGTTTTCTGGTTCAGGAAGAAAAACCGGCATGCGTTTGCTCT
H G T C R F L V Q E E K P A C V C S

  <Desc / Clms Page number 79>

 
EcoRI CATGGCTACACTGGAATTCGTTCGCAGCATGTTGTTCTGGTCGACTACCAGCGT H G Y T G I R C Q H V V L V D Y Q R
BamHI TAAGGATCC Ter 3. Preparation of pBM16 / NDP / TVV.



   In this constructed product, the synthetic modified TVV hybrid gene was expressed as the terminal part of a fusion protein containing the first 32 amino acids of the N gene at the N end. An aspartic acid-proline dipeptide labile in acid medium separates the two parts of the fusion product. The hybrid growth factor contained the amino acid sequence of human TGF in the N-terminal domain.



  The middle domain and the C-terminal domain originated from the truncated VGF sequence and ended with the YQR sequence. In addition, the synthetic gene includes the modification of GYACVC to GMYCRC. at. Preparation of the 4.3 kb NcoI-BglII fragment of pBM11 / NDP / VGFa.



   Plasmid pBM11 / NDP / VGFa was digested with NcoI and BglII and the 4.3 kb fragment was purified on a gel. The NcoI overhang is positioned at the aspartic acid-proline cleavage site just downstream of the first 32 amino acids of the N. b gene. Preparation of the 1.2 kb BamHI-BglII fragment of pBM11MS.



   Plasmid pBMIlMS was allowed to digest with
 EMI79.1
 BamHI and BglII and the 1.2 kb BamHI and p e fragment was purified on a gel. This fragment differs from the normal pBM11 fragment in that the NcoI site in the neomycin resistance gene has been eliminated; all

  <Desc / Clms Page number 80>

 Subsequent vectors free from this NcoI site are called pBM16. vs. Preparation of the synthetic TVV NcoI-BamHI gene of 170 bp.



   Plasmid pLEBAM / TVV was digested with NcoI and BamHI and the 170 bp NcoI-BamHI fragment was gel purified. This synthetic gene fragment comprises the NcoI site at the 5 ′ end and the BamHI site at the 3 ′ end. b. Ligation and isolation of pBM16 / NDP / TW.



   The 4.3 kb NcoI-BglII fragment of pBM11 / NDP / VGFa and the 1.2 kb BamHI-BglII fragment of pBMllM5 were ligated together as well as the 150 bp TW synthetic gene fragment NcoI-BamHI DNA ligase and the resulting mixture was used to transform competent HB101s. The transformants were selected on neomycin and sorted by restriction analysis and nucleotide sequencing according to the Sanger-dideoxy method. The plasmid is noted pBM16 to indicate the loss of the NcoI restriction site in the neomycin resistance gene. N gene.



   ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGGCTCAATGGA M D A Q T R R R E R R A E K Q A Q W K **** ClaI
AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATC
A A N P L L V G V S A K P V R I D P Ncol TGF
CCATGGTTGTTTCTCACTTTAACGACTGCCCGGACTCTCATACTCAGTTTTGCTT
M V V S H F N D C P D S H T Q F C F

  <Desc / Clms Page number 81>

 
KpnI VGF + SphI TCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCGTTTGCTCT HGTCIHARDIDGYACVCS
EcoRI
CATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCGT
H G Y T G I R C Q H V V L V D Y Q R
BamHI
TAAGGATCC
Ter C. Synthetic EGF.



  1. Preparation of pBMll / NDP / EGF.



   In this constructed product, the human EGF gene is expressed as part of a fusion product with the 32 N-terminal amino acids of the N gene which is found downstream of an Asp-Pro cleavage site. at. Preparation of a 5 kb NcoI fragment of pBMIl.



   Plasmid pBM11 / DP / VGFA was digested with Ncol and the S'-phosphates were removed by treatment with calf intestinal alkaline phosphatase. The 5 kb plasmid fragment was purified on a gel. This fragment includes an NcoI overhang at the Asp-Pro cleavage site downstream of the sequences coding for the first 32 amino acids of the N gene. The other NcoI site is located in the neomycin resistance gene. b. Preparation of a 0.6 kb NcoI-BamHI fragment of pBMIl.



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

  <Desc / Clms Page number 82>

 vs. Ligation and isolation of pBMIl / DP / EGF.



   The three sets of renatured EGF oligonucleotides were ligated together with a Nicol overhang at the 5 'end and a BamHI overhang at the 3' end, the 5 kb NcoI fragment of pBMIl and the fragment of 0.6 kb NcoI-BamHI of pBM11 using T4 DNA ligase and the resulting mixture was used to transform competent E. coli HB101. The transformants were selected on neomycin so that only colonies comprising a correctly reconstructed neomycin resistance gene could survive. The transformants were sorted by restriction analysis with EcoRI and BamHI and by DNA sequencing, as described above. N gene.



  ATGGATGCACAAACACGCCGCCGCGAACGTCGCGCAGAGAAACAGCGTCAATGGA
 EMI82.1
 M D A Q T R R R R R A A K Q A Q W K ** * AAGCAGCAAATCCCCTGTTGGTTGGGGTAAGCGCAAAACCAGTTCGGATCGATCC
A A N P L L V G V S A K P V R I D P EGFl + EcoRI EGF2 + CATGAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCATGAC
M N S D S E C P L S H D G Y C L H D EGF3 + NsiI SphI GGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTTGTTG G V C M Y I E A L D K Y A C N C V V G GCTACATCGGCGAACGTTGACGTGACCT
Y I G E R C Q Y R D L K W W E L R *

  <Desc / Clms Page number 83>

 
BamHI
AGGATCC EXAMPLE VI Preparation of the polypeptide of interest as a fusion protein with a modified alkaline phosphatase signaling sequence A. Preparation of pBM11 / PAD / EGF.



   Synthetic oligonucleotides were designed to allow insertion of DNA encoding a modified alkaline phosphatase signaling peptide and a linker region with 3 cloning sites (HindIII, SmaI and BamHI) into the expression vector pBMIl downstream of the PL promoter and the ribosomal binding site of the N gene. The nucleotide sequence has been optimized so that it is as similar as possible to the nucleotide sequence of the amino terminus of the N gene from lambda, because that the lambda N gene sequence has cleared with the sequence of its ribosomal binding site for efficient initiation and translation of the ribosome.

   In addition, the second amino acid in the alkaline phosphatase signaling sequence, namely lysine, which is a basic amino acid, was replaced by aspartic acid, which is an acidic amino acid.



  1. Preparation of the 0.17 kb EcoRI-BamHI fragment of EGF.
 EMI83.1
 



  Plasmid pBM11 / NDP / EGF (30 eg) was digested with 30 units of EcoRI, then treated with 4 units of the Klenow fragment of DNA polymerase to obtain blunt ends. The DNA was digested completely with 30 units of BamHI and the 0.17 kb fragment of the 11EGF gene was collected

  <Desc / Clms Page number 84>

 
 EMI84.1
 % electrophor% ese on ge after an electrophoresis on agarose gel. The DNA thus purified thus comprises an EcoRI site made non-sticky at the 5 ′ end and a BamHI overhang at the 3 ′ end. 2. Preparation of a 0.5 kb PvuI-HindIII fragment of pBMIl / PAD.
 EMI84.2
 



  Plasmid pBMI1 / PAD (ISAg) was digested with 30 units of HindIII and then treated with the Klenow fragment of DNA polymerase to make the ends non-sticky. The DNA was then digested with PvuI and the 0.5 kb PvuI-HindIII (franc) fragment was collected after agarose gel electrophoresis.



  3. Preparation of the 5.2 kb PvuI-BamHI fragment of pBMIl / PAD.



   Plasmid pBMIl / PAD (18 g) was put to digest with 30 units of PvuI, then 30 units of BamHI. The 5.3 kb fragment was collected after agarose gel electrophoresis.



  4. Ligation and isolation of pBM11 / PAD / EGF.



   The 0.17 kb EcoRI (franc) -BamHI fragment was ligated, the 0.5 kb PvuI-HindIII (franc) fragment and the 5.2 kb PvuI-BamHI fragment, and then the mixture was used. resulting to transform competent E. coli HB101. The transformants were sorted by DNA sequencing, as described above.

   The desired EGF / signaling sequence region has the following sequence:
Seq. ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTCACT
M D Q S T I A L A L L P L L F T

  <Desc / Clms Page number 85>

 
EGF CCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGAC
P V T K A N S D S E C P L S H D
NsiI
GGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTG
G Y C L H D G V C M Y I E A L
SphI GACAAGTACGCATGCAACTGCGTTGTTGGCTACATCGGCGAACGT DKYACNCVVGYIGER
BamHI
TGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTAAGGATCC C Q Y R D L K W W E L R *
The efficiency of production of the foreign protein in the pBM11 / PAD expression system and the possibility of purifying functionally active foreign proteins from the fusion product have been demonstrated by means of pBMll / PAD / EGF as

  example. After dimensional exclusion chromatography (TSK-250), 10.3 mg was collected
 EMI85.1
 op d of active EGF fusion polypeptide equivalents from 23 g (8 liters) of depressed E. coli to express the EGF gene. 40% of EGF activity came from EDF separated by cutting the signaling sequence.



  B. Preparation of pBMll / PAD / nVGFa.



   Synthetic oligonucleotides have been designed to link the synthetic gene VGFa with a modified alkaline phosphatase signaling sequence to provide an optimal signaling sequence cleavage site by coding for additional Nterminal residues that are immediately found

  <Desc / Clms Page number 86>

 downstream of the cleavage site of the signaling sequence in the natural VGF, noted extreme terminal domain above. The nVGFa sequence contains the modified GTC and GYACRC sequences instead of the GDC and GMYCRC sequences of the natural VGF and ends with the YQR sequence upstream of the natural PNT sequence. In this expression system, for the majority of molecules, the signaling sequence remains attached to nVGFa forming a fusion protein with nVGFa at the C end.



  1. Preparation of pBMIl / PAD digested with HindIII-PvuI of 0.5 kb.



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



  2. Preparation of the 5.2 kb PvuI-BamHI fragment of the plasmid pBM11.



   Plasmid pBM11 / NDP / VGFa was digested with PvuI and BamHI and the 5.2 kb fragment of the plasmid was purified on a gel.



  3. Preparation of the synthetic VGFa gene NcoI (franc) BamHI of 170 bp.



   Plasmid pBM11 / PAD / nVGFa was digested with NcoI and the overhangs were removed in 5 'by treatment with nuclease SI. This created a blunt or non-sticky end to the first codon of the truncated synthetic gene for VGFa. The DNA was then digested with BamHI and the 170 bp NcoI (franc) -BamHI fragment was purified on a gel.



  4. Ligation and isolation of pBMll / PAD / nVGFa.



   The oligonucleotides VGF105 and 106, the HindIIIPvuI fragment of 0.5 kb of pBM11 / PAD, the fragment PvuI-BamHI of

  <Desc / Clms Page number 87>

 5.2 kb of pBM11 and the synthetic gene VGFa NcoI (franc) -BamHI of 170 bp using DNA ligase and the resulting mixture was used to transform competent HB101. The transformants were selected on neomycin and sorted by restriction analysis and nucleotide sequencing according to the Sanger-dodeoxy method. A correct construct was isolated containing the alkaline phosphatase signaling sequence modified in frame with the nVGFa gene.



  Seq. of sign.



    ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA M D Q S T I A L A L L P L L F T P V T
VGFCAAAAGCTGACTCTGGTAACGCTATCGAAACTACTTCTCCGGAAATCACTAACGC
K A D S G N A I E T T S P E I T N A TACTACTGACATCCCGGCTATCCGTCTGTGCGGCCCGGAAGGCGACGGCTACTGC
 EMI87.1
 T T D I P A I R L C G P E G D G Y C KpnI SphI CTGCATGGTACCTGCATCCATGCACGTGACATCGACGGCTACGCATGCCGTTGCT LHGTCIHARDIDGYACRCS
EcoRI CTCATGGCTACACTGGAATTCGTTGCCAGCATGTTGTTCTGGTCGACTACCAGCG HGYTGIRCQHVVLVDYQR
BamHI TTAAGGATCC
Ter

  <Desc / Clms Page number 88>

 EXAMPLE VII Expression of the polypeptide of interest in the state of a fusion protein with an alkaline phosphatase signaling sequence.



  A. Preparation of pBM11 / PAK / nVGFa (alkaline phosphatase / nVGFa signaling sequence with N-terminus of natural VGF and of the GTC and GYACRC sequences.



   The plasmid pBM11 / PAD / nVGF was subjected to in vitro mutagenesis (Morinaga et al., Biotechnology (1984) 2: 636-643) so as to modify the codons coding for the second amino acid in the signaling sequence, c that is, to convert the Asp codon (D) back into the Lys codon (K), which is the residue found in the natural sequence. This mutagenesis also introduced a PvuI site into the signaling sequence.



   Seq. of sign.



   ATGGATCAATCTACAATCGCCCTCGCACTTCTCCCACTGCT M D Q S T I A L A L L P L L nVGFGTTCACTCCAGTGACAAAAGCTGACTCTGGTAACGCTATCGAAACTAC 'F T P V T K A D S G N A I E T T
TTCTCCGGAAATCACTAACGCTACTACTGACATCCCGGCTATCCG SPEITNATTDIPAIR
KpnI
TCTGTGCGGCCCGGAAGGCGACGGCTACTGCCTGCATGGT
L C G P E G D G Y C L H G

  <Desc / Clms Page number 89>

 
SphI
ACCTGCATCCATGCACGTGACATCGACGGCTACGCATG
T C I H A R D I D G Y A C
EcoRI
CCGTTGCTCTCATGGCTACACTGGAATTCGTTGCCAGCATGTTGT R C 5 H G Y T G l R C Q H V V
BamHI
TCTGGTCGACTACCAGCGTTAAGGATCC
L V D Y Q R Ter B. Preparation of pBMIl / PAK / EGF.



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



   The plasmid pBM11 / PAD / EGF was subjected to mutagenesis in vitro so as to modify the codons coding for the second amino acid in the signaling sequence in order to modify the coder Asp (D) into the codon for Lys (K) which is the residue found in the natural sequence. This mutagenesis also introduced a PvuI site into the signaling sequence sequence.

  <Desc / Clms Page number 90>

   PvuI
 EMI90.1
 Seq. of sign.



  ATGAAACAATCTACGATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA M K Q S T I A L A L L P L L F T P V T EGFCAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCA
K A N S D S E C P L S H D G Y C L H
NsiI SphI TGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTT
D G V C M Y I E A L D K Y A C N C V GTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC V G Y I G E R C Q Y R D L K W W E L R
BamHI GTTAAGGATCC * C. Preparation of TacPak / EGF (alkaline phosphatase / human EGF signaling sequence).



  1. Preparation of the plasmid fragments.



   Plasmid pl35-1 is derived from plasmid pDR540 (Pharmacia) and contains the SD of the Cro gene and a BglII site downstream of the SD lac. PDR540 is an expression vector containing the hybrid trp-lac promoter. P135-1 was digested with BglII and BamHI and treated with bacterial alkaline phosphatase.



   Plasmid cBM11 / PAK / EGF was digested with PvuII and BamHI and the fragment of about 230 bp encoding part of the alkaline phosphatase and human EGF signaling sequence was isolated.

  <Desc / Clms Page number 91>

 



  2. Preparation of the TacPakl and TacPak2 oligonucleotides.



   The synthetic oligonucleotides TacPakl and TacPak2 were developed with an overhang compatible with the BglII site of pl35-1 and a PvuII overhang compatible with the PvuII site in the PvuII / BamHI fragment of alkaline phosphatase / EGF. The oligonucleotides were synthesized in an apparatus for the synthesis of Applied Biosystems oligonucleotides.
 EMI91.1
 



  BglII Pvul i TacPakl 5'GATCTATGAAACAATCTACGAT 3 'TacPak2 3'ATACTTTGTTAGATGC 5' 3. Binding and isolation of the TacPak / EGF clone.



  Pl35-l digested with BglII-BamHI, the PAK / EGF fragment of 230 bp and the oligonucleotides TacPakl and TacPak2 were assembled using DNA ligase, then the product was transformed into competent HB101 cells and isolated. a product constructed correctly by DNA sequencing.



  (HindIII site of pDR540)
 EMI91.2
 I AAGCTTACTCCC trp-35 (16pb) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG) mRNA 5'Site fix. lacI lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC (AGGA) AACAGGATCACTA

  <Desc / Clms Page number 92>

 
PvuI croSD (llpb) BglII (AGGA) GGTTCAGATCT Seq.

   of sign.-> ATGAAACAATCTACGATCGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGA MKQSTIALALLPLLFTPVT
EGF-> CAAAAGCTAATTCTGACTCTGAATGCCCGCTGTCTCATGACGGCTACTGCCTGCA
K A N S D S E C P L S H D G Y C L H Nsil SphI TGACGGCGTATGCATGTACATCGAAGCTCTGGACAAGTACGCATGCAACTGCGTT
D G V C M Y I E A L D K Y A C N C V GTTGGCTACATCGGCGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC VGYIGERCQYRDLKWWELR
BamHI GTTAAGGATCC * EXAMPLE VIII Expression of a polypeptide of interest as a fusion protein with the alkaline phosphatase signaling sequence using an expression cassette containing a transcription termination region.
 EMI92.1
 



  A. Preparation of pTCPt / EGF Ç trp-35 16 bpC lac-10 Ç'IacSD Jll bp CATG / alkaline phosphatase signaling sequence / human EGF / term. trans.-NEO).



   This plasmid is designed to contain the elements of the tac promoter and to use the SD of cro aux

  <Desc / Clms Page number 93>

 for the purpose of expressing human EGF behind the alkaline phosphatase signaling sequence. It includes a structural background of pBR322 with the neomycin resistance gene.



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



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



  2. Preparation of the EcoRI (franc) -BamHI fragment of 2.8 kb of pBM16t / NDP / VGFa.



   PBM16t / NDP / VGFa was digested with EcoRI, then treated with the Klenow fragment to
 EMI93.1
 create blunt ends. The DNA was then digested with BamHI and the 2.8 kb fragment was isolated. This DNA fragment contains the origin of pBR322, the neomycin resistance gene from which the NcoI site has been deleted and the transcription terminator analogous to gene 32 downstream of the BamHI site.



  3. Ligation and isolation of pTCPt / EGF.



   The 2.8 kb EcoRI (franc) -BamHI fragment from pBM16t / NDP / VGFa was ligated with the HindIII (franc) -BamHI fragment from TacPak / EGF and the resulting DNA was used to transform JM109 cells. competent (lacIq). A correct construct was isolated by virtue of its resistance to neomycin and by DNA sequencing.

  <Desc / Clms Page number 94>

 (HindIII site of pDR540)
 EMI94.1
 I
AAGCTTACTCCC trp-35 (16pb) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG) mRNA 5'Site fix. lake! lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC (AGGA) AACAGGATCACTA PvuI croSD (llpb) BglII Seq.

   of sign.-> (AGGA) GGTTCAGATCT ATGAAACAATCTACGATCGCCCTCGCACTTCTCC
M K Q S T I A L A L L P
EGF-> CACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGTC
L L F T P V T K A N S D S E C P L S Nsil TCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGAC
H D G Y C L H D G V C M Y I E A L D
SphI AAGTACGCATGCAACTGCGTTGTTGGCTACATCGGC K Y A C N C V V G Y I G
BamHI GAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGA ERCQYRDLKWWELR *
Term. trans.



  CTAATTGGGGACCCTAGAGGTCCCCTTTTTTATTTTAAAACGATC

  <Desc / Clms Page number 95>

 B. Preparation of pTCPt / NVGFa u: trp-35 J 16 bp
 EMI95.1
 Ç lac-lOÇlacSD croSD 11 bpATGJ / alkaline phosphatase signaling sequence / N-terminal VGFa with GTC and GYACRC sequence) / term. trans.-NEO).



   This plasmid comprises the elements of the tac promoter and uses the cro SD to express the modified VGF gene with the N-terminal extension downstream of the alkaline phosphatase signaling sequence. This plasmid comprises a structural background of pBR322 with the neomycin resistance gene.



  1. Preparation of the 350 bp PvuI-BamHI fragment of pBM11 / PAK / nVGFa.



   Plasmid pBM11 / PAK / nVGFa was put to digest with PvuI and BamHI and by gel electrophoresis, the 350 bp fragment was isolated. This fragment contains most of the alkaline phosphatase signaling sequence and the nVGFa gene.



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



   Plasmid pTCPt / EGF was digested with PvuI and BamHI and the 2.8 kb fragment was isolated by gel electrophoresis.



  3. Ligation and isolation of pTCPt / nVGFa.



   The 2.8 kb fragment and the 350 bp fragment were ligated using DNA ligase and the DNA was used to transform competent JM109 cells (lacIq). By restriction analysis, a correct constructed product was isolated.

  <Desc / Clms Page number 96>

 



   (HindIII site of pDR540)
AAGCTTACTCCC trp-35 (16pb) lac-10 CATCCCCCTG [TTGACA] ATTAATCATCGGCTCG (TATAATG) mRNA 5'Site fix. lacI lacSD TGTGG / AATTGTG AGCGGATAACAATTTCACAC {AGGA} AACAGGATCACTA PvuI croSD (llpb) BglII Seq. de sign.-> {AGGA} GGTTCAGATCT ATGAAACAATCTACGATCGCCCTCGCACTTCTCCC M K Q S T I A L A L L P nVGF-> ACTGCTGTTCACTCCAGTGACAAAAGCTGACTCTGGTAACGCTATCGAAACTACT
L L F T P V T K A D S G N A I E T T TCTCCGGAAATCACTAACGCTACTACTGACATCCCGGCTATCCGTCTGTGCGGCC S P E I T N A T T D I P A I R L C G P KpnI CGGAAGGCGACGGCTACTGCCTGCATGGTACCTGATGATGATGATGATGATGATGATCCATCC
SphI EcoRI CGGCTACGCATGCCGTTGCTCTCATGGCTACACTGGAATTCGTTGCCAGCATGTT
G Y A C R C S H G Y T G I R C Q H V
BamHI Term.



  GTTCTGGTCGACTACCAGCGTTAAGGATCCGTGACTAATTGGGGACCCTAGAGGT
V L V D Y Q R * Trans.



  CCCCTTTTTTATTTTAAAACGATC

  <Desc / Clms Page number 97>

 C. Preparation of pTNPt / EGF (Çtrp-35 J 17 bp 1
 EMI97.1
 lac-10 nSD 0 lac-10 3 nSD 8 bp r, ATG 2 / alkaline phosphatase signaling sequence / human EGF / term. trans.-NEO).



   This plasmid is designed to possess the elements of the tac promoter and use the SD gene N to express human EGF behind the alkaline phosphatase signaling sequence. It includes a structural background of pBR322 with the neomycin resistance gene.



