WO1989010964A1

WO1989010964A1 - Novel bovine granulocyte-macrophage colony stimulating factor variant

Info

Publication number: WO1989010964A1
Application number: PCT/US1989/001422
Authority: WO
Inventors: Patrick W. Gray; Steven R. Leong
Original assignee: Genentech, Inc.
Priority date: 1988-05-06
Filing date: 1989-04-06
Publication date: 1989-11-16
Also published as: AU3532289A

Abstract

Novel bovine granulocyte-macrophage colony stimulating factor (GM-CSF) variant and the means and method for production. Composition comprising the variant bovine GM-CSF.

Description

NOVEL BOVINE GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR VARIANT Background of the Invention

This invention relates to bovine protein granulocyte- macrophage colony stimulating factor (GM-CSF) and a protein sequence variant of bovine GM-CSF. In particular, this invention relates to deoxyribonucleic acid (DNA) encoding a bovine GM-CSF sequence variant and methods for the use of such DNA for the production of protein. Colony stimulating factors are required for the proliferation and differentiation of a variety of lineages of hematopoietic stem cells (D. Metcalf, The Hemopoietic Colony Stimulating Factors [Elsivier Science Publishers, B.V. Amsterdam, 1984]; S.C. Clark and R. Ka en, "Science" 236: 1229 [1987]). These lymphokines also enhance the function of mature peripheral blood mononuclear cells and consequently are important in host defense mechanisms. At least four distinct colony stimulating factors (CSFs) have been isolated and identified by their ability to promote the proliferation and differentiation of bone marrow stem cells into neutrophilic granulocytes and macrophages in semi-solid media. Granulocyte-macrophage CSF (GM-CSF) stimulates the formation of granulocyte and macrophage colonies from progenitor cells (A. . Burgess et al. , "J.Biol.Chem." 252:1998 [1977]; N.M. Gough et al. , "Nature" 309:763 [1984]; G . G . Wong et al. , "Science" 228:810 [1985]; F.T. Lee et al. , "Proc.Natl.Acad.Sci.U.S.A." 82: 4360 [1985]; M.A. Cantrell et al.. "Proc.Natl.Acad.Sci.U.S.A." 82: 6250 [1985]). Granulocyte-CSF (G-CSF or CSF-j9) (M.A. Nicola et al. , "J.Biol.Chem." 258:9017 [1983]; S. Nagata et al. , "Nature" 319: 415 [1986]; M. Tsuchiya et al. , "Proc.Natl.Acad.Sci. U.S.A." 83:7633 [1986]) and macrophage-CSF (M-CSF or CSF-1) (S.K. Das and E.R. Stanley "J.Biol.Chem." 257: 13679 [1982]; E.S. Kawasaki et al. , "Science" 230:291 [1985]) are distinct factors which are more specific in generating only a single type of colony. Interle kin-3 (IL-3 or Multi-CSF) (J.N. Ihle et al. , "J.Immunol." 131:282 [1983]; M.C. Fung et al. , "Nature" 307:233 [1984]; Y.-C. Yang et al. , "Cell" 47:3 [1986]) stimulates the development of a variety of colony types: granulocytes, macrophages, mast cells, eosinophils, megakaryocytes, and erythroid cells. The nucleotide sequences for a number of these factors of both human (G.G. Wong et al. , "Science" £28:810 [1985]; F. Lee et al. , "Proc.Natl.Acad. Sci. U.S.A." 82:4360 [1985]; M.A. Cantrell et al. , "Proc.Natl. Acad.Sci.U.S.A." 82:6250 [1985]; S. Nagata et al. , "Nature" 319:415 [1986]; E.S. Kawasaki et al. , "Science" 230:291 [1985); Y.-C. Yang et al., "Cell" 47:3 [1986)) and murine (N.M. Gough et al.. "Nature" 309:763 [1984]; M. Tsuchiya £ al. , "Proc. Natl. Acad. Sci. U.S.A." 8 : 7633 [1986]; M.C. Fung et al. , "Nature" 307:233 [1984]) origin have recently been isolated and expressed in recombinant systems.

