NZ203661A - Recombinant dna method for production of human immune interferon - Google Patents

Description

Z 03 6 6 t Priority Date(s): Q,.. 9.;% P lomplete Specification Filed: .23.'.3. tw C/P/y/s/op.;. ... 1 J.03.}.. ...

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Publication Date: ......J JUL 1986] P.O. Journal, No: ...

Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION "NovellDNA and Use Thereof" -£,WE TAKEDA CHEMICAL INDUSTRIES, LTD., of 27, Doshomachi 2-chome, Higashi-ku, Osaka 541 Japan, a Japanese company, hereby declare the invention, for which £/we pray that a patent may be granted to me/us, arid the method by which it is to be performed, to be particularly described in and by the following statement:- ..\ ;-.sh.-C:v-PC:• j,.. ..... - 203661 - lfc- Novel recombinant DNA and Use thereof This invention relates to a DNA containing a base sequence coding for the human immune interferon polypeptide, a host transformed therewith, and a method of producing human immune interferon using said host, and various inter-5 feron products.

Background Art Interferons (hereinafter sometimes abbreviated to "IFs") are proteins produced by cells of higher animals in response to the stimulation of a virus, nucleic acid, etc., 10 and have antiviral, antitumor and other activities.

At present it is known that human IFs include three types, namely alpha, beta and gamma types, which are different in properties. The alpha and beta types are induced by a virus or nucleic acid and the gamma type is 15 usually induced by mitogens.

Comparatively, the study of alpha type IF (hereinafter referred to an "IF-ot") and beta type IF (hereinafter referred to as "IF-3") has considerably advanced, and the mechanisms of their production have been elucidated to a 20 considerable extent. Furthermore, advances in gene manipulation techniques have made it possible to produce IF-a and IF-6^ by growing Escherichia coli or to produce IF-a^ by growing a yeast; said IFs are thus obtainable in large amounts as physiologically active proteins [See D. V. 25 Goeddel et al., Nature, 287, 411 (1980); E. Yelverton et al. , Nucleic Acids Res., 9, 731 (1981); D. V. Goeddej " < 23AU6/98S 203661 Nucleic Acids Res., 8_, 4057 (1980); R. Hitzeman et al. , Nature, 293, ill (1981)]. Furthermore, attempts have been made to produce such IFs in amounts sufficiently large to enable commercial clinical use thereof.

Gamma type IF (hereinafter sometimes referred to as "IF-y") is also called immune interferon (hereinafter sometimes referred to as "IFI") because it is produced by immunocompetent cells under those conditions which may induce blast-transformation of lymphocytes or production of lymphoKine. IFI has greater antiproliferative and antitumor activities than IF-a and IF-3, and therefore is more promising from the viewpoint of clinical application. However, no efficient production system therefor has been established because fresh lymphocytes are necessary. Furthermore, the possibility has been suggested that different cell species produce IFIs of different molecular species in different experimental systems.

On the other hand, since IFs are highly species-specific, human-derived IFs must be used in application to humans. However, mass production of human immune interferon has been so difficult that its application in clinical practice has thus far been impossible. Development of a technique capable of producing large amounts of highly pure human IFI in a simple manner and at low cost has been eagerly waited for.

Making use of gene manipulation techniques, the present inventors have engaged in developing a technique which enables production of the desired IFI by cloning the human IFI gene, incorporating the resulting recombinant DNA molecule into a host, thereby causing expression of the human IFI gene in said host and recovering the human IFI.

Disclosure of the Invention This invention provides a DNA containing a base sequence coding for the human immune interferon polypeptide, a host transformed with said DNA, a method of pro^uci^ljt/>. ■ iomsvX:- W.»>wv.^I»wwwimi«W: 20366 human immune interferon or a polypeptide equivalent thereto, which method comprises growing a host transformed with said DNA, and novel interferon products.