  1. Preparation of the PvuI-BamHI fragment of 2.8 kb of pTNPt.



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



  2. Preparation of the 300 bp PvuI-BamHI fragment of pBMI1 / PAK / EGF.



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



  3. Liaison and isolation of pTNPt / EGF.



   The 2.8 kb fragment and the 300 bp fragment were ligated using DNA ligase and the DNA was transformed into JM109 (LacIq). A correct construct was isolated by restriction analysis and DNA sequencing.
 EMI97.2
 



  (EcoRI site of pBMll) 1 GAATTACTCCCCATCC SstI trp-35 (17pb) lac-10 5'lac CCCTG [TTGACA] ATTAATCATCGAGCTCG (TATAATG) TGTGG / AATTG

  <Desc / Clms Page number 98>

   BsmI mRNA-> n mRNA-> TGTGAGCGGATAACAATTTCACACAGCATTCAAAGCAGAAGGCTTTGGGGTGTGT GATACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGATTAGCTGCCAATGTG CAGCACGGACACGACACGAC

   of sign.-> CTGAC (AGGA) GAATCCAG ATGAAACAATCTACGATCGCCCTCGCACTTCTC
M K Q S T I A L A L L
EGF-> CCACTGCTGTTCACTCCAGTGACAAAAGCTAATTCTGACTCTGAATGCCCGCTGT P L L F T P V T K A N S D S E C P L S
NsiI CTCATGACGGCTACTGCCTGCATGACGGCGTATGCATGTACATCGAAGCTCTGGA
H D G Y C L H D G V C M Y I E A L D
SphI CAAGTACGCATGCAACTGCGTTGTTGGCTACATCGGCGAACGTTGCCAGTACCGT
K Y A C N C V V G Y I G E R C Q Y R BamHI BamHI GACCTGAAATGGTGGGAACTGCGTTAAGGATCCGTGACTAATTGGGGA D L K W W E L R * (BamHI site of pBMll) Term. trans. 1 CCCTAGAGGTCCCCTTTTTTATTTTAAAACGATCC

  <Desc / Clms Page number 99>

 EXAMPLE IX Assembly of growth factor genes for expression in eukaryotic cells.



  A. Preparation of the plasmid pRSV / VGF.



   Transfection of this plasmid into Chinese hamster ovary cells led to the production of natural VGF.



  (a) Preparation of the HindIII (franc) -BglII (franc) fragment of pRSV.



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



  (b) Preparation of a DdeI fragment containing the VGF gene.



   A genome fragment subcloned from vaccinia DNA was subjected to digestion with DdeI, the projecting ends were blunted with the Klenow fragment and a 550 bp DdeI fragment was gel purified.



  (c) Ligation and isolation of pRSV / VGF.



   The HindIII (franc) -BglII (franc) fragment of 4 bb of pRSV and the DdeI fragment (franc) of 550 bp of vaccinia DNA were ligated with DNA ligase, and then selected the transformants on ampicillin and sorted by restriction analysis. A correct construct, pRSV / VGF, was isolated.



  (d) Transfection of CHO cells with pRSV / VGF.



   The plasmid pRSV / VGF was cotransfected with a plasmid pRSV by calcium phosphate precipitation in Chinese hamster ovary (CHO) cells.



  The transfectants were sorted by Southern hybridization

  <Desc / Clms Page number 100>

 point blot and a positive clone noted pRSV / VGF52 was isolated.



    B. Preparation of the plasmid pAc / VGF.



   An additional expression system which is used for the recombinant VGF protein is an insect system. See Maeda et al. and Carbonell et al., above. In such a system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus multiplies in Spodoptera frugiperda cells. The envelope gene can be cloned into nonessential regions (eg, the polyhedrin gene) of the virus and is brought under the control of an AcNPV promoter (eg, the polyhedrine promoter).

   Effective insertion of the constructed product of the VGF gene results in the inactivation of the polyhedrin gene and in the production of non-occluded recombinant virus (that is to say the virus lacking the protein envelope encoded by the polyhedrine gene) . This recombinant virus is then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.



   The VGF gene of interest is brought under the control of an appropriate promoter for an insect cell system. Plasmid pAc610 contains the polyhedrin gene cloned into a vector plasmid having an ampicillin resistance marker. In this gene are inserted polylinker segments which are 50 bases downstream from the site of transcription start of the polyhedrin gene and 7 bases before the first ATG codon. The VGF gene is cloned into an appropriate polylinker segment site, so that it is under the control of the polyhedrin promoter. The initiation codon ATG methionine and the end codons

  <Desc / Clms Page number 101>

 translation are those of the VGF gene; the transcription start sites and the polyadenylation signals are those of the polyhedrin gene.



  (a) Preparation of SmaI-BamHI pAc610.



   Plasmid pAc610 was put to digest with SmaI and BamHI.



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



   Plasmid pRSV / VGF was digested with HindIII and the projecting ends were blunted using the Klenow fragment. The DNA was then digested with BglII and the 560 bp fragment was purified on a gel.



  (c) Ligation and isolation of pAc / VGF.



   The SmaIBamHI fragment from pAc610 and the 560 bp fragment from pRSV / VGF containing the VGF gene were ligated using DNA ligase and the transformants were sorted by restriction analysis. A correct constructed product denoted pAc / VGF was isolated.



  (d) Transfection of Sf9 cells with pAc / VGF.



   Plasmid pAc / VGF was cotransfected with wild-type baculovirus DNA AcNPV in Sf9 insect cells (Spodoptera frugiperda). Transfectants were sorted by occlusion of the negative phenotype in agar assays. A negative occlusion plate was isolated and after five successive rounds of plate purification, a high titer recombinant virus stock was prepared.



  C. Preparation of pAc / SFGF.



   Expression of the growth factor of the natural Shope fibroma virus in a baculovirus.



  (a) Preparation of pAc610 digested with BamHI.



   Plasmid pAc610 was put to digest with BamHI and SmaI. The SmaI site in this plasmid is located downstream of the promoter of the polyhedrin gene of

  <Desc / Clms Page number 102>

 baculovirus.



  (b) Preparation of a 430 bp HincII-Sau3a fragment of the genomic DNA of the Shope fibroma virus.



   The 3.7 kb BglII fragment was cloned from the 19 kb BamHI C fragment of Shope fibroma virus genomic DNA into a plasmid SP64 digested with BamHI and it was noted SP64 / 3.7. Plasmid SP64 / 3.7 was digested with HincII and the 1 kb HincII fragment was gel purified. The 1 kb fragment was then digested with Sau3a and the 430 bp HincII-Sau3a fragment was gel purified.



  (c) Ligation and isolation of pAc / SFGF.



   PAc610 digested with BamHI-SmaI and the 430 bp HincII-Sau3a fragment containing the SFGF gene was ligated, then the resulting mixture was transformed into competent E. coli HB101. The transformants were sorted by restriction analysis and a correct constructed product denoted pAc / SFGF was isolated.



  EXAMPLE X Preparation of polypeptides.



  A. Solid phase synthesis of VGF and TGF.



  1. The fragment of the synthetic VGF.



   According to the usual protocol, the sequence corresponding to the truncated VGF, which begins with the sequence DIPAIR and ends with the sequence LVDY, was assembled on an apparatus for the synthesis of Applied Biosystems Model 430A peptides. Treatment of the resin-peptide in the final stage with liquid HF under normal "weak-strong" conditions gave the crude deprotected peptide. The peptide was subjected to chromatofocusing on PBE94 (Pharmacia). The partially purified peptide was eluted at pH 6.5. Brief treatment with 0.2N sodium hydroxide removed the protective radicals from cysteine (ethylcarbamoyl) and the peptide was oxidized in the presence of glutathione

  <Desc / Clms Page number 103>

 oxidized and reduced.

   Purification by gel filtration and HPLC gave highly purified VGF having the
 EMI103.1
 0 next sequence.



  DIPAIRLCGPEGDGYCLHGDCIHARDIDGMYCRCSHGYTGIRCQHVVLVDY 2. rGF-1. human and synthetic rat.



   Human and rat TGF-OL were synthesized essentially as described above with respect to VGF and were also acquired from Penninsula Labs.



  B. Isolation of recombinant polypeptides prepared in prokaryotic cells.



  1. The growth factors produced from pBM-based vectors by means of the PL promoter and the CI repressor of ts.



   E. coli B (HB101) containing the plasmids pBM11 / NDP / growth factor was cultured in Luria broth at 30 "C. The density of the culture was measured at 550 nm and at the moment when the density reached a absorbance from 0.7 to 0.9, the synthesis of the growth factor fusion protein was induced by raising the temperature to 42 C. The culture was incubated at this temperature for 5 to 20 hours, then isolated the bacteria by centrifugation and were frozen at -70 ° C until needed.



   For isolation of the recombinant protein, the cells were thawed in a buffer containing 0.05 M NaH2PO4 pH 7.2, 0.5 M NaCl and 0.01 M EDTA. 150 were used. ml of buffer for a preparation from 50 g of bacteria. The cells were ruptured by exposure to ultrasound on ice for 15 minutes with a 6.4 mm probe at 50% pulses and a power of 60 W. After the cells were ruptured, the insoluble proteins were collected.

  <Desc / Clms Page number 104>

 by centrifugation at 12,000 rpm in a GSA rotor for 90 minutes. The pellet containing the insoluble proteins was then resuspended in 50 ml of 6 M guanidine hydrochloride. The insoluble fraction was collected by centrifugation for 2 hours at 25,000 rpm in a Beckman type 30 ultracentrifugation rotor.

   The supernatant was collected and stored at -20 ° C until needed.



   The purification of the fusion protein was carried out on a column of Sephacryl S300 or of Fractogel HW-55 brought to equilibrium with guanidine hydrochloride 1 M. The fractions containing the fusion protein were identified as those containing a polypeptide of a molecular weight compatible with the molecular weight of the polypeptide encoded by the synthetic gene, as determined on a 15% polyacrylamide-urea gel.



   To obtain an active form of the recombinant growth factor, the fusion protein was allowed to fold by incubating in 50 mM TrisHCl buffer pH 8.7 containing 1 M guanidine hydrochloride, reduced glutathione 1.25 mM and 0.25 mM oxidized glutathione at 40C for 3 to 10 days. The biological activity of the growth factor was monitored by an assay for competitive binding to the receptor, as described above (see Example I. C). When maximum activity was reached, the protein was dialyzed against distilled water and lyophilized to dryness.



   When it was desirable to eliminate the signal sequence, the protein was cut either by resuspending it in 70% formic acid and incubating it at 400C for 3 days, or by incubating it until overnight until the temperature

  <Desc / Clms Page number 105>

 ambient in a 100-fold molar excess of cyanogen bromide. The cleavage product was dialyzed against distilled water and lyophilized to dryness.



   To further purify the recombinant growth factor, it was resuspended in 40% acetonitrile with 0.1% trifluoroacetic acid and purified by HPLC on a TSK-250 BioRad column. The growth factor containing fractions were pooled and further purified by reverse phase HPLC either on jBondapak C-18 Waters or on Dynamax C-8 Rainin. The eluent formed a linear gradient of 20 to 40% acetonitrile containing 0.1% trifluoroacetic acid. The fractions containing binding activity on the receptor were pooled, then lyophilized and stored at -20 ° C until used.



  (a) TGF and modified TGF.



  (i) N / TGF.



   Using plasmid pBM11 / N / TGF, recombinant modified human TGF containing 33 amino acids of the N-terminus N gene and the modified QEEK sequence were produced instead of the natural human sequence QEDK.



  (b) The modified and truncated VGF.



  (i) PAD / nVGFa.