Both human {G.G. Wong et al. , "Science" j>28.:810 [1985]; F. Lee et al., "Proc. Natl. Acad. Sci. U.S.A." 82:4360 [1985]; M.A. Cantrell et al. , "Proc. Natl. Acad. Sci. U.S.A." i$2:6250 [1985]; Patent Cooperation Treaty [PCT] International Publication No. WO 86/00639, WO 86/03225, and WO 87/02060) and murine (N.M. Gough et al. , "Nature" 209:763 [1984]; PCT International Publication No. WO 85/04188; European Patent Application No. 183,350) GM-CSF cDNAs have recently been cloned. Natural GM-CSF from both sources is an acidic glycoprotein of molecular weight 23,000 and is required for the continuous culture of granulocyte and macrophage progenitor cells in vitro. GM-CSF is active at low concentrations (10"^_ 10^"12 M) (D. Metcalf, The Hemopoietic Colony Stimulating Factors [Elsevier Science Publishers, B.V.Amsterdam, 1984]; F. Walker and A.W. Burgess "EMBO J." 4:933 [1985]; J.C. Gasson et al. , "Proc. Natl. Acad. Sci. U.S.A." 83:669 [1986]) and its specific cell surface receptor has been partially characterized (F. Walker and A.W. Burgess, "EMBO J." 4:933 [1985]; J.C. Gasson et al. , "Proc. Natl. Acad. Sci. U.S.A." 83.:669 [1986]). Stimulation of T lymphocyte clones with antigen, concanavalin A, or interleukin 2 results in the rapid synthesis of GM-CSF mRNA (A. Kelso et al. , "J.Immunol." 136:1718 [1986]; T.R. Mosmann et al. , "J.Immunol." 136.:2348 [1986]). In addition to promoting growth of granulocytes and macrophages, GM-CSF also stimulates the proliferation of some T cell lines (T. Kupper et al. , "J.Immunol." 138:4288 [1987]; A. Woods et al- . "J.Immunol." 138:4293 [1987]) and modulates the activity of mature neutrophilic granulocytes (J.C. Gasson et al- » "Science" £26:1339 [1984]).

The medical applications for CSFs lie in three general areas: restoration of hematopoietic dysfunction by raising cell counts from suppressed to normal levels; augmentation of host defense against infection; and, possibly, in malignant disease, stimulating the hyperproduction of functionally primed effector cells (J.C. Gasson, I.S.Y. Chen, CA. Westbrook, D.W. Golde, in Normal and Neoplastic Hematopoiesis. D.W. Golde and P.A. Marks, Eds. [Liss, New York, 1983], p.129]).

It is an object of this invention to obtain a DNA sequence encoding a protein comprising the amino acid sequence of mature native bovine GM-CSF and to produce useful quantities of that protein using recombinant DNA techniques. Another object of this invention is to obtain DNA encoding a protein comprising substitutions, insertions and deletions within the sequence of mature native bovine GM-CSF and having improved therapeutic and/or physicochemical properties and to utilize recombinant DNA techniques to produce useful quantities of that protein. Summary of the Invention

A bovine GM-CSF amino acid sequence variant is obtained by a method comprising

(a) constructing a vector comprising (i) a DNA sequence encoding a protein sequence comprising a substitution, insertion or deletion within the sequence of mature native bovine GM-CSF and (ii) operably ligated thereto, a DNA sequence encoding a signal capable of secreting the protein;

(b) transforming a host cell with the vector;

(c) culturing the transformed host cell; and

(d) recovering the protein from the periplasm of the cell or cellular extracts. Nucleic acid is provided that encodes a bovine GM-CSF variant. This nucleic acid, which may or may not encode mature native bovine GM-CSF, is used to probe cDNA libraries and to identify nucleic acid capable of encoding protein with sequence comprising substantial portions of the sequence of mature native bovine GM- CSF.