Thus, the DNA provided by the present invention is a having the ! 2_ base sequence represented by the formula (5') TGT TAC TGC CAG GAC CCA TAT GTA AAA GAA GCA GAA AAC CTT AAG AAA TAT TTT AAT GCA GGT CAT TCA GAT GTA GCG GAT AAT GGA ACT CTT TTC TTA GGC ATT TTG AAG AAT TGG AAA GAG GAG AGT GAC AGA AAA ATA ATG CAG AGC CAA ATT GTC TCC TTT TAC TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA AAG AGT GTG GAG ACC ATC AAG GAA GAC ATG AAT GTC AAG TTT TTC AAT AGC AAC AAA AAG AAA CGA GAT GAC TTC GAA AAG CTG ACT AAT TAT TCG GTA ACT GAC TTG AAT GTC CAA CGC AAA GCA ATA CAT GAA CTC ATC CAA GTG ATG GCT GAA CTG TCG CCA GCA GCT AAA ACA GGG 140 AAG CGA AAA AGG AGT CAG ATG CTG TTT CGA GGT CGA 146 AGA GCA TCC CAG -X (31) (I) wherein X is TAA, TGA or TAG.

The DNA of formula (I) codes for the polypeptide of the formula (N) Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Yal Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys 25 lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin ^ Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu 30 Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arlg Arg Ala Ser Gin (C) (II) or a polypeptide equivalent thereto in immunological or 35 biological activities.

The above DNA (I) may have at the 51-end thereof 203661 4 ' ATG AAA TAT ACA AGT TAT ATC TTG GCT TTT CAG CTC TGC ATC GTT TTG GGT TCT CTT GGC 31 (III) or ATG (IV) .

When the DNA (I) has the DNA of formula (III) at the 5' end, it codes not only for the polypeptide (II) but also for the polypeptide having Met Lys Tyr Thr Ser Tyr lie Leu Ala Phe Gin Leu Cys lie Val Leu Gly Ser Leu Gly added to the N-terminus or a polypeptide equivalent thereto in activities. When the DNA (I) has the DNA of formula (IV) at the 5'-end, it codes not only for the polypeptide (II) but also for the polypeptide having Met added to the N-terminus or a polypeptide equivalent thereto in activities The DNA (I) is preferably connected at a site downstream from a promoter. Suitable promoters therefor include the tryptophan (trp) promoter, the bacteriophage lambda P promoter or other known promoters such as the lactose (lac) promoter, and the protein chain elongation factor Tu (tufB) promoter. Preferable among them are the trp promoter and the P^ promoter. The P^ promoter is a very strong promoter that can be efficiently and conveniently controlled by the A cl repressor. The gene encoding the repressor carries a mutation, clts2 or clts857, which renders the repressor temperature-sensitive. At 30°Cthe repressor functions normally, and from about 37°C to about 42°C it is inactivated. Thus the PT promoter is repressed (turned-off) at 30°C and derepressed (turned-on) at 42°C. This feature is desirable herein in that the gene product " of interest may be toxic to the cell or that if present in large quantity it would be detrimental to cell growth. The ability to control the PL promoter allows one to grow the culture at about 30°C to about 36°C without expressing the gene product and at an optimum time, shift the temperature to about 37°C to about 4 2°C to produce the desired gene product.

In general formula (I), X, which is an oligodeoxy-ribonucleotide, is preferably TAA. ■>• im»>*./»». w t»?w~* HeJS .v7;i<WtetiW.«lS(*S'.:' 20366( The symbols as used herein in the specification, drawings and claims have the meanings shown in Table 1.

Table 1 DNA = deoxyribonucleic acid 5 A = adenine T. = thymine G = guanine C = cytosine RNA = ribonucleic acid 10 dATP = deoxyadenosine triphosphate dTTP = deoxythymidine triphosphate dGTP = deoxyguanosine triphosphate dCTP = deoxycytidine triphosphate ATP = adenosine triphosphate 15 EDTA = ethylenediamine tetraacetate SDS = sodium dodecyl sulfate Gly = glycine Ala = alanine Val = valine 20 Leu = leucine lie = isoleucine Ser = serine Thr = threonine Cys = cysteine 25 Met = methionine Glu = glutamic acid Asp = aspartic acid Lys = lysine Arg = arginine 3 0 His = histidine Phe = phenylalanine Tyr = tyrosine Trp = tryptophan Pro = proline 35 Asn = asparagine = glutamine Gin RBS = ribosome binding site o 203661 The DNA of the present invention having the base sequence represented by the formula (I) can be produced, for example, by (a) growing human IFI-secreting cells, for example, human 5 peripheral blood lymphocytes, (b) isolating from the cells a messenger (m) RNA coding for human IFI( (c) synthesizing a single-stranded complementary (c) DNA with the use of said messenger RNA and reverse transcriptase, (d) converting said (c) DNA to a double-stranded DNA, (e) inserting said double stranded DNA into a plasmid, (f) transforming a host, e.g. Escherichia coli or Bacillus subtilis, with said recombinant plasmid. (g) isolating a plasmid containing the required DNA from the culture of said host, (h) if desired, excising out the cloned DNA from the plasmid, and (i) if desired, connecting said cloned DNA at a site 20 downstream from a promoter in a vehicle.