   Recombinant modified VGF was produced using the plasmid pBM11 / PAD / nVGFa containing the extreme N-terminal sequence of VGF and the modified sequences GTC and GYACRC instead of the sequences of the natural VGF GDC and GMYCRC. The nVGFA fragment was expressed as a fusion protein with an alkaline phosphatase signaling sequence modified at the N-terminus and truncated to the YQR sequence at the C-terminus.

  <Desc / Clms Page number 106>

 



  (ii) NDP / VGFa.



   We produced using the plasmid
 EMI106.1
 pBM11 / NDP / VGFa of the modified recombinant VGF starting at e has the sequence DIPAIR and ending at the sequence YKQR in the VGF. It includes the modified GTC and GYACRC sequences instead of the natural GDC and GMYCRC sequences of VGF. The VGFα fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N terminus and the dipeptide aspartic acid-proline labile in acid medium.



  (iii) VGFa.



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



  (iv) NDP / VGFA.



   Using the plasmid pBM11 / NDP / VGFA, recombinant modified VGF was produced starting at the sequence DIPAIR and ending at the sequence YKQR in VGF and comprising the modified GTC and GYACVC sequences instead of the natural GDC and GMYCRC sequences of VGF . The VGFA fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N end and the dipeptide aspartic acid-proline labile in acid medium.



  (c) The chimeric TGF / VGF hybrids.



  (i) N / TTV (TGF / TGF / VGF).



   By means of pBM11 / N / TTV, recombinant modified TTV containing the amino acid sequence of human TGF was produced in the aminoterminal two-thirds of the gene with the exception of the sequence QEEK which was modified relative to the sequence Natural human QEDK. The carboxyl terminus originated from the amino acid sequence of VGF and ended with the sequence YQR upstream of the natural sequence PNT.

  <Desc / Clms Page number 107>

 



  The TTV fragment was expressed as a fusion protein with 33 amino acids of the N gene at the N terminus.



  (ii) NDP / TTV.



   Using plasmid PBM11 / N / TTV, modified recombinant TTV was produced as described in (a), except that the TTV fragment was expressed as a fusion protein with 32 amino acids of the N gene at the end N and the dipeptide aspartic acid-proline labile in an acid medium.



  (iii) NDP / VTV.



   Using the plasmid pBM11 / NDP / VTV, recombinant modified VTV containing the amino acid sequence of human TGF in the middle domain was produced with the amino acid sequence QEEK replacing the natural sequence QEDK. The N-terminal domain and the C-terminal domain originated from the truncated VGF sequence, starting with the DIPAIR sequence and ending with the YQR sequence. The VTV fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N end and the dipeptide aspartic acid-proline labile in acid medium.



  (iv) NDP / TVV.



   Using the plasmid pBMI1 / NDP / TW, recombinant modified VTV was produced containing the amino acid sequence of human TGF in the N-terminal domain of the gene. The middle domain and the Cterminal domain originated from the truncated VGF sequence, ending with the YQR sequence. In addition, the synthetic gene includes the modification GYACVC instead of GMYCRC. The TW fragment was expressed as a fusion protein with 32 amino acids of the N gene at the N end and the dipeptide aspartic acid-proline labile in an acid medium.

  <Desc / Clms Page number 108>

 



  * 2. Growth factors produced in vectors including the tac or lac promoters.



   Bacterial hosts containing expression cassettes which include tac or lac promoters from 30 to 370C up to an A600 optical density of 0.2 to 0.8 have been grown in LB broth or in a chemically defined medium, such as M9 medium supplemented with thiamine and glucose. An appropriate antibiotic was added to the growth medium to select hosts containing the expression cassette. The bacterial cultures were induced with a concentration of 100 to 1000 mM in IPTG and the culture was carried out at 30 ° C. for 16 to 24 hours. For expression cassettes free of the lacI gene, the bacterial hosts carried a factor F with the lacqI gene, such as JM109, XL1, JM103, etc.

   For expression cassettes carrying the lacI gene, examples of bacterial hosts are HB101, DH1, DH5, etc. In the case where the bacterial host comprises a functional lac operon (lac +), the expression cassette can be induced by 1% of lactose. After induction, growth factors can be isolated either from the medium or from the collected cells by centrifugation.



  (a) PAK / EGF.



   Human EGF produced by the TacPal / EGF and pTcCPt / EGF expression cassettes was isolated from the medium in an active form free of the alkaline phosphatase signaling sequence. About 85% of the active EGF was found in the medium, the rest being associated with the cell pellet. These expression cassettes produced 4 mg of active EGF per liter. The cells were separated from the medium by centrifugation, then the medium was passed through an Amicon SY30 spiral filter, cutting for a Mr of 30,000, and then in a Q-Sepharose column,

  <Desc / Clms Page number 109>

 after which the purified human EGF was eluted in 20 mM NaH2PO4 of pH 7 with an NaCl gradient from 0 to 0.5 M.

   Alternatively, the growth factors of the cell pellet can be isolated by osmotic shock or exposure to ultrasound, and then purified substantially as described above.



  (b) PAK / nVGFa.



   Using the pTCPt / nVGFa expression cassette, recombinant nVGFa was produced. By exposure to ultrasound, nNGFa was isolated from the cell pellet, which was found to constitute approximately 40% of the total bacterial protein.



  C. Isolation of VGF and SFGF produced by expression of natural genes in eukaryotes.



  1. Preparation of VGF in monkey kidney cells.



   Natural VGF was produced by infection of monkey kidney cells with vaccinia virus. We infected cell monolayers
 EMI109.1
 Cercopithecal kidney (BSC-1) with 15 plaque-forming units (pfu) per purified W cell (WR strain grown in Hela cells and purified by sucrose density gradient sedimentation (Moss, B., (1981) in Gene Amplification and Analysis, editors Chirickjian, JG and Papis, TS



    (Elsevier / North-Holland, NY), volume 2, pages 253-266)). The infected cells were incubated at 37 GC with approximately 1 ml of Eagle's base medium supplemented with 2% fetal calf serum per 2 × 106 cells. Dummy infected cells were treated identically. The supernatants of the cell cultures were clarified by low speed centrifugation and lyophilized. The residue was then resuspended in IN acetic acid and dialyzed extensively against 0.2M acetic acid. We have

  <Desc / Clms Page number 110>

 
 EMI110.1
 The insoluble material was separated by centrifugation and the supernatant was lyophilized, then resuspended in one hundredth of its original volume of 1M acetic acid and stored at 4OC.



  2. Preparation of VGF in CHO cells.



   Natural VGF was also produced by transfection of the pRSV / VGF plasmid containing the VGF gene fragment into Chinese hamster ovary cells. The transfected cells were made to multiply and the VGF was sought in the medium and the cell pellet by immunoprecipitation using an antiserum against the N-terminal peptide of the VGF.



  3. Preparation of VGF in the silkworm with a baculovirus promoter.



  (a) Transformation.



   The host cell for AcNPV is Spodoptera frugiperda (sf9). To produce a recombinant virus stock, the plasmid containing VGF is mixed with DNA from AcNPV and the transfection is carried out in sf9 cells according to the calcium phosphate technique. The recombinant viruses are isolated from the medium and purified on a plate with sf9 cells. Recombinant plaques are identified by hybridization using radiolabeled VGF DNA as a probe.



  (b) Expression.



   After identifying the recombinant virus, it was multiplied on sf9 cells. The recombinant virus stock was then used to infect sf cells, then the cells were lysed and the supernatants were searched for the produced VGF protein which was purified as described above with regard to the mammalian cells infected with the virus. vaccine.



   The expected sequence of VGF produced in insect cells is as follows:

  <Desc / Clms Page number 111>

 
DSGNAIETTSPEITNATTDIPAIRLCGP
EGDGYCLHGDCIHARDIDGMYCRCSHGYTGIR
CQHVVLMYQRSENPNTTTSYIPSPGIMLVLVGI
IIITCCLLSVYRFTRRTKLPIQDMVVP A possible glycosylation site appears to asparagine at position 15 from the Nterminal end of the polypeptide.



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



   The expected molecular weight of the 121 residue peptide is 13,304, excluding carbohydrates. The apparent molecular weight of the VGF produced by the baculovirus is 17,000, and therefore appreciably lower than that to which the cells infected with VV lead, which indicates that the two forms were probably treated differently. The VGF produced by baculovirus may be free of an additional fraction of the N-terminal sequence, or part of the C-terminal sequence, or both, and / or may differ in the type and extent of glycosylation.



  4. Preparation of SFGF in the silkworm using a baculovirus promoter.



  (a) Transfection of Sf9 cells with pAc / SFGF.



   Plasmid pAc / SFGF was cotransfected with wild-type baculovirus DNA AcNPV in Sf9 insect cells (Spodoptera frugiperda). The transfectants were sorted after occlusion of the phenotype

  <Desc / Clms Page number 112>

 negative in agar assays. A negative occlusion plate was isolated and after five successive rounds of plate purification, a stock of high titer recombinant virus was prepared. The recombinant virus was revealed in the Southern analysis to contain the SFGF gene.



  D. natural EGF.



  1. The natural EGF was isolated from the salivary glands of the mouse or it was acquired from Collaborative Research.



  EXAMPLE XI.- Activity tests.



  A. Mitogenic test.



   The mitogenesis tests were performed as follows: diploid human fibroblasts from explants of newborn foreskin were subcultured at a density of 3 × 104 cells per well (96 well plates, Nunclon, Roskilde, Denmark) and they were cultivated until confluence in Eagle medium modified according to Dulbecco (GIBCO) / 10% of newborn calf serum. The cultures were then introduced into a medium containing 0.2% of newborn calf serum and two days later, the EGF (10 ng per ml) or the growth factor to be tested (10 ng d was added). '' EGF equivalents per ml).

   After 8 hours, cultures were labeled with 5-C 125] iodo-2'-deoxy-
 EMI112.1
 i uridine (Amersham, $ 10 Ci per ml, 5 Ci per mg; 1 Ci = 37 GBq) and the amount of the isotope incorporated in the fraction insoluble in trichloroacetic acid was determined, as described elsewhere (Twardzik and al., Proc. Natl. Acad. Sci. USA (1985) 182: 5300-5304).



  B. Stimulation test for the growth of colonies on soft agar.



   0.5 ml of base coat was added.

  <Desc / Clms Page number 113>

 0.5% agar (Agar Noble; Difco Laboratories, Detroit, Michigan) in growth medium in 24-cup Costar tissue culture plates. 0.5 ml of 0.3% agar in growth medium containing 1 to 1.5 × 10 4 cells per ml of NRK cells or another cell line of interest was applied over the agar base layer. various concentrations of the factor to be examined. The plates were incubated at 370C in a humid atmosphere at 5% CO 2 in air and they were re-fed after 7 days by the addition of 0.5 ml of 0.3% agar in growth medium containing the same growth factor concentrations to be examined. Unfixed and uncolored colonies were counted.

   The number of colonies with more than 6 cells was noted.



  C. Inhibition test of binding to the EGF receptor.



   The radiolabelled receptor assays were carried out in the following manner: the binding of the 125I-labeled EGF (125I-EGF) to its receptor on A431 cell monolayers was modified compared to the procedure described by Cohen and Carpenter, Proc. Natl. Acad. Self. USA (1975) 72: 1317-1321. Cells (1x103 per well) were fixed on 24 well plates (Linbro, Flow Laboratories) with 10% formaldehyde in phosphate buffered saline before testing . Cells attached to formalin do not detach from the plates as easily as non-attached cells and the values measured repeatedly are thus more reproducible.

