HUMAN INTERLEUKIN-3 PROTEINS

Abstract: Purified recombinant human IL-3 (hIL-3), mutant analog proteins having hIL-3 biological activity, recombinant expression vectors comprising nucleotide sequences encoding hIL-3, microbial expression systems comprising appropriate host organisms transformed with the foregoing recombinant expression vectors, and related processes.

Description: TITLE Human Interleukin-3 Proteins BACKGROUND OF THE INVENTION The present invention relates generally to colony stimulating factors (CSFs), and particularly to purified recombinant human interleukin-3 (IL-3) protein compositions. The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony stimulating factors (CSFs). In murine and human systems, these proteins include granulocyte-macrophage colony stimulating factor (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow, and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Other CSFs include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, and burst promoting activity (BPA), which induces development of erythroid cell progenitors into hemoglobin-containing cells. An additional CSF, isolated first in murine systems, and more recently from human cell sources, has been designated IL-3 or multi-CSF. Murine IL-3 was originally identified by Ihle et al., J. Immunol. 126:2184 (1981) as a factor which induced expression of a T cell associated enzyme, 20a-hydroxysteroid dehydrogenase. The factor was purified to homogeneity and shown to regulate the growth and differentiation of numerous subclasses of early hematopoietic and Lymphoid progenitor cells. cDNA clones corresponding to murine IL-3 were first isolated by Fung et al., Nature 307:233 (1984) and Yokota et al., Proc. Natl. Acad. Sci. USA 81:1070 (1984). Gibbon and human genomic DNA homologues of the murine IL-3 sequence were disclosed by Yang et al., Cell 47:3 (1986). The deduced amino acid sequence of the human IL-3 homologue was approximately 29% homologous to murine IL-3; however, biological activity was reported only for expression products of the gibbon sequence. Ihle and Weinstein, Adv. Immunol. 39:1 (1986) provide a comprehensive review of publications relating to the biological activities of IL-3. In view of its potential clinical utility as a therapeutic agent in treatment of various cytopenias, there is interest in IL-3 in the hematology and oncology communities. Therapeutic compositions having human IL-3 activity could be employed to potentiate immune responsiveness to infectious pathogens, or to assist in reconstituting normal blood cell populations following viral infection or radiationor chemotherapy-induced hematopoietic cell suppression. SUHNARY OF THE INVENTION The present invention concerns recombinant human IL-3 Proteins which are expressed and secreted in high yields in microbial systems. The invention also concerns recombinant expression vectors comprising nucleotide sequences encoding the proteins, related microbial expression systems, and processes for making the proteins using the microbial expression systems. BRIEF DESCRIPTION OF THE DRAWINGS The present invention relates generally to colony stimulating factors (CSFs), and particularly to purified recombinant human interleukin-3 (IL-3) protein compositions. The differentiation and proliferation of hematopoietic cells is regulated by secreted glycoproteins collectively known as colony stimulating factors (CSFs). In murine and human systems, these proteins include granulocyte-macrophage colony stimulating factor (GM-CSF), which promotes granulocyte and macrophage production from normal bone marrow, and which also appears to regulate the activity of mature, differentiated granulocytes and macrophages. Other CSFs include macrophage CSF (M-CSF or CSF-1), which induces the selective proliferation of macrophages, and burst promoting activity (BPA), which induces development of erythroid cell progenitors into hemoglobin-containing cells. An additional CSF, isolated first in murine systems, and more recently from human cell sources, has been designated IL-3 or multi-CSF. FIG. 1 indicates the nucleotide sequence and corresponding amino acid sequence of a human IL-3 protein having a proline residue at position 8 of the mature polypeptide (hIL-3[P81). DETAIL-Rn DESCRIPTION OF THE INVENTION A DNA segment encoding human IL-3 was isolated from a cDNA library prepared by reverse transcription of polyadenylated RNA isolated from human peripheral blood T-lymphocytes (PBT). Synthetic oligonucleotide probes having sequence