The human peripheral blood lymphocytes to be used in the practice of the invention may be either uninduced cells or induced cells.

The IFI inducer to be used in practicing the inven-25 tion may be any agent capable of inducing human lymphocytic cells to produce IFI and includes, among others, phytohemag^glutinirfPHA) , Concanavalin A (ConA) , staphylococcal enterotoxin A (SEA), neuraminidase-galactose oxidase (NAGO), 12—0—tetradecanoylphorbol—13—acetate (TPA) 30 and combinations thereof.

Induction of IFI can be effected by growing the cells in a per se known medium, such as RPMI-164 0 or MEM medium, preferably in RPMI-164 0 medium containing foetal bovine serum. The cultivation is carried out at a temperature 35 of 35-39°C, desirably 36-38°, for 12-72 hours, desi: 22-26 hours. The cells are collected (by a per s^k^qwt^ 2' 203661 method) and, after addition, for example, of guanidine thiocyanide, bentonite, heparin., .pqlyyinyl sulfate, aurintricarboxylic ,acid, vanadium complex, diethyl pyrocarbonate (DEPC)or sodium dodecyl sulfate (SDS) as the RKase inhibitor, lysed for RNA extraction by adding sodium N-lauroylsarcosinate t Tween 80 or Nonidet P-40, for example. After repeated extraction as necessary by adding phenol, phenol-chloroform or the like to the RNA-containing extract, RNAs are collected by precipitation with ethanol. Since many of mRNAs present in the cytoplasm of eukaryotic cells are known to contain a poly (A) sequence at the 3' end, poly (A) + RNAs are collected with the use of oligo (dT) cellulose, poly(U) sepharose or the like and fractionated by the sucrose density gradient centrifugation method. Aliquots of each fraction obtained are injected into oocytes of Xenopus laevis (a frog species) for translation. IJ. B. Gurdon, et al., Nature, 233, 177 (1971)]. The produced protein is assayed for antiviral activity. In this manner, the mRNA for IFI can be obtained. The mRNA can also be purified by formamide-polyacrylamide gel electrohoresis or by agarose gel electrophoresis or by agarose gel electrophoresis using glyoxal.

Using the thus-obtained mRNA as the template, the single-stranded complementary DNA (cDNA) chain is synthesized by a per se known method, for instance, with the use of a reverse transcriptase. The cE^a is then converted to the double-stranded DNA [T. Maniatis et al., Cell, 8, 163 (1976)].

This DNA is inserted, for example, into the plasmid pBR322, e.g., at the PstI or SphI restriction endonuclease cleavage site, for example by the dG-dC or dA-dT homopolymer tailing method described in T. S. Nelson [Methods in Enzymology, 68, 41 (1979), Academic Press Inc., New York], The recombinant plasmid is then introduced, for example, into Escherichia coli xl^76. bv transformation.

The transformants can be selected as 2 03 6 6 1 resistant or ampicillin-resistaht colonies. Separately, a probe oligonucleotide having a base sequence supposedly corresponding to at least part of the amino acid sequence of the IFI polypeptide is synthesized chemically and labeled 32 with P. Using the labeled oligonucleotide as the probe, the desired clones are secondarily screened out from among the already-obtained tetracycline- or ampicillin-resistant transformants, for example by the per se known colony hybridization method [M. Grunstein and D. S. Hogness, Proc. Natl. Acad. Sci. USA, 72, 3961 (1975)].

For confirming the presence of the IFI gene, the base sequence of the clones showing the positive colony hydridization result is determined, for example, by the Maxam-Gilbert method [A. M. Maxam and W. Gilbert, Proc.