   Under these operating conditions, the 125I-EGF (lux1010 counts per minute per nanomole) saturates
 EMI113.1
 0 the fixation test at 3 nM; the tests were performed at 10% of the saturation value. Growth factor concentrations are expressed

  <Desc / Clms Page number 114>

 in nanograms of EGF equivalents per ml, that is to say by the quantity required to produce an inhibition of the binding of 125I-EGF which is equivalent to that produced by a known quantity of EGF.



  D. Radioimmunoassay.



   Each aliquot of 50 pitres of reaction medium contains: 20 mM sodium phosphate
 EMI114.1
 pH 7, 4, 200 mM NaCl, 40 mM dithiothreitol, 0.1% beef serum albumin, 0.1% Nana, 125 I-labeled peptide (2 x 04 shots per minute) corresponding to the 17 residues 1 'carboxy-terminals <TGF (Linsley et al., Proc. Natl. Acad. Soi. USA (1985) 82: 356-369); of the antiserum at a final dilution of 1: 5000 and the other specified additions. The reaction was started by addition of antiserum and the operation was continued at 230C for 90 minutes.

   An equal volume of 10% formalin fixed S. aureus (Pansorbin, Calbiochem) was then added, followed by further incubation for another 30 minutes at 23 "C. The immunoadsorbent was separated by sedimentation, followed by the quantity of fixed peptide labeled with 125I was measured The quantity of fixed peptide, corrected for non-specific binding measured in the absence of antibodies (less than 5% of the total), is expressed as a percentage of the maximum binding.



  E. Wound healing.



  1. Thermal lesion in the middle layer of the dermis.



   Thermal lesions were caused in the middle layer of the dermis on the dorsal part of the thorax of anesthetized Yorkshire sows (15 kg), the back of which was shaved and the hair removed with a commercial depilatory cream. A brass block (3 cm x 3 cm, 147 g) was brought to equilibrium in a 700C water bath and placed in firm contact with the skin for exactly 10 seconds. We then removed

  <Desc / Clms Page number 115>

 the resulting bulb. Five burns were made in the average thickness of the dermis on each side of the spine, at a distance of about 25 m from each other. The burns were treated twice daily with approximately 3 ml of a creamy excipient (Silvadene R), either as it was or with growth factor added, while others were left untreated.

   After 9 or 10 days of treatment with natural VGF, the sows were anesthetized and the pressure ulcer detached from the burns. Biopsies were taken from each burn in areas where the epithelium had reformed.



  2. Lesion by donor graft in the middle layer of the dermis.



   Dwarf pigs were anesthetized with 20 mg per kg of ketamine and 2 mg per kg of Rompum
 EMI115.1
 aged 5 less than a weight of 20, 5 kg. After shaving the thorax in the dorsal part, it was brushed with betadine and rinsed thoroughly with physiological saline solution. A series of 6 donor sites of 5 cm × 5 cm was produced on each flank of the dorsal part of the thorax by means of a Padgett dermatome at 1.52 mm by taking two passes at 0.76 mm. The topical treatment was made with 1 ml of physiological saline solution in 20 g of Silvadene in uniform distribution between the six wounds on the left flank. The right side was treated with 1 ml of the growth factor to be examined in 20 g of Silvadene, uniformly distributed among the six wounds. Each wound was covered with a large burn dressing.

   The animal was anesthetized, as described above, on days 1, 2.3, 4.7, 8.9, 10 and 11 after the procedure. We removed the bandages. The wounds were then carefully brushed with betadine and rinsed thoroughly with physiological saline.

  <Desc / Clms Page number 116>

 



  The appropriate agent was applied and the wounds covered again, as previously described.



  EXAMPLE XI.



  Biological activity of recombinant growth factors prepared in prokaryotic cells.



  A. Attachment to the EGF receptor.



   The present test makes it possible to determine the ability of a molecule to bind to the EGF receptor by measuring its ability to prevent EGF itself from binding to this receptor. All growth factors and hybrid growth factors, modified or truncated, isolated to date have been shown to be active in the test for inhibition of binding to the EGF receptor. A summary of the results is given below.



   BOARD
Attachment of recombinant growth factors to the EGF receptor.
 EMI116.1
 
 <tb>
 <tb>



  Peptide <SEP> Cassette <SEP> Purity <SEP> Fixation
 expression <tb> <SEP> on <SEP> on
 <tb> receiver
 <tb> of <SEP> EGF
 <tb> N / TGF-protein
 <tb> of <SEP> fusion <SEP> truncated
 <tb> modified <SEP> pBMIl / N / TGF <SEP> 95% <SEP> Yes
 <tb> PAD / nVGFa-protein
 <tb> of <SEP> fusion <SEP> truncated
 <tb> modified <SEP> pBMll / PAD / nVGFa <SEP> 95% <SEP> Yes
 <tb> NDP / VGFa-protein
 <tb> of <SEP> fusion <SEP> truncated
 <tb> modified <SEP> pBMll / NDP / VGFa <SEP> 95% <SEP> Yes
 <tb> NDP / VGFA-protein
 <tb> of <SEP> fusion <SEP> truncated
 <tb> modified <SEP> pBMll / NDP / VGFA <SEP> 95% <SEP> Yes
 <tb>
 

  <Desc / Clms Page number 117>

 
 EMI117.1
 
 <tb>
 <tb> N / TTV-protein
 <tb> of <SEP> fusion <SEP> hybrid
 <tb> truncated <SEP> modified <SEP> pBMll / N / TTV <SEP> 95% <SEP> Yes
 <tb> NDP / TTV-protein
 <tb> of <SEP> fusion <SEP> hybrid
 <tb> truncated <SEP> modified <SEP> pBMll / NDP / TTV <SEP> 95%

   <SEP> Yes
 <tb> NDP / VTV-protein
 <tb> of <SEP> fusion <SEP> hybrid
 <tb> truncated <SEP> modified <SEP> pBMll / NDP / VTV <SEP> 95% <SEP> Yes
 <tb> NDP / TVV-protein
 <tb> of <SEP> fusion <SEP> hybrid
 <tb> truncated <SEP> modified <SEP> pBMIl / NDP / TVV <SEP> 95% <SEP> Yes
 <tb> PAK / EGF <SEP> pBMll / PAK / EGF <SEP> 95% <SEP> Yes
 <tb> EGF <SEP> pBMll / PAK / EGF <SEP> 95% <SEP> Yes
 <tb> PAD / EGF <SEP> pBMIl / PAD / EGF <SEP> 95% <SEP> Yes
 <tb> EGF <SEP> pBMll / PAD / EGF <SEP> 95% <SEP> Yes
 <tb> PAK / EGF <SEP> TacPak / EGF <SEP> 95% <SEP> Yes
 <tb> EGF <SEP> TacPak / EGF <SEP> 95% <SEP> Yes
 <tb> PAK / EGF <SEP> pTCPt / EGF <SEP> 95% <SEP> Yes
 <tb> EGF <SEP> pTCPt / EGF <SEP> 95% <SEP> Yes
 <tb>
 
A comparison of the binding inhibition curves for natural mouse EGF and the recombinant hybrid growth factor N / TTV expressed in a bacterium (fusion polypeptide of 32

  N-terminal amino acids of the lambda N gene and modified truncated hybrid TGF / VGF) suggests that there is no difference in binding activity.



  B. Mitogenic activity.



   The activity of various purified growth factors was determined, which was in all cases comparable to that of EGF. The envisaged compounds are indicated below.

  <Desc / Clms Page number 118>

 



   TABLE Mitogenic activity of growth factors
 EMI118.1
 
 <tb>
 <tb> Peptide <SEP> Activity <SEP> mitogenic *
 <tb> TTV-hybrid <SEP> truncated <SEP> modified <SEP> Yes
 <tb> N / TTV-protein <SEP> from <SEP> fusion <SEP> hybrid
 <tb> truncated <SEP> modified <SEP> Yes
 <tb>
 * Measured by incorporation of 3H-thymidine or 125IIdu.



  C. Wound healing.



  1. Lesion in the middle layer of the dermis.



   The effect of TGF, EGF and natural or synthetic VGF, as well as that of recombinant growth factors on lesions of the middle layer of the dermis was evaluated, as described in example VE1. By computer-assisted telemetry, the healed fraction of the initial extent of the burn was measured in percent and the percentage of epithelial reconstruction on the burn was determined. The reconstitution of the epithelium was approximately 15% for untreated burns. Treatment with Silvadene alone or with Silvadene supplemented with EGF led to a reconstitution of approximately 50% of the epithelium, while treatment with synthetic TGF or natural VGF led to a reconstitution of approximately 90% of the epithelium.

   The most favorable concentration for reconstitution of the epithelium is 1-10 M, g per ml for EGF, but synthetic TGF and natural VGF give a maximum response at 0.1 kg per ml.



   Experiments similar to those described above were performed to assess the healing effect of TGF, a modified truncated form of VGF (VGFa), or a hybrid truncated fusion form

  <Desc / Clms Page number 119>

 modified TGF and VGF (TTV) all obtained by recombinant technology in bacteria. These recombinant growth factors and hybrid growth factors accelerated wound healing to the same extent as synthetic TGF or natural VGF, with an optimal concentration of 0.1 g per ml.



  2. Lesion by donor graft in the middle layer of the dermis.



   The suitability of the truncated modified VGFa was assessed
 EMI119.1
 to accelerate the healing of a wound in an access model with donor graft. This mode of treatment is that indicated above (see example I) with 1 ml of VGFa, at
 EMI119.2
 5 e 5 1R per ml, in 20 g of Silvadene. The wounds were photographed daily. The summary of the results is as follows.

  <Desc / Clms Page number 120>

 



   TABLE Effect of recombinant VGFa on wound healing.
 EMI120.1
 
 <tb>
 <tb>



  State <SEP> from <SEP> the <SEP> wound
 <tb> Processing * <SEP> JAI ** <SEP> 7 <SEP> JAI <SEP> 8 <SEP> JAI <SEP> 9 <SEP> JAI <SEP> 10
 <tb> Solution <SEP> Very <SEP> Open <SEP> Almost <SEP> Cicaphysiological <SEP> open <SEP> with <SEP> scar
 <tb> salty <SEP> some <SEP> sorted
 <tb> scarring
 <tb> VGF <SEP> a <SEP> - <SEP> Open <SEP>; <SEP> Almost <SEP> Cica-Cicatronqué <SEP> training <SEP> scar <SEP> sorted
 <tb> changed <SEP> apparent <SEP> sorted
 epithelium <tb>
 <tb>
 * The excipient is Silvadene.



  ** JAI = Day after the intervention.



   The modified truncated VGFa accelerates wound healing, compared with the control excipient. The photographs (not attached) to the 5th day after the intervention reveal significant differences between the physiological saline solution and the VGFa.



  EXAMPLE XXIII Biological activity of the VGF prepared in eukaryotic cells.



  A. VGF prepared in monkey kidney cells (BSC-1).



  1. In the medium originating from BSC-1 cells, 24 hours after infection with W, the presence of a material which could compete with 125I-EGF was sought for binding to cells of human epidermal carcinoma rich in EGF receptors (A431). The cells infected with W give off a powerful activity which competes with EGF, which activity is called VGF.

  <Desc / Clms Page number 121>

 Control medium from control cultures of dummy infected BSC-1 cells has minimal activity in competition with EGF.



   Monitoring of VGF production at the earliest envisaged time, two hours after infection, revealed increased levels of VGF in the culture medium. At 12 hours, maximum amounts of this activity were observed in the culture supernatant, the increase being only slight after 24 hours.



   The rate of production of VGF has been shown to be a function of the multiplicity of viral infection, as shown in the following table.