homology to N-terminal and C-terminal regions of the native human genomic DNA sequence were emu wed to screen the library by conventional DNA hybridization techniques. DNA was isolated from those clones which hybridized to the probes and analyzed by restriction endonuclease cleavage, agarose g s ml trophoresis, and additional hybridization experiments (8"Southern blots") involving the electrophoresed fragments. After isolating several clones which hybridized to each of the probes, the hybridizing segment of one hIL-3 clone was subcloned and sequenced by conventional techniques. The segment was also transcribed into RNA in vitro, and the resulting message capped, polyadenylated, and injected into Xenopus oocytes. The resulting oocyte translation products were then tested for the capacity to induce hematopoietic cell proliferation in a human bone marrow assay, described below. Sequencing of several clones isolated from the PBT library indicated the presence in each of a proline residue at position 8 of the mature hIL-3 sequence. One of the hIL-3 sequences was inserted into a yeast expression vector under control of a particular promoter. The vector was used to transform an appropriate yeast expression strain, which was grown in culture under conditions promoting deregression of the yeast promoter. The resulting yeast-conditioned culture supernatant is assayed to confirm high-level expression of hIL-3tP8]. Definitions "Human interleukin-3" and "hIL-3" refer to a human endogenous secretory protein which induces proliferation of granulocyte, macrophage, and erythrocyte progenitors from populations of multipotent hematopoietic stem cells. As used throughout the specification, the term means a protein having IL-3 biological activity and an amino acid sequence which is substantially homologous to the sequence set forth in FIG. 1. "Substantially homologous," which can refer both to nucleic acid and amino acid sequences, means that a particular subject sequence, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which do not result in an adverse functional dissimilarity between reference and subject sequences. For purposes of the present invention, sequences having greater than 90 percent homology, equivalent biological activity, and equivalent expression characteristics are considered substantially hxmalogous. Sequences having lesser degrees of homology, comparable biaactivity, and equivalent expression characteristics are considered eqplvalents. "Mutant amino acid sequence" refers to a polypeptide encoded by-a nucleotide sequence intentionally made variant from a native sequence. "Mutant protein or "mutein" means a protein comprising a mutant amino acid sequence. "Native sequence" refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a gene or protein. The terms "KEX2 protease recognition site" and "N-glycosylation site" are defined below. The term "inactivate", as used in defining particular aspects of the present invention, means to alter a selected KEX2 protease recognition site to retard or prevent cleavage by the KEX2 pro tease of Saccharomyces cerevisiae, or to alter an N-glycosylation site to preclude covalent bonding of oligosaccharide moieties to particular amino acid residues. "Recombinant," as used herein, means that a protein is derived from recombinant microbial (e.g., bacterial or fungal) expression systems. As a product, this defines a human protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Protein expressed in bacterial cultures will be free of polysaccharide; protein expressed in yeast will have a glycosylation pattern different from that expressed in mammalian cells. "Crude yeast-conditioned culture supernatant" refers to media withdrawn from yeast cultures which has not been subjected to concentration or purification procedures. "Purified", as used in the context of this disclosure, refers to a recombinant protein in the form of a protein composition having a specific activity in a human bone marrow proliferation assay of at least 1 x 106 units/mg. The efficiency of the microbial expression systems disclosed herein permit production of sufficient quantities of human IL-3 to permit quantitative purification. Specific activities in the range 107 to 108 units/mg are contemplated as projected final product criteria. "DNA segment" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., in. a quantity or concentration enabling identification, man-ipuladtlo" and recovery of the segment and its component nucleotide sequence by standard biochemical