In a specific embodiment, the signal sequence DNA is obtained from the J _ coli STII heat shock protein gene and the host cell is E. coli bacteria. The variant bovine GM-CSF protein is expressed and recovered from cultured transformed host cells and comprises at least one substitution, insertion or deletion within the protein sequence of mature native bovine GM-CSF and exerts at least one known or inherent biological activity of mature native bovine GM-CSF. Brief Description of the Drawings

Figure 1 discloses the sequence of the synthetic DNA fragments used as probes for the isolation and identification of bovine GM- CSF cDNA.

Figure 2 discloses the cDNA sequence of and the corresponding amino acid sequence of an expression protein derived from plasmid pBovGM CSF-3.4. The sequence is translated from the first ATG encountered. The first 17 amino acids (presented in low case) represent a presumed signal sequence.

Figure 3 discloses the detail of the construction of plasmid pST2-gamma.

Figure 4 discloses the detail of the construction of plasmid pAPSTII-BoGMCSF-1.

Figure 5 discloses the nucleotide and amino acid sequences corresponding to the synthetic DNA fragments used in construction of the variant bovine GM-CSF expression and secretion plasmid pGM- D3. The variant DNA and amino acid residues are designated by underline. The indicated amino acid numbers refer to the variant GM-CSF amino acid sequence.

Figure 6 discloses the DNA sequence of and the corresponding amino acid sequence of an expression protein derived from plasmid pGM-D3.

Detailed Description of the Invention

For the purpose of this invention, mature native bovine GM- CSF is defined as the class of proteins or polypeptides which is biologically active and which has the amino acid sequence set forth in Figure 2. Variant bovine GM-CSF is defined as the class of proteins or polypeptides which is biologically active and which comprise amino acid sequence variations from that of mature native bovine GM-CSF as set forth in Figure 2.

Biologically active means that the mature native bovine GM- CSF protein or polypeptide, or the variant bovine GM-CSF protein or polypeptide, qualitatively exerts at least one known or inherent activity of the bovine GM-CSF having the amino acid sequence of Figure 2 or antagonizes such known or inherent activity. The variant protein or polypeptide typically exhibits the same qualitative biological activity as the naturally occurring protein or polypeptide, although variants are also selected in order to modify the biological activity characteristics of bovine GM-CSF. One known biological activity of mature native bovine GM-CSF is the stimulation of colony formation. In particular, mature native bovine GM-CSF is known to stimulate the formation of colonies in agar from bone marrow progenitor cells. An assay of this activity typically is performed by (i) plating bovine bone marrow cells at a concentration of 2 x 10-* cells per mL in 35 mm petri dishes with 20% fetal bovine serum, MEM-α media (GIBCO) , 0.6% agar (Difco) and the recombinant protein obtained from diluted extracts of the transformed cultured cells, or a control, (ii) incubating the assay culture at 37°C in 5% CO2 for 14 days and (ϋi) examining the culture for colony formation. A qualitative comparison then is made between the biological activities of the recombinant bovine GM-CSF variant and mature native bovine GM-CSF, together with suitable controls such as T-cell stimulated and unstimulated control media, and cultured nontransformed host cell media. Variant bovine GM-CSF is further defined as the class of proteins or polypeptides other than native alleles and GM-CSFs of other species which comprise amino acid sequence variations of mature native bovine GM-CSF selected from the variation classes known as substitution, insertion or deletion. Ordinarily, the variant GM-CSF will contain stibstantial blocks of amino acid sequence entirely homologous with mature native bovine GM-CSF. Preferably, the amino acid sequence variations in the sequence of mature native bovine GM-CSF occur within the range of residues Pro- (5) and Pro-(89). More preferably, the variations occur within the range of residues Pro-(5) and Pro-(51) and most preferably within the range of residues Pro-(12) and Lys-(20). One such variant bovine GM-CSF has the amino acid sequence as set forth in Figure 6.

Amino acid sequence substitution is that variation class in which at least one amino acid residue in the mature native bovine GM-CSF protein, and typically and preferably only one residue, has been removed and a different residue Inserted in its place. Such substitutions generally are made in accordance with the following Table I when it is desired to modulate finely the characteristics of mature native bovine GM-CSF.