Natl. Acad. Sci. USA, 74, 560 (1977)] or the dideoxy-nucleotide synthetic chain termination method using phage M13 [J. Messing et al. , Nucleic Acids Res., 9_, 309 (1981)]. The whole or part of the IFI gene may also be excised from the clones obtained and then connected downstream from an adequate promoter and SD (Shine-Delgarno) sequence, for introduction into an adequate host.

The promoter includes the above-mentioned promotors and the host includes such bacteria as Escherichia coli or Bacillus subtilis, and other microorganisms, such as the yeast Saccharomyces cerevisiae. Preferred as the host is Escherichia coli, such as E. coli strains 294 or W3110, especially E. coli 294.

Escherichia coli 294 is a known bacterial strain [K. Backman et al., Proc. Natl. Acad. Sci. USA, 73, 4174 (1976)] and is also on deposit in the Institute for Fermentation, Osaka (Deposit No. IFO-14171).

The transformation of a host with the DNA of the invention is carried out, for example, by the known method of S. N. Cohen, et al. [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)].

The compound of the formula: w*M*»sr"w •' - ' f Z •4 4 • i 203661 Y-Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Wet 5 Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val 10 Gin Arg Lys Ala lie His Glu Leu lie Gin Val Wet Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin (II') wherein Y is (a) hydrogen, (b) Met or (c) Met Lys Tyr Thr Ser 15 Tyr lie Leu Ala Phe Gin Leu Cys lie Val Leu Gly Ser Leu Gly can be produced, for example, by growing thus obtained host, accumulating compound (II') in the culture broth and recovering the same.

The host is grown in a per se known medium. | 20 The medium is, for example, M9 medium containing ! glucose and casamino acids [J. Miller, Experiments in f Molecular Genetics, 4 31-433, Cold Spring Harbor Laboratory, New York, 1972]. For efficient working of the promoter, - a derepressing agent such as $-indolylacrylic acid may be \ 25 added as necessary.

; The incubation is carried out generally at 15-43°C for jw 3-24 hours, if necessary with aeration and/or stirring.

When a transformant having a X cl repressor and an expression vector containing the PT promoter is used, Li lower temperatures are used for incubation, i.e., approximately 30° to about 36°C and the X cl repressor may advantageously be inactivated under incubation at about 37° to 42°C.

After the incubation, the cells are collected by a 35 known method and, after suspending in a buffer sojjjtiign, for instance, lysed, for example, by ultrasonia^f^realLfterit, 2 036 61 by lysozyme, and/or freezing and thawing, or other methods [e.g., Gunsalus, "Extraction of Enzymes from Microorganisms", Methods in Enzymology 1:51 (1955). The supernatant is recovered by centrifugation.

The isolation of human IFI from said supernatant may be carried out by commonly known methods for protein purification.

The IFI, or polypeptide equivalent thereto in immunological or biological activities, as produced in accordance with the invention has immunological and biological activities equivalent to those of the IFI species produced by previously known methods and can be used in the same manner and for the same purposes as the known IFI species.

IFI has exhibited antiviral, antitumor, growth inhibition and immunosuppression activity. The compound can be mixed with a sterile pharmaceutically acceptable carrier such as sterile water, human serum albumin, (HSA), normal saline solution, or other carriers well known in the art, and administered orally or topically. Daily dosages for intravenous treatment range from about 10 million units to about 100 million units per day for normal adults, preferably about 5 0 to 60 million units per day. The pharmaceutical product may contain other pharmaceutically acceptable ingredients, such as salts, diluents, adjuvants, other carriers, buffers, binders, surface active agents, preservatives, etc. Preparations for parenteral use could be provided as an ampoule of a sterile solution or suspension of the product with water or another pharmaceutically acceptable liquid as the carrier therefore, or an ampoule of sterile powder (preferably obtained by lyophilization of a solution of IFI) for dilution'-withpharmaceutically acceptable liquid.

Moreover, the product of the present invention may contain other pharmaceutically active components which are suitably administerable with IFI, including for example lymphocyte or fibroblast interferon, in amounts 2 03 6 6 1 ranging from about 1-99%, based on the amount of IFI.