methods, for example, using a cloying vector. nNucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. "Recombinant expression vector" refers to a plasmid comprising a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, and (2) a structural or coding sequence which is transcribed into mRNA and translated into protein. Preferably, the transcriptional unit includes a leader sequence enabling extracellular secretion of translated protein by a host cell. "Recombinant expression system" means a combination of an expression vector and a suitable host microorganism. Yeast expression systems, preferably those employing Saccharomyces cerevisiae, are employed in production of the proteins of the present invention. Assays for hIL-3 Biological Activity Assays used to measure hIL-3 biological activity are described below. LL Human Bone Marrow Proliferation Assay Freshly isolated human bone marrow cells are preincubated for 2 hours. at 370C, 5% C02, in tissue culture flasks containing 2 x 106 cells per ml pre-warmed, pre-gassed serum-free RPMI 1640 medium (Gibco, Chagrin Falls, OH, USA) containing 50 units/ml penicillin, 50 vg/ml streptomycin, and 300 vg/ml fresh L-glutamine (hereinafter "assay medium"). After preincubation, nonadherent cells are removed by pipetting the media gently over the surface of the flask. Nonadherent cells are collected by centrifugation at 1000 rpm for 10 minutes at 40C, resuspended in a small volume of assay medium containing 10% fetal bovine serum (FBS), and counted using Trypan blue for viability and Turks stain for recovery of white cells. Cells are kept at about 40C in assay medium containing 10% FBS until added to assay plates. 50 l assay medium are added to each well of a 96 well flat bottom tissue culture plate. 50 ul of sample diluted in assay medium are added to the first well of each row, and serial dilutions are made across each row in the usual manner. 1.25 x 104 bone marrow cells, in asvo-lume of 100 u1, are then added to each well. Plates are incubated final 4 dazes at 37 C, 5X C02, in a plastic box containing sterile distilled H2O to prevent desiccation. On day 4, 25 ul of assay medium containing 5% FBS and 80 pCi/ml [JH]-thymidine (80 Ci/mmol) are added to each well and the plate incubated 5 hr at 370C, 5% CO2, in a plastic box. After incubation, cells are harvested onto glass fiber filters, washed, and tested for incorporated radioactivity using a scintillation counter. Units of hIL-3 activity are calculated by reference to the quantity of hIL-3 which induces 50X of maximal thymidine incorporation. For example, if a 100 ul sample generates one-half maximal thymidine incorporation at a dilution of 1:20, one unit is defined as the activity contained in 1/20 of 100 ul, or 5 pl. The sample would therefore contain 1000 divided by 5, or 200 units per milliliter (U/ml) of hIL-3 activity. 2. Human Bone Marrow Colony Assays In this assay, 50 ul samples are plated in appropriate wells in log-2 dilution series. A 1.4% agar suspension is prepared by Heating in a boiling water bath, and then held at 400C prior to use. An incubation medium is prepared by mixing seven parts nutrient medium [(&alpha;-minimum- essential medium (oMEM) supplemented with vitamins, 28.5t RESr 0'..7 x 104 M 2-mercaptoethanol, 0.12 mg/ml asparagine, 0.7 mg/ml glutamine, 150 U/ml penicillin G, and 150 U/ml streptomycin] and three parts agar suspension, and held at 370C. Percoll treated bone marrow cells are warmed to 370C and added to the incubation medium to provide a final concentration of approximately 1 x 105 cells/ml. The resulting mixture is kept at 370C while dispensing 250 ul aliquots into each well. Plates are held at about 230C until the agar solidifies, then incubated at 370C in plastic boxes containing distilled water to prevent desiccation. Colonies having 50 or more cells each are counted on days 7 or 10 and 14. Earlier counts are better for granulocyte colonies, while later counts are better for macrophage and mixed colonies. In each assay, several wells are plated without hematopoietic growth factor samples to obtain a background colony count. hIL-3 activity, expressed in colony forming units per milliliter ("CFU/ml"), is defined as that sample dilution providing one-half of the maximum colonies formed by 1 x 10 bone marrow cells, multiplied by the number of colonies observed in the half maximal case. Cell types in colonies are determined by staining individual cells with a stain consisting of 0.6X Orcein and 60X acetic acid. The Native hIL-3 Sequence