Table I. original residue exemplary substitutions

Ala gly; ser Arg lys

Asn gin; his

Asp glu

Cys ser

Gin asn Glu asp

Gly ala; pro

His asn; gin

He leu; val

Leu ile; val Lys arg; gin; glu

Met leu; tyr; ile

Phe met; leu; tyr

Ser thr

Thr ser Trp tyr

Tyr trp; phe

Val ile; leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table I, i.e. selecting residues that differ more significantly in their effect on maintaining (a) the structure of the protein or polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions that in general are expected to produce the greatest changes in mature native bovine GM-CSF will be those in which (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (c) a cysteine or proline residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine.

Amino acid sequence insertions include amino- and/or carboxyl- terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions (i.e., insertions within the mature native bovine GM-CSF sequence) may range generally from about 1 to 10 residues, more preferably 1 to 5. An excellent example of a single terminal insertion is mature native bovine GM-CSF having an N-terminal methionyl residue. This variant is an artifact of the direct expression of bovine GM-CSF in recombinant cell culture, i.e. , expression without a signal sequence to direct the secretion of mature native bovine GM-CSF. Other examples of terminal insertions include fusions of signal sequences, whether heterologous or homologous, to the N-terminus of mature native bovine GM-CSF to facilitate the secretion of mature native bovine GM-CSF from recombinant hosts.

Amino acid deletions generally range from about 1 to 30 residues, more preferably 1 to 10 residues, and typically are contiguous.

Amino acid sequence variants of mature native bovine GM-CSF include, for example, substitutions or insertions of, or deletions from, residues within the mature native bovine GM-CSF amino acid sequence shown in Figure 2. Substitution, insertion, deletion or any combination thereof may be combined to arrive at a final construct.

A protein or polypeptide variant ordinarily is prepared by site specific mutagenesis of nucleotides in the DNA encoding the mature, native protein, thereby producing DNA encoding a variant protein, and thereafter expressing the DNA in recombinant cell culture. The mutations that will be made in the DNA encoding- the variant bovine GM-CSF obviously must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see European Patent Application No. 75,444A). The DNA sequence encoding the variants of this invention are produced by conventional site-directed mutagenesis of a DNA sequence encoding mature native bovine GM-CSF or its analogs or variants. Such methods of mutagenesis include the M13 system of Zoller and Smith, "Nucleic Acids Res." 10:6487- 500 (1982); "Methods Enzymol." 100:468 (1983); and "DNA" 3:479-88 (1984). An exemplary oligonucleotide used in accordance with the M13 method to effect changes in sequence of mature bovine GM-CSF and produce variant bovine GM-CSF with the sequence of Figure 6 is 5'-CCATGGCAGCCATTATCAGTTAAGGAGGC. It is understood, of course, that DNA encoding other variants of mature native bovine GM-CSF are analogously produced by one skilled in the art through site- directed mutagenesis using an appropriately chosen oligonucleotide.

The variant bovine GM-CSF which is the subject of this invention also is produced by means of in vitro synthesis, although in most instances this is not preferable and particularly is not preferred when the variant exceeds about 100-500 amino acid residues in length.

These amino acid sequence variants are characterized by the predetermined nature of the variation, a feature which distinguishes the variant from naturally occurring allelic or interspecies variations of the GM-CSF sequence.

Operably ligated is defined to mean that a DNA sequence is influenced by other prior DNA sequences. This generally means that the first DNA sequence controls either the transcription or translation of the DNA to which it is operably ligated. For example, DNA encoding a promoter such as the alkaline phosphatase promoter is operably ligated to DNA encoding a preprotein or prepolypeptide such as a secretion signal-mature protein fusion protein when it affects the rate of translation of DNA encoding that preprotein or prepolypeptide. In a second example, DNA encoding a secretion signal such as the E^. coli STII secretion signal is operably ligated to DNA encoding mature native bovine GM- CSF when it is placed in reading frame with the DNA encoding mature native bovine GM-CSF. Ligation refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated (T. Maniatis et al. , "Molecular Cloning: A Laboratory Manual" at 146 [Cold Spring Harbor, Cold Spring Harbor, N.Y. , 1982]).