Brief Description of the Drawings Fig. 1 shows the restriction enzyme cleavage map of the plasmid pHIT3709 as obtained in Example 1 (vii) hereof, the portion Y/////A indicating the portion coding for the peptide supposed to be the signal peptide and the portion K\\\\\\V{ indicating the portion coding for the IFI polypeptide. Fig. 2 shows the primary structure (base sequence) of the plasmid pHIT3709 as obtained in Example 1 (vii). Fig. 3 shows the construction scheme for Example 2.

Fig. 4 is a partial restriction map of a 1200 bp Bgl II - Bam H 1 fragment containing a PL promoter. Fig. 5 illustrates the construction of an expression vector pRC14, which contains the lambda PT promotor on a 350 bp insert.

Xj Fig. 6 illustrates the scheme used to isolate the 82 bp fragment containing the SD sequence for the int gene of bacteriophage lambda for construction of expression vector pRC15.

Fig. 7 illustrates the construction of the expression vector pRC15. Fig. 8 illustrates the construction of expression vectors pRC21 and pRC2 2. Figure 9 illustrates the construction of expression vector pRC23 with a P^ promoter and a synthetic RBS.

Figure 10 shows the scheme used to insert the gene coding for immune interferon into pRC22, and the sequence of the promoter-gene junction.

Fig. 11 illustrates the insertion of the immune interferon gene into expression vector pRC2 3, and the sequence of the promoter-gene junction and its modification.

Example 1 (i) Isolation of mRNA coding for IFI Lymphocytes prepared from human peripheral blood were incubated in RPMI-164 0 medium (containing 10% foetal bovine serum) containing 15 ng/ml of 12-0-tetradecanoylphorbol-13-acetate (TPA) and 4 0 yg/ml of Concanavalin A at 37°C for IFI induction. After 24 hours of incubation, the thus- *1 J 263661 induced human lymphocytes (1 * 10"^ cells) vere destroyed and denatured in a thioguanidine s.olution. {-5 M guanidine thiocyanate, 5\ mercaptoethanol, 50 mM Tris HC1 (pH 7.6), 10 mM EDTA) in a Teflon homogenizer. Then sodium N-lauroyl sarcosinate was added in the concentration of 41 and the mixture, after homogenization, was layered on 6 ml of 5.7 M cesium chloride solution (5.7 M cesium chloride, 0.1 M EDTA) and centrifuged at 15°C and 24,000 rpm for 30 hours using a Beckman SW27 rotor to give an RNA precipitate .

This RNA precipitate was dissolved in 0.25% N-lauroyl sarcosinate and then precipitated vith ethanol to give 8.3 mg of RNA. This RNA was allowed to be adsorbed, in a high concentration salt solution (0.5 M NaCl, 10 mM Tris HC1 (pH 7.6), 1 xnM EDTA, 0.3% SDS) , on an oligo (dT) cellulose column and the poly (A)-containing mRNA was eluted with a low concentration salt solution (10 mM Tris HC1 (pH 7.6), 1 mM EDTA, 0.3% SDS). There was collected 700 pg of mRNA.

This mRNA was again precipitated with ethanol, then dissolved in 0.2 ml of a solution (10 mM Tris HCl (pH 7.6), 2 mM EDTA, 0.3% SDS), treated at 65°C for 2 minutes and fractioned to 22 portions by 10-35% sucrose density gradient centrifugation at 20°C and 25,000 rpm for 21 hours using a Beckman SW 27 rotor. Aliquots of each fraction were injected into Xenopus laevis oocytes and the proteins synthesized were assayed for interferon activity, i.e., assayed for antiviral activity as assayed by the inhibition test of the cytopathic effect of the vesicular stomatitis virus against human amnion-derivea VJISK cells [W. E.