The nucleotide and deduced amino acid sequences of the hIL-3 cDNA isolated as described below are set forth in FIG. 1. In FIG. 1, nucleotides are numbered beginning with the GCT codon corresponding to the N-terminal alanine of the mature native protein. Similarly, amino acids are numbered from this alanine residue. The native polypeptide includes a leader sequence which is cleaved upon secretion to provide mature protein. A recombinant DNA segment encoding the amino acid sequence of hIL-3 can be obtained by screening of appropriate cDNA libraries or by assembly of artificially synthesized oligonucleotides. As a matter of choice, hIL-3 sequences incorporating codons specifying proline or serine at position 8 of the mature sequence can be assembled. Protein Expression in Recombinant Yeast Systems Yeast systems may be used for expression of the recombinant proteins of this invention. Preferred expression vectors can be derived from pBC102 K22 (ATCC 67,255) which contains DNA sequences from pBR322 for selection and replication in E. coli (Apr gene and origin of replication) and yeast DNA sequences including a glucose-repressible alcohol dehydrogenase 2 (ADH2) promoter. The ADH2 promoter has been described by Russell et al., J. Biol. Chem. 258:2674 (1982) and Beier et al., Nature 300:724 (1982). Plasmid pBC102 K22 also includes a Trpl gene as a selectable marker and the yeast 2 p origin of replication. Adjacent to the promoter is the yeast factor leader sequence enabling secretion of heterologous proteins from a yeast host. The factor leader sequence is modified to contain, near its 3' end, an Asp718 (KpnI and Asp718 are isoschizomers) restriction site to facilitate fusion of this sequence to foreign genes. A sequence coding for the Glu-Ala-Glu-Ala amino acids was omitted to allow efficient processing of secreted protein, as described by Brake et al., Proc. Natl. Acad. Sci. USA 81:4642 (1984). Alternative expression vectors are yeast vectors which cfflmprise an factor promoter, for example pYoBuGM (ATCC 53157), which bears the wild-type human GM-CSF gene. Others are known to those skilled in the art. The construction of pYoBuGM is described in published European Patent Application No. 183,350 (8530682.7), the disclosure of which is incorporated by reference herein. The choice of appropriate yeast strains for transformation will be determined by the nature of the selectable markers and other features of the vector. Appropriate S. cerevisiae strains for transformation by expression vectors derived from pBC102 K22 or pYaHuGM include strains X2181-1B, available from the Yeast Genetic Stock Center, Berkeley, CA, USA tsee below, having the genotype trpl gall adel his2; J17 (ATCC 52683; a his2 adel trpl metl4 ura3); and IL166-5B (ATCC 46183; a hisl trpl). A particularly preferred expression strain for use with pBC102-K22, XV2181, is a diploid formed by mating two haploid strains, X2181-1B, available from the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California, Berkeley, CA 94702, USA; and XV617-1-3B, available from the Department of Genetics, University of Washington, Seattle, WA 98105, USA, or Immunex Corporation, 51 University Street, Seattle, WA 98101, USA. A suitable transformation protocol is that described by Hinnen, et al., Proc. Natl. Acad. Sci. USA 75:1929 (1978), selecting for Trp+ transformants in a selective medium consisting of 0.67% yeast nitrogen base, 0.5% casamino acids, 2D glucose, 10 ug/ml adenine and 20 ug/ml uracil. Host strains transformed by vectors comprising the ADH2 or a-factor promoters are grown for expression in a rich medium consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ug/ml adenine and 80 vg/ml uracil. Derepression of the ADH2 promoter occurs upon exhaustion of medium glucose. Crude yeast supernatants are harvested by filtration and frozen or held at 40C prior to further purification. Purification of IL-3 Recombinant human IL-3 resulting from fermentation of yeast strains can be purified by single or sequential reversed-phase HPLC steps on a preparative HPLC column, by methods analogous to those described by Urdal et al., J. Chromatog. 