   BOARD
Effect of multiplicity of infection on the release of VGF.
 EMI121.1
 
 <tb>
 <tb>



  Multiplicity <SEP> VGF <SEP> cleared
 <tb> viral <SEP> ng <SEP> eq. EGSE <SEP> <SEP> by <SEP> ml
 <tb> upf <SEP> by <SEP> cell
 <tb> 0 <SEP> -
 <tb> 10 <SEP> 2.7
 <tb> 20 <SEP> 4.5
 <tb> 40 <SEP> 6, <SEP> 1
 <tb> 80 <SEP> 10.1
 <tb>
 BSC-1 cell cultures were infected at the indicated upf-cell ratio, and the supernatants were harvested (approximately 1 ml for 2 x 106 cells) 24 hours after infection. Each sample was acidified and lyophilized, then the radiolabelled EGF receptors were assayed in duplicate, as described elsewhere (Delarco and Todaro, Proc. Natl. Acad. Sci. USA (1978) 75: 401-405). (ng eq. = nanograms of equivalents).

  <Desc / Clms Page number 122>

 



  2. Partial purification of the VGF.



   Competitive activity with EGF found in W-infected BSC-1 cells was subjected to partial purification from the supernatants of the acid-extracted liquors, 24 hours after infection, as described above. The polypeptides solubilized in acid medium (10.5 mg) from the supernatants were deposited on a column of BioGel P10 brought to equilibrium with 1 M acetic acid and the competitive activity with EGF was sought in samples of all fractions. The major active peak (fraction 42) eluted with an apparent Mr of 25,000 shortly after carbonic anhydrase which is a marker of a Mr of 29,000.

   Molecular weight was confirmed using tandem-mounted Bio-Sil TSK 250 dimensional HPLC columns, where all activity eluted at a major peak in the protein marker region of a Mr of. 25,000. The VGF in an amount of 1 microgram is the equivalent of 90 nanograms of EGF in a competition test for the radiolabelled receptor.



  3. Further purification of the VGF.



   Combined fractions from the gel filtration column (Nos 25 to 35) were concentrated by vacuum centrifugation, then the solid was resuspended in 0.05% trifluoroacetic acid, the mixture clarified and it was injected into a 3.9 mm x 30 cm JLBondapak C18 column (Waters, Milford, MA). Peptides were eluted with a linear gradient of 20 to 60% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 1.0 ml per minute at 22 C. In aliquots of each fraction, measured the competitive activity of EGF for a radiolabelled receptor.

   The peptide corresponding to the peak activity was collected and diluted

  <Desc / Clms Page number 123>

 with 0.05% trifluoroacetic acid, then injected again into an LBondapak column which has been eluted under isocratic conditions. The acetonitrile concentration was about 22-25%.



   The table below gives a comparison of the rate of production of VGF by BSC-1 cells infected with 15 upf of W per cell with that of ÓTGF produced by cells transformed by a retrovirus.



   BOARD
 EMI123.1
 Comparison of the biological activity of VGF P. g q with that of the TGF of EGV.
 EMI123.2
 
 <tb>
 <tb>



  Postman <SEP> Fixation2 <SEP> (a) <SEP> Induction3 <SEP> (b) <SEP> Stimulation4 <SEP> (c)
 <tb> of <SEP> growth
 <tb> None-1. <SEP> 779 <SEP> <20
 <tb> VGF <SEP> 2.3 <SEP> 3,760 <SEP> 108
 <tb> TGF-Ó <SEP> 0.2 <SEP> NT <SEP> 294
 <tb> EGF-8. <SEP> 482 <SEP> 346
 <tb>
 NT = Not tested
 EMI123.3
 (a) Fixation on the EGF receptor, in ng of EGF equivalents per ml of medium. (b Induction of DNA synthesis, in C 125I J Idu incoporated (counts per minute per dish).



  (c) Stimulation of cell growth independent of anchoring, in colonies on soft agar per dish.



  1 VGF was purified (90 ng of EGF equivalents per
 EMI123.4
 microgram of protein) by gel filtration, then by elution from a Ctg Bondapak column (Waters Associates). TGF was purified from embryonic cells of Fisher rats transformed with the feline snyder-Theilen sarcoma virus as described by Marquardt et al., Proc. Natl. Acad. Sci. USA (1983) 80: 4684-4688; EGF was purified from the mouse submandibular gland (Cohen et al., J. Biol. Chem.



  (1980) 255: 41834-41842).

  <Desc / Clms Page number 124>

 2 The quantitative definition of EGF equivalents is based on a standard 125I-EGF binding competition curve, as described.



  3 Mitogenesis tests were performed as described; 10 ng of purified EGF per ml or of VGF in the same number of EGF equivalents per ml were added to calm cultures of diploid human fibroblasts. The values for the incorporation of C-Iiododeoxyuridine (125r ardu) represent the average of measurements made in triplicate.



  4 The number of colonies on soft agar is the average number of colonies containing a minimum of
20 NRK cells for six random low magnification fields, 10 days after transplanting (1.5 x 104 cells per ml) with purified EGF (5 ng per ml) or VGF in the same number of equivalents EGF per ml and 2.0 ng of TGF-j per ml purified from human platelets, horn described by Assoian et al., J. Biol. Chem. (1983) 258: 7155-7160. Agars carrying NRK cells treated with TGF-pn did not give colonies.



  B. VGF prepared in CHO cells.



   The biological activity of the VGF prepared in CHO cells was evaluated by applying the binding test on the EGF receptors. The culture growth medium was used without further purification. The growth factor attached to the EGF receptors.



  C. The VGF prepared in the silkworm.



   The biological activity of partially purified VGF (approximately 50%) prepared in the silkworm was evaluated by applying the EGF receptor binding test which revealed that there is binding to the receptors. of the EGF.

  <Desc / Clms Page number 125>

 



  BOARD
 EMI125.1
 Percentage of reconstitution of epithelium g on wounds after treatment with growth factor.
 EMI125.2
 
 <tb>
 <tb>



  Pork <SEP> 1
 <tb> (9 <SEP> days <SEP> after <SEP> the <SEP> burn) <SEP> VGF <SEP> natural
 <tb> g <SEP> by <SEP> ml
 <tb> Flank <SEP> right <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0.1 <SEP> 1.0
 <tb> 15% <SEP> 0% <SEP> 70% <SEP> 65%
 <tb> h-EGF
 <tb> g <SEP> by <SEP> ml
 <tb> Flank <SEP> left <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0.1 <SEP> 0.5 <SEP> 1.0
 <tb> 75% <SEP> 55% <SEP> 60% <SEP> 70% <SEP> 30%
 <tb> Pork <SEP> 2
 <tb> (10 <SEP> days <SEP> after <SEP> the <SEP> burn) <SEP> VGF *
 <tb> 1'- <SEP> 9 <SEP> by <SEP> ml
 <tb> Flank <SEP> right <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0.1 <SEP> 0, <SEP> 5 <SEP> 1.0
 <tb> 50% <SEP> 0% <SEP> 70% <SEP> 95% <SEP> 60%
 <tb> Flank <SEP> left <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0, <SEP> 1 <SEP> 0, <SEP> 5 <SEP> 1,

  0
 <tb> 50% <SEP> 0% <SEP> 60% <SEP> 75% <SEP> 40%
 <tb> Pork <SEP> 3
 <tb> (9 <SEP> days <SEP> after <SEP> the <SEP> burn) <SEP> TGF <SEP> from <SEP> rat
 <tb> u, <SEP> g <SEP> by <SEP> ml
 <tb> Flank <SEP> right <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0.1 <SEP> 1.0 <SEP> 1.0
 <tb> 20% <SEP> 15% <SEP> 90% <SEP> 65 <SEP> 90%
 <tb> TGF <SEP> human
 <tb>; <SEP> g <SEP> by <SEP> ml
 <tb> Flank <SEP> left <SEP> Silvadene <SEP> No <SEP> processed <SEP> 0, <SEP> 1 <SEP> 1, <SEP> 0 <SEP> 1, <SEP> 0
 <tb> 25% <SEP> 5% <SEP> 90% <SEP> 85% <SEP> 65%
 <tb>
 * VGF: natural VGF prepared from cells infected with the vaccinia virus.



  D. Immunological comparison of VGF and TGF.



   During the radioimmunoassay described above,

  <Desc / Clms Page number 126>

 a 50% antigen shift from the antibody to the 17 carboxy-terminal amino acids of the 1 'molecule was observed <Rat C-TGF, at an antigen concentration of approximately 0.2 to 0.3 ng of EGF equivalents, when 125-labeled L-TGF competes with CL-TGF. No competition was observed during a test with VGF in equivalent concentration.



  In a competitive radioimmunoassay for native EGF, preparations of VGF, even taken at 50 ng of EGF equivalents per ml, show minimal displacement ( <10%) of 125I-EGF from a polyclonal antibody to native EGF.



   It is evident from the results above that VGF is a powerful agent for reconstituting the epithelium which undergoes in a satisfactory manner the comparison with other growth factors of which previous tests have demonstrated mitogenic activity. The polypeptides of the invention can be used with different hosts to induce an immunogenic response, promote cell proliferation, heal wounds, and so on. Wounds are observed to form an epithelium and a vascularization, as well as a rapid reconstitution of the solidity in the wound, that is to say the resistance to separation or tearing. The hosts can be mammals, such as rodents, domestic animals, primates or humans.



   The subject of the invention is new compositions having wide utility in numerous fields, for example for diagnosis, for exerting effects in vitro and in vivo on cells, for acting as mitogens, for serving as additives in media. nutritious, to serve as agonists or antagonists for EGF and TGF, to act as

  <Desc / Clms Page number 127>

 immunogens or as therapeutic agents, to aid in wound healing, and so on.

   In addition, due to the presence of a small oligopeptide having a binding activity, the oligopeptide can be used as it is or in conjunction with other polypeptides, or be fused with other polypeptides to modify the properties of these. other polypeptides, which leads to the binding of the polypeptide to cells carrying receptors for growth factor. It is therefore possible to reversibly attach different polypeptides to cells carrying receptors for growth factor and thus to modify the properties of the cells in a predetermined manner.



   Although various embodiments and details have been described to illustrate the invention, it goes without saying that it is susceptible of numerous variants and modifications without departing from its scope.