Deoxyribonucleic acid (DNA) which encodes mature native bovine GM-CSF is obtained by chemical synthesis or by screening reverse transcripts of mRNA from bovine peripheral blood lymphocytes which are stimulated by a suitable agent such as concanavalin A. Stimulation of murine T-cells by concanavalin A at similar concanavalin A concentration but over a shorter time period has been reported (T.R. Mosmann et al. , "J.Immunol." 136:2348 [1986]). Plasmids are designated by a low case p followed by capital and/or low case letters and/or numbers. The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures. Other equivalent plasmids are known in the art and will be apparent to the ordinarily skilled worker in the field.

Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant. Unless otherwise provided, the method used herein for transformation of J . coli is the C Cl2 method of M.

Mandel and A. Higa, "J.Mol.Biol." £3:159 (1970).

Oligonucleotides are short length single or double stranded polydeojcyribonucleotides which are chemically synthesized by known methods and then purified on polyacrylamide gels.

Example 1. Construction of a Bovine Lymphocyte cDNA Library. Bovine peripheral blood lymphocytes were stimulated in vitro with concanavalin A (10 μg/mL) for 72 h. Total RNA was isolated by homogenizing the cells in 5 M guanidinitun thiocyanate and then precipitating the RNA with 4 M lithium chloride (G. Cathala et al.. "DNA" 2:329 [1983]). Polyadenylated RNA was prepared by oligo-dT cellulose (Collaborative Research, Lexington, MA) chromatograph . The mRNA was used to synthesize cDNA (U. Gubler and B.J. Hoffman, "Gene" 25:263 [1983]; cDNA synthesis kit, Amersham, Arlington Heights, IL). Complimentary synthetic DNA fragments comprising a 19 nucleotide fragment and a 15 nucleotide fragment of sequence 5'- AATTCTCGAGCTCACCTGC and 5'-GCAGGTGAGCTCGAG, respectively, were ligated to the bovine cDNA to provide EcoRI endonuclease sites at both ends. The cDNA was then fractionated on an 8% polyacrylamide gel and subsequently ligated to EcoRI digested λgtlO (T. Huynh et al. , "Approaches In Biochemistry" [1985]). The recombinant DNA was packaged into phage particles using an in vitro packaging system (Promega, Madison, WI) . A library of approximately 5 x 10° phage was generated from 10 ng of cDNA.

Example 2. Isolation of Mature Native Bovine GM-CSF cDNA.

Two 70 base synthetic oligonucleotide probes were prepared (B.C. Froehler et al. ,"Nuc. cids Res." 14:5399 [1986]). The sequences of these probes are presented in Figure l.a.(l) and l.a.(2). The bovine lymphocyte cDNA library was screened independently with these two probes; 1.1 x 10° plaques were plated on twenty 15-cm plates and duplicate nitrocellulose filters were blotted from each plate (T. Maniatis et a . , "Cell" 15:687 [1978]). One set of filters was screened with probe 1 (Figure l.a.fl]) and the other set of filters was screened with probe 2 (Figure l.a.[2]). The probes were kinased with

(Amersham) and then added to the filters which were previously bathed for four hours in hybridization solution: 20% formamide, 0.75 M sodium chloride, 0.075 M sodium citrate, 1% polyvinyl pyrolidone (Sigma Chemical Co., St. Louis, MO), 1% Ficoll, 1% bovine serum albumin (Sigma; fraction V), 0.05 M sodium phosphate, pH 6.5, and 50 ng/ml sonicated salmon sperm DNA (Sigma) . Following overnight hybridization at 42°C, the filters were washed extensively: filters hybridized with probe 1 were washed in 0.06 M sodium chloride, 0.006 M sodium citrate, 0.1% sodium dodecyl sulfate at room temperature and filters hybridized with probe 2 were washed in 0.03 M sodium chloride, 0.003 M sodium citrate, 0.1% dodium dodecyl sulfate at 37"C. Approximately 100 plaques in the library hybridized independently with both probes. Twenty clones were plaque purified (T. Maniatis et al. , "Cell" 15:687 [1978]). Phage DNA was subsequently hybridized with a third synthetic DNA probe. The sequence of this probe is presented in Figure l.b. Four of the cloned DNAs hybridized with all three probes and one of these clones, clone ABovGMCSF, contained an EcoRI insert of approximately 800 bp in length, as determined by electrophoresis on a 5% polyacrylamide gel. ABovGMCSF was digested by EcoRI. the 800 bp insert was isolated, and the insert was subcloned into pUC119 (J. Vieira and J. Messing, "Methods in Enzymol." 151:3 [1987]) to provide plasmid pBovGM CSF-3. . The cDNA sequence of plasmid pBovGM CSF-3.4 was determined by the dideoxy chain termination method (A.J.H. Smith "Enzymol." 65:560 [1980]) and is presented in Figure 2.