296:171 (1984), and Grabstein et al., J. Exp. Med. 163:1405 (1986). For example, yeast-conditioned medium containing hIL-3 can be filtered through a 0.45 p filter and pumped, at a flow rate of 100 ml/min, onto a 5 cm x 30 cm column packed with 10-20 p reversed phase silica (Vydac, The Separations Group, Hesperia, CA, USA). The column can be equilibrated in 0.1% trifluoroacetic acid in water (Solvent A) prior to the application of the yeast-conditioned medium and then flushed with this solvent following application of the medium to the column until the optical absorbance at 280 nm of the effluent approaches baseline values. At this time, a gradient of 0.1% trifluoroacetic acid in acetonitrile (Solvent B) can be established that leads from 0 to 60-100% Solvent B at a rate of change of 1-2% per minute and at a flow rate of 100 ml/min. At a suitable time (10-20 minutes) following initiation of the gradient, one minute fractions are collected and aliquots of the fractions analyzed for protein content by polyacrylamide gel electrophoresis and fluorescamine protein determination. If desired, fractions containing hIL-3 can be pooled, concentrated, and reapplied to a similar HPLC column in 0.9 M acetic acid plus pyridine to pH 4.0, for an additional elution step mediated by gradient of 0.9 M acetic acid, pyridine (pH 4.5) and 60% n-propanol. Fractions eluting from the column can be analyzed for protein concentration by fluorescamine analysis, and hIL-3 activity determined by appropriate assay. Additional HPLC steps can be employed if indicated. Inactivation of KEX2 Processing Sites The native hIL-3 protein includes an Arg-Arg pairing at position 52 and an Arg-Arg-Lys triplet beginning at position 106, both of which are susceptible to cleavage by the KEX2 protease of Saccharomyces cerevis iae. Site-specific mutagenesis procedures can be employed to inactivate KEX2 pro tease processing sites by deleting, adding, or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys Airings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. The resulting muteins are less susceptible to cleavage by the KEX2 pro tease at locations other than the yeast factor leader sequence, where cleavage upon secretion is intended. Inactivation of N-glycosylation Sites Many secreted proteins acquire covalently attached carbohydrate units following translation, frequently in the form of oligosaccharide units linked to asparagine side chains by N-glycosidic bonds. Both the structure and number of oligosaccharide units attached to a particular secreted protein can be highly variable, resulting in a wide range of apparent molecular masses attributable to a single glycoprotein. hIL-3 is a secreted glycoprotein of this type. Attempts to express glycoproteins in-recombinant systems can be complicated by the heterogeneity attributable to this variable carbohydrate component. For example, purified mixtures of recombinant glycoproteins such as human or murine granulocyte-macrophage colony stimulating factor (GM-CSF) can consist of from 0 to 50% carbohydrate by weight. Miyajima et al., EMBO Journal 5:1193 (1986) reported expression of a recombinant murine GM-CSF in which N-glycosylation sites had been mutated to preclude glycosylation and reduce heterogeneity of the yeast-expressed product. The presence of variable quantities of associated carbohydrate in recombinant secreted glycoproteins complicates purification procedures, thereby reducing yield. In addition, should the glycoprotein be employed as a therapeutic agent, a possibility exists that recipients will develop allergic reactions to the yeast carbohydrate moieties, requiring therapy to be discontinued. For these reasons, biologically active, homogeneous analogs of immunoregulatory glycoproteins having reduced carbohydrate are desirable for therapeutic use. Functional mutant analogs of hIL-3 having inactivated N-glycosylation sites can be produced by oligonucleotide synthesis and ligation or by site-specific mutagenesis techniques as described below. These analog proteins can be produced in a homogeneous, reduced-carbohydrate form in good yield using yeast expression systems. The present invention contemplates analog forms of human IL-3 comprising an amino acid sequence homologous to the native sequence of hIL-3, but comprising at least one amino acid substitution, deletion, or insertion inactivating at least one N-glycosylation site. N-glycosylation sites in eukaryotic proteins are characterized by the amino acid triplet Asn-Al-Z, where A1 is any amino acid except Pro, and Z is Ser or Thr. In this sequence, asparagine provides a side chain amino group for covalent attachment of carbohydrate. Such a site can be eliminated by substituting another amino acid for Asn or for residue Z, deleting Asn or Z, or inserting a non-Z amino acid between A and Z, or an amino acid other than Asn between Asn and A1. Preferably, substitutions are made