    

Claims (1)

 EMI128.1   R E V E N D I C A T I O N S CLAIMS 1. - DNA sequence, characterized in that it encodes a hybrid polypeptide which: (1) binds to an EGF receptor (epidermal growth factor); (2) has substantially the same loop structure as a natural growth factor which binds to an EGF receptor ("natural ligand"), and (3) has at least about 30% homology with a ligand natural, where this hybrid polypeptide comprises three domains, each of which is an amino acid sequence having at least 30% homology with a fragment of the same natural ligand or of a different natural ligand, and having the same relative position as of natural ligand fragment,  with the restriction that said DNA sequence which codes for the hybrid polypeptide does not encode a natural ligand and provided that there are not two contiguous domains derived from the same natural ligand, said domains are defined as follows: a first domain d '' at least about 11 N-terminal amino acids starting approximately at amino acid aa-6 to anal and ending approximately at amino acid aa to aa, comprising one of the following residual amino acid sequences: - 5-11 5 10 15 IIKRIKLCNDDYKNYCLNNGTC; IVKHVKVCNHDYENYCLNNGTC; DIPAIRLCGPEGDGYCLH-GDC; VVSHFNDCPDSHTQFCFH-GTC; VVSHFNKCPDSHTQYCFH-GTC; NSDSECPLSHDGYCLHDGVC; NSYPGCPSSYDGYCLNGGVC;  NSYPGCPPSYDGYCLNGGVC; QDAPGCPPSHDGYCLHGGVC; or NRKKKNPCNAEFQNFCIH-GEC;  <Desc / Clms Page number 129>  a second domain of at least approximately 11 amino acids of central fragment starting approximately at amino acid aa 11 to aa 15 and ending approximately at amino acid aa25 to aa comprising one of the following residual amino acid sequences: 15 20 25 30 LNNGTC-FTVALNNVSLNPFCACHI; LNNGTC-FTIALDNVSITPFCVCRI; LH-GDCIH - ARDID - GMYCRCSH; FH-GTCRFLVQEDK ---- PACVCHS; FH-GTCRFLVQEEK - PACVCHS; LHDGVCMYIEALDK ---- YACNCVV; LNGGVCMHIESLDS - YTCNCVI;  LNGGVCMYVESVDR - YVCNCVI; LHGGVCMHIESLNT ---- YACNCVI / or IH-GECKYIEHLEA - VTCKCQQ; a third domain of at least about 14 C-terminal amino acids starting about at amino acid aa25 to aa29 and ending about at amino acid aa40 to aa53, comprising one of the following amino acid sequences 25 30 35 40 45 FCACHINYVGSRCQFINLITIK; FCVCRINYEGSRCQFINLVTY; YCRCSHGYTGIRCQHWLVDYQRSENPNTTTS; ACVCHSGYVGARCEHADLLAWAASQKKQAIT; ACVCHSGYVGVRCEHADLLAWAASQKKQAIT; ACNCVVGYIGERCQYRDLKWWELR; TCNCVIGYSGDRCQTRDLRWWELR; VCNCNIGYIGERCQHRDLR; ACNCVIGYVGERCEHQDLDLWE; or TCKCQQEYEGERCGEK.  2. DNA sequence according to claim 1,  <Desc / Clms Page number 130>  characterized in that it further codes a fourth domain of at least about 6 extreme N-terminal amino acids starting approximately at amino acid at -7 to axa 1 and ending approximately at amino acid aa- 45 to aa-7, comprising one of the following residual amino acid sequences - 20-15-10-5-1;  EMI130.1  MVPRDLVATLLCAMCIVQA ----- TMPSLDNYLYIIKRIK; MATRNLVASL.  LCIMYAVHA ------- MNDYLYIVKHVK; MSMKYLMLLFAAMIIRSFA-DSGNAIETTSPEITNATTDIPAIR; MVPSAGQLALPALGIVLAACQAENSTSPLSADPPVAAAVVSHFN-; MVPAAGQLALLALGILVAVCQAENSTPPLSADSPVAAAVVSHFN; NSDS; NSYP; NSYP; QDAP; or SVRVEQVVKPPQDKTESENTSDKPKRKKKGGKNGKNRRNRKKKN; united at the amino terminus of this first domain, with the restriction that, when the hybrid polypeptide comprises an amino acid sequence identical to a natural ligand, the DNA sequence coding for the fourth domain is present and codes for something else than the extreme N-terminal amino acids of the natural ligand.  3.-DNA sequence according to any one of claims 1 and 2, characterized in that the ligand  EMI130.2  natural is VGF, TGF-alpha, MGF, EGF, SFGF or amphiregulin.  4.-DNA sequence according to any one of the preceding claims, characterized in that at least one of the domains is homologous for at least about 30%  EMI130.3  with a fragment of VGF, TGF-alpha, EGF or SFGF. 5.-DNA sequence according to any one of the preceding claims, characterized in that it also codes for an amino acid sequence selectively cut and inserted at the carboxy terminal of the fourth domain in  <Desc / Clms Page number 131>  wherein said amino acid sequence is Asp-Pro or Met-X, where X represents an amino acid residue.  6.-DNA sequence according to any one of the preceding claims, characterized in that it further codes a signal sequence, united at the amino terminus of this first domain, where the signal sequence is an N fragment -terminal of a lambda N protein, of a lambda Cro protein or of amphiregulin or a signaling sequence comprising an alkaline phosphatase, a monkey TGF-beta signaling sequence or a killer toxin signaling sequence by K. lactis.  7.-DNA sequence according to any one of the preceding claims, characterized in that it comprises at least one alteration of the sequence which codes for a natural ligand giving the following changes in the amino acid sequence: HisGlyAsp to HisGlyThr ; GlyMetTyrCys to GlyTyrAlaCys; ArgCysSer to ValCysSer; CysLeuHisAspCys to CysLeuHisGlyThrCys; and GlyMetTyrCysArgCys to GlyTyrAlaCysValCys.    8. - DNA sequence, characterized in that it encodes a hybrid polypeptide which comprises three domains, the sequence of each domain corresponding at least substantially to the sequence of a fragment of a natural growth factor capable of binding on an EGF receptor ("natural ligand") and having the same relative position as these fragments of the natural ligand, with the restriction that said DNA sequence does not code for a natural ligand and provided that there is not two contiguous domains derived from the same natural ligand, said domains being at least:  a first domain of at least approximately 11 N-terminal amino acids starting approximately at amino acid aa-6 to anal and ending approximately at amino acid aa to aa, comprising one of the following residual amino acid sequences:  <Desc / Clms Page number 132>    - 5-11 5 10 15 IIKRIKLCNDDYKNYCLNNGTC; IVKHVKVCNHDYENYCLNNGTC; DIPAIRLCGPEGDGYCLH-GDC; VVSHFNDCPDSHTQFCFH-GTC; WSHFNKCPDSHTQYCFH-GTC; NSDSECPLSHDGYCLHDGVC; NSYPGCPSSYDGYCLNGGVC;  NSYPGCPPSYDGYCLNGGVC; QDAPGCPPSHDGYCLHGGVC; or NRKKKNPCNAEFQNFCIH-GEC; a second domain of at least about 11 amino acids of central fragment starting approximately at amino acid aa to aa15 and ending approximately at amino acid aa25 to aa29, comprising one of the following residual amino acid sequences: 15 20 25 30 LNNGTC-FTVALNNVSLNPFCACHI; LNNGTC-FTIALDNVSITPFCVCRI; LH-GDCIH-ARDID-GMYCRCSH; FH-GTCRFLVQEDK ---- PACVCHS FH-GTCRFLVQEEK - PACVCHS; LHDGVCMYIEALDK ---- YACNCVV; LNGGVCMHIESLDS - YTCNCVI;  LNGGVCMYVESVDR - YVCNCVI; LHGGVCMHIESLNT ---- YACNCVI; or IH-GECKYIEHLEA - VTCKCQQ; a third domain of at least about 14 C-terminal amino acids starting approximately at amino acid aa25 to aa29 and ending approximately at amino acid aa40 to aa47, comprising one of the following residual amino acid sequences:  <Desc / Clms Page number 133>   25 30 35 40 45 FCACHINYVGSRCQFINLITIK; FCVCRINYEGSRCQFINLVTY; YCRCSHGYTGIRCQHWLVDYQRSENPNTTTS; ACVCHSGYVGARCEHADLLAWAASQKKQAIT; ACVCHSGYVGVRCEHADLLAWAASQKKQAIT; ACNCVVGYIGERCQYRDLKWWELR; TCNCVIGYSGDRCQTRDLRWWELR; VCNCNIGYIGERCQHRDLR; ACNCVIGYVGERCEHQDLDLWE; or TCKCQQEYEGERCGEK;  and further coding a signal sequence of 8 to 45 N-terminal amino acids of an N protein or Cro protein of bacteriophage lambda, of amphiregulin, of gene T4 of a bacterial alkaline phosphatase signaling sequence , joined to the amino terminus of this first domain.  9. DNA sequence according to claim 8, characterized in that it also codes for an amino acid sequence selectively cut and inserted at the carboxy terminal of said signal sequence and of the amino terminal of said polypeptide in which said sequence the amino acid is Asp-Pro or Met-X, where X represents an amino acid residue.    10. DNA sequence according to any one of claims 8 and 9, characterized in that the signal sequence is approximately 32 or 33 amino acids of the N-amino acid termini of the protein N.    11. - DNA sequence according to claim 10, characterized in that the polypeptide of interest is TGF-, VGF, VGFA, VGFa or EGF.    12. DNA sequence according to any one of claims 8 to 10, characterized in that the signal sequence is an alkaline phosphatase signaling sequence or a modified alkaline phosphatase signaling sequence.  <Desc / Clms Page number 134>      13. DNA sequence according to claim 12, characterized in that the polypeptide of interest is VGFa, nVGFa or EGF.    14.-DNA sequence according to claim 10, characterized in that the polypeptide of interest has the following amino acid sequence: M D I P A I R L C G P E G D G Y C L H G T C R F L V Q E E K P A C V C S H G Y T GIRCQHVVLVDYQR 15.-DNA sequence according to claim 10, characterized in that the polypeptide of interest has the following amino acid sequence: M V V S H F N D C P D S H T Q F C F H G T C I H A R D I D G Y A C V C S H G Y T G I RCQHVVLVDYQR 16.-DNA sequence according to any one of claims 8 to 15, characterized in that the signal sequence is around 21 N-terminal amino acids of the Cro protein.    17. A method for producing an expression cassette, characterized in that it comprises, in the direction of transcription, a regulatory region for initiation of transcription and for functional translation in a host cell; a DNA sequence encoding a polypeptide which: (1) binds to an EGF receptor;  (2) has substantially the same loop structure as a natural growth factor capable of binding to an EGF receptor ("natural ligand"), and (3) has at least about 30% homology with a natural ligand, where this hybrid polypeptide comprises three domains, each of which is an amino acid sequence having at least 30% homology with a fragment of the same natural ligand or of different natural ligands, and having the same position  <Desc / Clms Page number 135>  relative that these fragments of natural ligands, with the restriction that said DNA sequence which codes for the hybrid polypeptide does not code for a natural ligand and provided that there are not two contiguous domains derived from the same natural ligand, said domains are defined as follows:  a first domain of at least approximately 11 N-terminal amino acids starting approximately at amino acid aa-6 to anal and ending approximately at amino acid aa to aa, comprising one of the following residual amino acid sequences: - 5-11 5 10 15 IIKRIKLCNDDYKNYCLNNGTC; IVKHVKVCNHDYENYCLNNGTC; DIPAIRLCGPEGDGYCLH-GDC; WSHFNDCPDSHTQFCFH-GTC; VVSHFNKCPDSHTQYCFH-GTC; NSDSECPLSHDGYCLHDGVC; NSYPGCPSSYDGYCLNGGVC;  NSYPGCPPSYDGYCLNGGVC; QDAPGCPPSHDGYCLHGGVC; or NRKKKNPCNAEFQNFCIH-GEC; a second domain of at least about 11 amino acids of central fragment starting approximately at amino acid aa11 to aa15 and ending approximately at amino acid aa25 to aa29, comprising one of the following residual amino acid sequences:  <Desc / Clms Page number 136>   15 20 25 30 LNNGTC-FTVALNNVSLNPFCACHI; LNNGTC-FTIALDNVSITPFCVCRI; LH-GDCIH-ARDID-GMYCRCSH; FH-GTCRFLVQEDK - PACVCHS; FH-GTCRFLVQEEK - PACVCHS; LHDGVCMYIEALDK - YACNCW; LNGGVCMHIESLDS - YTCNCVI;  LNGGVCMYVESVDR - YVCNCVI; LHGGVCMHIESLNT - YACNCVI; or IH-GECKYIEHLEA - VTCKCQQ; a third domain of at least about 14 C-terminal amino acids starting about at amino acid aa25 to aa29 and ending about at amino acid aa40 to aa53, comprising one of the following amino acid sequences 25 30 35 40 45 FCACHINYVGSRCQFINLITIK; FCVCRINYEGSRCQFINLVTY; YCRCSHGYTGIRCQHVVLVDYQRSENPNTTTS; ACVCHSGYVGARCEHADLLAVVAASQKKQAIT; ACVCHSGYVGVRCEHADLLAWAASQKKQAIT; ACNCVVGYIGERCQYRDLKWWELR; TCNCVIGYSGDRCQTRDLRWWELR; VCNCNIGYIGERCQHRDLR; ACNCVIGYVGERCEHQDLDLWE; or TCKCQQEYEGERCGEK as well as a regulatory region for transcription termination and functional translation in the host cell.    18.