Example 3. Construction of E. coli Expression and Secretion Plasmid PSTH-Y

Plasmid pIFN-gamma (tetra-Ser) (de la Maza et al. , "Infection and Immunity" 55:2727 [1987]) was digested with Aval and then partially digested with Ndel (Figure 3) . The vector was treated with the Klenow fragment of DNA polymerase I to repair the sticky ends and then isolated and ligated to itself to provide pTetraSer ΔAN. Plasmid pAPH-1 (P. Gray et al. , "Gene" 1£: 247 [1985]) was digested with EcoRI and Xbal. the 400 basepair EcoRI-Xbal fragment was isolated and ligated into EcoRI and Xbal digested pTetraSer ΔAN to provide plasmid pAP tetraSer. Plasmid pAP tetraSer was digested with Xbal and Ndel and the vector was isolated. Oligonucleotide fragments of sequence shown in Figure 3. were kinased, annealed and ligated into the Xbal-Ndel vector obtained from plasmid pAP tetraSer to provide plasmid pST2-gamma which contains a promoter from the alkaline phosphatase gene (Y. Kikuchi et al. , "Nuc.Acids Res." .9:5671 [1981]) and the J _ coli secretory signal of heat signal entertoxin II (R.N. Picken et al. , "Infection and Immunity" 42:269 [1983]).

Example 4. Construction of an Expression and Secretion

Plasmid for Mature Native Bovine GM-CSF.

Plasmid pST2-gamma was digested with Mlul and Bglll and the vector fragment was isolated on a 1% agarose gel (Figure 4) .

Plasmid pBovGM CSF-3.4 was digested with Ddel and Sau3A and a 353 basepair fragment was isolated which contained the nucleotide sequence encoding the 104 carboxy terminal amino acids of mature native bovine GM-CSF. Four oligonucletides with the DNA sequence shown in Figure 4 were kinased with ATP and annealed to provide a 77 basepair oligonucleotide fragment which encoded the 22 amino terminal residues of mature native bovine GM-CSF. The 77 basepair oligonucletide fragment and the 353 basepair fragment obtained from pBovGM CSF-3.4 were ligated into the isolated pST2- amma-derived vector fragment to provide plasmid pAPSTII-BoGMCSF-1.

Example 5.

Construction of an Expression and Secretion Plasmid for Variant Bovine GM-CSF. Example 4 was repeated except that four oligonucletides with the DNA sequence shown in Figure 5 were kinased and annealed to provide a 74 basepair oligonucleotide fragment which encoded the 21 amino terminal residues of variant GM-CSF. The 74 basepair oligonucletide fragment and the 353 basepair fragment obtained from pBovGM-CSF-3.4 were ligated into the isolated pSTII-7-derived vector fragment to provide plasmid pGM-D3.

Example 6.

Expression and Secretion of Mature Native Bovine GM-CSF.