conservatively; i.e., the most preferred substitute amino acids are those having physicochemical characteristics resembling those of the residue to be replaced. Similarly, when a deletion or insertion strategy is adopted, the potential effect of the deletion or insertion upon biological activity should be considered. Thus, an analog hIL-3 lacking N-glycosylation sites is a protein having a mutant amino acid sequence which is substantially homologous to the native sequence set forth in FIG. 1, wherein at least one occurrence Asn-A1-Z in the native sequence has been replaced in the mutant sequence by Asn-A2-Y or X-A2-A3, where A1, A2 and A3 are the same or different and can be any amino acid, X is any amino acid not Asn; Y is any amino acid not Z; and Z is Ser or Thr. Preferably, all occurrences of Asn-A -Z in the native sequence are 2 23 replaced in the mutant sequence by Asn-A2-Y or X-A2-A3. Referring now to the sequence of hIL-3 set forth in FIG. 1, it can be seen that the native protein contains two putative N-glycosylation sites, the first being the triplet AsnCysSer beginning at residue 15,. and the second being AsnAlaSer beginning at residue 68. Appropriately conservative substitute amino acids for Asn include Asp, Gin',, Glu, Ala, Gly, Ser, and Thr, of which Asp, Gln, and Glu are preferre*. Where Z is Ser, appropriate substitutes are Met, Leu, Ile, Val, Asp, Gln, Glu, or Asn; of which Met, Leu, Ile, and Val are preferred. Other conservative amino acid substitutions could be made to provide protein lacking N-glycosylation sites. Construction of Analog Sequences and Muteins In addition to the particular muteins described above, numerous DNA constructions including all or part of the nucleotide sequences depicted in FIG. 1, in conjunction with oligonucleotide cassettes comprising additional useful restriction sites, can be prepared as a matter of convenience. Expression vectors and systems comprising such constructions, as well as hIL-3 muteins produced by such systems, are within the scope of the present invention. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native squence. Following ligation, the resulting reconstructed sequence ennde-s a mutein having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, January 1985, 12-19; Smith et al., Genetic Engineering: Principles and Methods (Plenum Press, 1981); and U. S. Patent 4,518,584 disclose suitable techniques, and are incorporated by reference herein. For either approach, conventional techniques for oligonucleotide synthesis are suitable, for example, the triester synthesis procedures disclosed by Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978). In site-specific mutagenesis, a strand of the gene to be altered is cloned into an M13 single-stranded phage or other appropriate vector to provide single-stranded (ss) DNA comprising either the sense or antisense strand corresponding to the gene to be altered This DNA is then hybridized to an oligonucleotide primer complementary to the sequence surrounding the codon to be altered, but comprising a codon (or an antisense codon complementary to such codon) specifying the new amino acid at the point where substitution is to be effected. If a deletion is desired, the primer will lack the particular codon specifying the amino acid to be deleted, while maintaining the correct reading frame. If an insertion is desired, the primer will include a new codon, at the appropriate location in the sequence, specifying the amino acid to be inserted. Preferably, the substitute codon, deleted codon, or inserted codon is located at or near the center of the oligonucleotide. The size of the oligonucleotide primer employed is determined by the need to optimize stable, unique hybridization at the mutation site with the 5' and 3' extensions being of sufficient length to avoid editing of the mutation by exonucleases. Thus, oligonucleotides used in accordance with the present invention will usually contain from about 15 to about 25 bases. Oligonucleotides of greater size are not needed. In a preferred approach (see Walder et al., supra), the resulting oligonucleotide/ss vector hybrid is directly transformed into yeast. Alternatively, a mutagenic primer is hybridized to a gapped duplex having a single-stranded template segment containing the gene to be altered. In the latter case, the primer is extended along the template strand by reaction with DNA polymerase I (Klenow fragment), T4 DNA polymerase, or other suitable DNA polymerase, providing a resulting double stranded DNA