-A method according to claim 17, characterized in that said expression cassette further comprises an additional DNA sequence further coding a fourth domain of at least about 6 acids  <Desc / Clms Page number 137>  extreme N-terminal amino acids starting approximately at amino acid aa-7 to aa-1 and ending approximately at amino acid aa-45 to ara-7, comprising one of the following residual amino acid sequences: - 20- 15-10-5-1;  EMI137.1  MVPRDLVATLLCAMCIVQA ----- TMPSLDNYLYIIKRIK; MATRNLVASLLCIMYAVHA -------- MNDYLYIVKHVK;  MSMKYLMLLFAAMIIRSFA-DSGNAIETTSPEITNATTDIPAIR; MVPSAGQLALPALGIVLAACQAENSTSPLSADPPVAAAWSHFN-; MVPAAGQLALLALGILVAVCQAENSTPPLSADSPVAAAWSHFN; NSDS; NSYP; NSYP; QDAP; or SVRVEQVVKPPQDKTESENTSDKPKRKKKGGKNGKNRRNRKKKN; joined to the amino terminus of this first domain, with the restriction that, when the polypeptide has an amino acid sequence identical to a natural ligand, a DNA sequence encoding the fourth domain is present and codes for something other than the extreme N-terminal amino acids of the natural ligand.    19. A method according to claim 18, characterized in that said expression cassette comprises an additional DNA sequence coding for an amino acid sequence capable of being selectively cut and inserted at the carboxyl end of this fourth domain and joined to the amino terminal of this first domain in which said amino acid sequence is Asp-Pro or Met-X, where X represents an amino acid residue.    20.-A method for producing a hybrid polypeptide, characterized in that it comprises a host cell comprising an expression cassette which comprises, in the direction of transcription, a regulatory region for initiation of transcription and functional translation  <Desc / Clms Page number 138>  in a host cell; a DNA sequence encoding a polypeptide which: (1) binds to an EGF receptor;  (2) has substantially the same loop structure as a natural growth factor capable of binding to an EGF receptor ("natural ligand"), and (3) has at least about 30% homology with a natural ligand, where this hybrid polypeptide comprises three domains, where this polypeptide comprises three domains, each of which is an amino acid sequence having at least 30% homology with fragments of the same natural ligand or of different natural ligands, and having the same relative position as these fragments of natural ligands, with the restriction that said DNA sequence which codes for the hybrid polypeptide does not code for a natural ligand and provided that there are not two contiguous domains derived from the same natural ligand, said areas are defined as follows:  a first domain of at least approximately 11 N-terminal amino acids starting approximately at amino acid aa-6 to anal and ending approximately at amino acid aa to aa, comprising one of the following residual amino acid sequences: - 5-11 5 10 15 IIKRIKLCNDDYKNYCLNNGTC; IVKHVKVCNHDYENYCLNNGTC; DIPAIRLCGPEGDGYCLH-GDC; WSHFNDCPDSHTQFCFH-GTC; VVSHFNKCPDSHTQYCFH-GTC; NSDSECPLSHDGYCLHDGVC; NSYPGCPSSYDGYCLNGGVC;  NSYPGCPPSYDGYCLNGGVC; QDAPGCPPSHDGYCLHGGVC; or NRKKKNPCNAEFQNFCIH-GEC; a second domain of at least about 11 acids  <Desc / Clms Page number 139>  central fragment amines starting approximately at amino acid aa 11 to aa 15 and ending approximately at amino acid aa25 to aa29, comprising one of the following residual amino acid sequences: 15 20 25 30 LNNGTC-FTVALNNVSLNPFCACHI; LNNGTC-FTIALDNVSITPFCVCRI; LH-GDCIH - ARDID - GMYCRCSH; FH-GTCRFLVQEDK - PACVCHS; FH-GTCRFLVQEEK - PACVCHS; LHDGVCMYIEALDK ---- YACNCVV; LNGGVCMHIESLDS ---- YTCNCVI;  LNGGVCMYVESVDR - YVCNCVI; LHGGVCMHIESLNT ---- YACNCVI; or IH-GECKYIEHLEA ---- VTCKCQQ; a third domain of at least about 14 C-terminal amino acids starting about at amino acid aa25 to aa29 and ending about at amino acid aa40 to aa53, comprising one of the following amino acid sequences 25 30 35 40 45 FCACHINYVGSRCQFINLITIK; FCVCRINYEGSRCQFINLVTY; YCRCSHGYTGIRCQHWLVDYQRSENPNTTTS; ACVCHSGYVGARCEHADLLAVVAASQKKQAIT; ACVCHSGYVGVRCEHADLLAWAASQKKQAIT; ACNCVVGYIGERCQYRDLKWWELR; TCNCVIGYSGDRCQTRDLRWWELR; VCNCNIGYIGERCQHRDLR; ACNCVIGYVGERCEHQDLDLWE;  or TCKCQQEYEGERCGEK as well as a regulatory region for transcription termination and functional translation in the cell  <Desc / Clms Page number 140>  host in an appropriate culture medium so that the polypeptide is expressed and, isolating said hybrid polypeptide.    21. A method according to claim 20, characterized in that said expression cassette comprises an additional DNA sequence encoding a fourth domain of at least about 6 extreme N-terminal amino acids starting approximately at amino acid aa- 7 to aa-1 and ending approximately in amino acid aa-45 to aa-7, comprising one of the following residual amino acid sequences - 20-15-10-5-1;  EMI140.1  MVPRDLVATLLCAMCIVQA ----- TMPSLDNYLYIIKRIK; MATRNLVASLLCIMYAVHA ------- MNDYLYIVKHVK; MSMKYLMLLFAAMIIRSFA-DSGNAIETTSPEITNATTDIPAIR; MVPSAGQLALPALGIVLAACQAENSTSPLSADPPVAAAVVSHFN-;  MVPAAGQLALLALGILVAVCQAENSTPPLSADSPVAAAVVSHFN; NSDS; NSYP; NSYP; QDAP; or SVRVEQVVKPPQDKTESENTSDKPKRKKKGGKNGKNRRNRKKKN; joined to the amino terminus of this first domain, with the restriction that, when the polypeptide has an amino acid sequence identical to a natural ligand, a DNA sequence encoding the fourth domain is present and codes for something other than the extreme N-terminal amino acids of the natural ligand.    22. A method according to claim 21, characterized in that said expression cassette comprises an additional DNA sequence coding for an amino acid sequence capable of being selectively cut and inserted at the carboxyl end of this fourth domain and joined to the amino terminus of this first domain in which said amino acid sequence is Asp-Pro or Met-X, where X  <Desc / Clms Page number 141>  represents an amino acid residue.  23.-Hybrid polypeptide, characterized in that: (1) binds to an EGF receptor; (2) has substantially the same loop structure as a natural growth factor capable of binding to an EGF receptor ("natural ligand"), and (3) has at least about 30% homology with a natural ligand, where this hybrid polypeptide comprises three domains, where this polypeptide comprises three domains, each of which is an amino acid sequence having at least 30% homology with fragments of the same natural ligand or of different natural ligands, and having the same relative position as these fragments of natural ligands, with the restriction that said DNA sequence which codes for the hybrid polypeptide does not code for a natural ligand and provided that there are not two contiguous domains derived from the same natural ligand,  said domains are defined as follows: a first domain of at least about 11 N-terminal amino acids starting approximately at amino acid aa-6 to anal and ending approximately at amino acid aa to aa; a second domain of at least about 11 amino acids of central fragment starting about at amino acid aa to aa and ending about at amino acid aa25 au29; a third domain of at least about 14 C-terminal amino acids starting at about amino acid aa25  EMI141.1  29 40 53 to aa29 and ending approximately in amino acid aa40 to aa53. 24.-Hybrid polypeptide according to claim 23, characterized in that it further comprises a fourth domain of at least about 6 extreme N-terminal amino acids starting approximately at amino acid aa-7 to aa-1 and ending approximately at amino acid aa-45 to aa-7, comprising one of the following residual amino acid sequences:  <Desc / Clms Page number 142>  - 20-15-10-5-1; MVPRDLVATLLCAMCIVQA ----- TMPSLDNYLYIIKRIK; MATRNLVASLLCIMYAVHA ------- MNDYLYIVKHVK; MSMKYLMLLFAAMIIRSFA-DSGNAIETTSPEITNATTDIPAIR; MVPSAGQLALPALGIVLAACQAENSTSPLSADPPVAAAWSHFN-; MVPAAGQLALLALGILVAVCQAENSTPPLSADSPVAAAVVSHFN; NSDS; NSYP; NSYP; QDAP;  or SVRVEQWKPPQDKTESENTSDKPKRKKKGGKNGKNRRNRKKKN; united at the amino terminus of this first domain, with the restriction that, when the polypeptide has an amino acid sequence identical to a natural ligand, the fourth domain is present and is something other than the extreme N-terminal amino acids of the natural ligand.  25.-hybrid polypeptide according to claim 24, characterized in that it further comprises an amino acid sequence capable of being selectively cut and inserted at the carboxyl end of this fourth domain, and united with the amino terminus of this first domain in which said amino acid sequence is Asp-Pro or Met-X, where X represents an amino acid residue.  26.-Hybrid polypeptide, characterized in that it comprises the following amino acid sequence or a fragment thereof of at least 37 amino acids comprising at least approximately amino acids 1 to 37:  <Desc / Clms Page number 143>    EMI143.1   <tb> <tb> aa-24-20-15-10-5-1 <tb> D <SEP> S <SEP> G <SEP> N <SEP> A <SEP> I <SEP> E <SEP> T <SEP> T <SEP> S <SEP> P <SEP> E <SEP > I <SEP> T <SEP> N <SEP> A <SEP> T <SEP> T <SEP> D <SEP> I <SEP> P <SEP> A <SEP> I <SEP> R <tb> 1 <SEP> 5 <SEP> 10 <SEP> 15 <SEP> 20 <tb> L <SEP> C <SEP> G <SEP> P <SEP> E <SEP> G <SEP> D <SEP> G <SEP> Y <SEP> C <SEP> L <SEP> H <SEP > X1 <SEP> G <SEP> D <SEP> C <SEP> I <SEP> H <SEP> X2 <SEP> A <SEP> R <SEP> D <tb> 25 <SEP> 30 <SEP> 35 <SEP> 40 <tb> I <SEP> D <SEP> G <SEP> X3 <SEP> M <SEP> Y <SEP> C <SEP> R <SEP> C <SEP> S <SEP> H <SEP> G <SEP > Y <SEP> T <SEP> G <SEP> I <SEP> R <SEP> C <SEP> Q <SEP> H <SEP> V <SEP> V <tb> 45 <SEP> 50 <SEP> 53 <tb> L <SEP> V <SEP> D <SEP> Y <SEP> Q <SEP> R <SEP> S <SEP> E <SEP> N <SEP> P <SEP> N <SEP> T <tb>    where Xl is a bond or an amino acid, X2 is a bond or 1 or 2 amino acids, X3 is a bond or 1 to 4 amino acids and Xl, X2 and X3 can be the same or different.