Mature native bovine GM-CSF was obtained in shake cultures using plasmid pAPSTII-BoGMCSF-1 from Example 4. J _ coli 294 (D.V. Goeddel et.al., "Nature" 281: 544-48 [1979]) was transformed with plasmid pAPSTII-BoGMCSF-1 and innoculated into 10-

20 ml of Luria broth (LB) medium with 5 g/ml ampicillin in a 50 or

125 ml shake flask. The flask was cultured for 12-24 hours at 37°C without the addition of any further medium, after which the cells were recovered by centrifugation. The cells were frozen, then thawed and suspended in buffer to separate the periplasmic protein from cell debris by low-speed centrifugation or by filtration. The supernatant solution was concentrated by ultrafiltration using, for example, an Amicon hollow-fiber apparatus, and mature native bovine GM-CSF was obtained using techniques well-known and standard in the field, including for example, ion exchange and size exclusion chromatography using, for example, DEAE-Ultrogel and AcA44 Ultrogel resins, respectively (G. G. Wong e_t al. , "Science" 228: . 810 [1985J).

Example 7. Expression and Secretion of Variant Bovine GM-CSF. Variant bovine GM-CSF Is obtained in the same fashion as described in Example 6 except that plasmid pGM-D3 is cultured and variant bovine GM-CSF is recovered from the cultured fluids.

Claims

Claims :

1. A method to obtain a variant bovine GM-CSF comprising

(a) constructing a vector comprising (i) a DNA sequence encoding said variant GM-CSF sequence comprising a substitution, insertion or deletion within the sequence of mature native bovine GM-CSF and (ii) operably ligated thereto, a DNA sequence encoding a signal capable of secreting the protein; (b) transforming a host cell with the vector;

(c) culturing the transformed host cell; and

(d) recovering the protein from the periplasm of the cell or cellular extracts.

2. A method of claim 1 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro-(89).

3. A method of claim 1 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro-(51).

4. A method of claim 1 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(12) and Lys-(20).

5. A method of claim 1 wherein the sequence is that in Figure 6.

6. A method of claim 1 wherein the host vector further comprises the alkaline phosphatase promoter operably ligated to the DNA sequences encoding the secretion signal and protein.

7. A method of claim 1 wherein the host vector further comprises the alkaline phosphatase promoter operably ligated to the DNA sequences encoding the E_^ coli STII secretion signal and protein.

8. A method of claim 1 wherein the host cell is a gram negative organism.

9. A method of claim 1 wherein the host cell is E^. coli.

10. A DNA sequence (i) encoding a protein sequence comprising a substitution, insertion or deletion within the sequence of mature native bovine GM-CSF and (ii) operably ligated thereto, a DNA sequence capable of secreting the protein.

11. The DNA sequence of claim 10 wherein the DNA sequence capable of secreting the protein encodes the Ej. coli STII secretion signal.

12. The DNA sequence of claim 10 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro- (89).

13. The DNA sequence of claim 10 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro- (51).

14. The DNA sequence of claim 10 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(12) and Lys- (20).

15. The DNA sequence of claim 10 wherein the protein sequence is that in Figure 6.

16. A replicable vector comprising the DNA sequence of claim 10.

17. The replicable vector of claim 16 wherein the DNA sequence is operably ligated to the alkaline phosphatase promoter.

18. A fusion protein comprising the STII leader and an amino acid sequence comprising a substitution, insertion or deletion within the sequence of mature native bovine GM-CSF.

19. A fusion protein of claim 18 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro- (89).

20. A fusion protein of claim 18 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro-

(51).

21. A fusion protein of claim 18 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(12) and Lys- (20).

22. A fusion protein of claim 18 wherein the amino acid sequence is that in Figure 6.

23. A composition comprising a variant bovine GM-CSF wherein at least one residue of the native amino acid sequence of mature bovine GM-CSF has been deleted or substituted, or into which at least one residue has been inserted.

24. The variant of claim 23 wherein the substitution, insertion or deletion within the sequence of mature native,bovine GM-CSF is in the region of residues Pro-(5) and Pro-(89).

25. The variant of claim 23 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(5) and Pro-(51).

26. The variant of claim 23 wherein the substitution, insertion or deletion within the sequence of mature native bovine GM-CSF is in the region of residues Pro-(12) and Lys-(20).

27. The variant of claim 23 wherein the amino acid sequence is that in Figure 6.