which is circularized and used to transfect a suitable host strain. In both cases, replication of the heteroduplex by the host provides progeny of both strands. In E. coli, transfected cells are plated to provide colonies, which are screened using a labeled oligonucleotide corresponding to that used in the mutagenesis procedure. If yeast are transformed directly, transformants are pooled, DNA isolated and transformed into E. coli. The resulting colonies are screened by hybridization. Conditions are employed which result in preferential hybridization of the primer to the mutated DNA but not to the progeny of the parent strand. DNA cnutai'nin the mutated gene is then isolated and spliced into a s =t t expression vector, and the vector used to transform a host strain-. The host strain is then grown in culture to provide the analog protein. Construction of Fusion Proteins In one embodiment of the present invention, the amino acid sequence of mature hIL-3 is linked to a yeast factor leader sequence via an N-terminal fusion construct comprising a nucleotide encoding the peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK). The latter sequence is highly antigenic and provides an epitope reversibly bound by specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this pep tide are also resistant to intracellular degradation prTar to secretion. Example: Isolation of cDNA encoding hIL-3 and Yeast Expression of Active Protein Two oligonucleotides were synthesized, with sequences complementary to selected 5' and 3' sequences of the hIL-3 gene. The 5' probe, complementary to a sequence encoding part of the hIL-3 leader, had the sequence 5t-CAGGACGAGGACGAGGTTGAG-3'. The 3' probe, corresponding to a region encoding amino acids 123-130 of the mature protein, had the sequence 5'-TGCTGAAACTCGGAGCGCTAG-3'. The method of synthesis was a standard automated triester method substantially similar to that disclosed by Sood et al., Nucleic Acids Res. 4:2557 (1977) and Hirose et al., Tet. Lett. 28:2449 (1978). Following synthesis, oligonucleotides were deblocked and purified by preparative gel electrophoresis. For use as screening probes, the oligonucleotides were terminally radiolabeled with 32P-ATP and T4 polynucleotide kinase using techniques similar to those disclosed by Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory 1982). A cDNA library was constructed by reverse transcription of polyadenylated mRNA isolated from total RNA extracted from human peripheral blood T lymphocytes (PBT) stimulated with phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA). The cDNA was rendered double-stranded using DNA polymerase I, blunt-ended with T4 DNA polymerase, methylated with EcoRI methylase to protect EcoRI cleavage sites within the cDNA, and ligated to EcoRI linkers. The resulting constructs were digested with EcoRI to remove all but one copy of the linkers at each end of the cDNA, and ligated to EcoRI-cut and dephosphorylated arms of bacteriophage XgtlO (Huynh et al., DNA Cloning: A Practical Approach, Glover, ed., IRL Press, pp. 49-78). The ligated DNA was packaged into phage particles to generate a library of recombinants. 500,000 recombinants were plated on E. coli strain C600hfl- and screened by standard plaque hybridization techniques. Eleven clones were isolated from the library which hybridized to both probes. These were plaque purified and used to prepare bacteriophage DNA which was digested with EcoRI. The digests woe electrophoresed on an agarose gel, blotted onto nylon filters, and retested for hybridization. One clone was partially digested with EcoRI followed by preparative agarose gel electrophoresis, then subcloned into an EcoRI-cut derivative (pGEMBL) of the standard cloning vector pBR322 containing a polylinker having a unique EcoRI site, a BamH1 site and numerous other unique restriction sites. An exemplary vector of this type is described by Dente et al., Nucleic Acids Research 11:1645 (1983). Restriction mapping indicated the presence of restriction sites corresponding to those previously reported for human IL-3. Sequencing revealed the presence of a codon (CCC) encoding proline in position 8 of the mature protein. Sequencing corresponding cDNAs isolated from three additional clones confirmed this variation from the sequence reported by Yang et al., Cell 47:3 (1986). Injection of RNA transcribed from the putative hIL-3 sequence into Xenopus oocytes resulted in expression of significant activity in the bone marrow proliferation assay. A yeast expression vector was constructed by digesting pBC102 K22 (ATCC 67,255) with Asp718 and SpeI, removing a fragment comprising the mature sequence of human G-CSF, and ligating the following oligonucleotide polylinker to the vector fragment: Asp718 QNcoI DStuI GTACCAGCTAGCTAGCTAGCTCCATGGATCCAGGCCTA GTCGATCGATCGATCGAGGTACCTAGGTCCGGATGATC tBamHI SpeI The resulting vector was designated pBC115. cDNA encoding hIL-3 was excised from the pGEMBL cloning vector by digestion with HpaI and BamHI, providing a fragment extending from amino acid 14 of mature hIL-3 to a site downstream of the coding sequence. Oligonucleotides were synthesized and assembled to provide a fragment encoding (1) the C-terminal 5 amino acids of the yeast factor leader peptide, beginning with the Asp718 site and terminating in a KEX2 pro tease recognition site; (2) an 8 codon sequence encoding a synthetic N-terminal "flag" identification peptide (DYKDDDDK; see above); and (3) a short sequence encoding the N-terminal 14 amino acids of mature hIL-3 protein up to and including an HpaI blunt end. The sequence of this 84 base pair KpnI-HpaI fragment, which was constructed from 4 oligomers of approximately 40 nucleotides each, is set forth below: 5'-GTA CCT TTG GAT AAA AGA GAC TAC AAG GAC GAC GAT GAC AAG GA AAC CTA TTT TCT CTG ATG TTC CTG CTG CTA OTG TTC Val Pro Leu Asp Lys Arg Asp Tyr Lys Asp Asp Asp Asp Lys < -----&alpha; ;-factor leader--- ---identification peptide----ol -GCT COO ATG ACC CAG ACG AOG COO TTG AAG ACC AGC TGG GTT-3' -CGA GGG TAC TGG GTC TGC TGC AGG AAC TTC TGG TCG ACO CAA Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp Val hIL-3 > The foregoing fragment, the Asp718-BamHI vector fragment derived from pBC115, and the HpaI-BamHI hIL-3 fragment were ligated together to provide an expression plasmid, designated pBC125. Plasmid DNA from an E. coli clone containing the pBC125 vector was isolated and used to transform yeast strain XV2181 for expression of the hIL-3 gene product. The transformed yeast strain was grown in shake flask culture under conditions promoting derepression of the ADH2 promoter. Yeast-conditioned supernatants were collected by centrifugation and assayed for bone marrow proliferation-inducing activity. These experiments indicated high level expression of the hIL-3 protein.

Claims: CLAIHS What is claimed is: 1. Purified recombinant human interleukin-3 (hIL-3). 2. A crude yeast-conditioned culture supernatant comprising hIL-3 at a protein concentration of' at least 1.0 ug/ml. 3. A crude yeast-conditioned culture supernatant according t° Claim 2, comprising hIL-3 at a protein concentration of at least 10 Wmll . 4. A protein according to Claim 1, comprising an amino acid sequence which is substantially homologous to the amino acid sequence of FIG. 1. 5. A protein according to Claim 4, comprising a mutant amino acid sequence comprising at least one amino acid substitution, deletion, or insertion inactivating an N-glycosylation site or inactivating a KEX2 pro tease recognition site. 6. A recombinant expression vector comprising a DNA segment encoding hIL-3. 7. A recombinant expression vector comprising a DNA segment encoding a protein according to Claim 5. 8. A yeast recombinant expression vector according to Claim 6. 9. A yeast recombinant expression vector according to Claim 8, pBC125. 10. A recombinant expression system comprising a vector according to Claim 6. 11. A recombinant expression system comprising a vector according to Claim 7. 12. A recombinant expression system comprising a vector according to Claim 8. 13. A yeast recombinant expression system comprising a vector according to Claim 9. 14. A process for preparing purified hIL-3 or an analog thereof, comprising culturing a system according to Claim 10 under conditions promoting expression. 15. A process for preparing purified hIL-3 or an analog thereof, comprising culturing a system according to Claim 11 under conditions promoting expression. 16. A process for preparing purified hIL-3 or an analog thereof, comprising culturing a system according to Claim 12 under conditions promoting expression. 17. A process for preparing purified hIL-3 or an analog thereof, comprising culturing a system according to Claim 13 under conditions promoting expression.

Inventors: ANDERSON DIRK M, COSMAN DAVID J, PRICE VIRGINIA L, ANDERSON, DIRK, M., COSMAN, DAVID, J., PRICE, VIRGINIA, L.
Applicants: IMMUNEX CORP, IMMUNEX CORPORATION
Application year: 1988
Main classification: C12P21
IPC codes: A61K38, A61P7, A61P37, C07K1, C07K14, C12N5, C12P21, C12R1