Stewart, The Interferon System 11-26, Springer-Verlag, New York (1979)]. In this manner, it was revealed that fraction 12 (the sedimentation coefficient being 12-14S) had an activity of 195 units (international 3FN units) per microgram of RNA. The mRNA in the thus-obtained fraction 12 weighed about 2 0 vg. ,f> { 2 03661 (ii) Synthesis of single-stranded DNA Using the above mRNA and a reverse transcriptase, 100 yl of a reaction mixture (5 yg of mRNA, 50 yg of oligo (dT), 100 units of reverse transcriptase, 1 mM each of dATP, dCTP, dGTP and dTTP, 8 mM MgCl2, 50 mM KCl, 10 mM dithiothreitol and 50 mM Tris-HCl (pH 8.3) were incubated at 42°C for an hour, then deproteinized with phenol, and treated with 0.1 N NaOH at 70°C for 20 minutes for degradation of RNA. (iii) Synthesis of double-stranded DNA The thus-synthesized single-stranded complementary DNA was subjected to reaction in 50 VI of a reaction mixture (the same mixture as above-mentioned except that the mRNA and oligo (dT) were absent) at 42°C for 2 hours for synthesizing the double-stranded DNA. (iv) Addition of dC tails The above double-stranded DNA was treated with nuclease SI in 50 yl of a reaction mixture (double stranded DNA, 0.1 M sodium acetate (pH 4.5), 0.25 M NaCl, 1.5 mM ZnSO^, 60 units SI nuclease) at room temperature for 30 minutes. The reaction mixture was deproteinized with phenol, the DNA was precipitated with ethanol and subjected to terminal transferase reaction in a mixture of double-stranded DNA, 0.14 M potassium cacodylate, 2 mM dithiothreitol, 1 mM CoCl2» 0.15 mM dCTP, 3 0 units terminal transferase in 0.3 M Tris (base) (pH 7.6), at 37°C for 3 minutes for addition to the double-stranded DNA of about 2 0 deoxycytidine chains at each 3'-end of the DNA.

This series of reactions gave about 300 ng of a double-stranded deoxycytidine-chain-containing DNA. (v) Cleavage of Escherichia coli plasmid and addition of dG tails Separately, 10 yg of Escherichia coli plasmid pBR322 DNA was treated with restriction enzyme PstI in 50 pi of a reaction mixture containing 10 yg pBR322 DNA, 50 mM NaCl, 6 mM MgC^, 6 mM 2-mercaptoethanol, 100 yg/ml bovine 2036 6i serum albumin, and 20 units PstI in 6 mM Tris HC1 (pH 7.4), at 37°C for three hours, for cleavage at the one PstI recognition site present in the pBR322 DNA. The reaction mixture was then deproteinized with phenol and the DNA was further treated with terminal transferase in 50 yl of a reaction mixture containing 10 yg DNA, 0.14 M potassium cacolylate, 2 mM dithiothreitol, 1 mM C0CI2, 0.15 mM dGTP, and 3 0 units terminal transferase in 0.3 M Tris (base) (pH 7.6) at 37°C for 3 minutes for addition of about 8 deoxyguanosine residues at each 3'-end of the above plasmid pBR322 DNA. (vi) Annealing of cDNA and transformation of Escherichia coli The annealing was effected by heating 0.1 yg of the thus-obtained dC-tailed synthetic double-stranded DNA and 0.5 yg of the above dG-tailed plasmid pBR322 in a solution containing 0.1 M NaCl, 50 mM Tris HC1 (pH 7.6) and 1 mM EDTA at 65°C for 2 minutes and then at 45°C for 2 hours, followed by gradual cooling. The transformation of Escherichia coli was performed by the method of Enea et al. [J. Mol. Biol., 96, 495 (1975)]. (vii) Isolation of plasmid containing cDNA About 8500 tetracycline-resistant colonies were thus isolated, and the DNA of each colony was fixed to a nitrocellulose filter [M. Grunstein and D. S. Hogness, Proc. Natl. Acad. Sci. USA, 72 3961 (1975)].

Separately, based on the amino acid sequence of IFI as reported by D. V. Goeddel et al. [Nature, 295, 503 (1982)], two base sequences 5' TCCTGGCA^TA^C 3' and 5' r CTT AC^TTCAT^TC^TC^T 3', presumably corresponding to amino acids Nos. 1-5 (Cys-Tyr«Cys•Gin•Asp) and amino acids Nos. 77-82 (Lys-Gln-Asp-Met*Asri-Val) of said IFI sequence, respectively, were chemically synthesized by the triester method described in R. Crea et al. [Proc. Natl. Acad. Sci USA, 75, 5765 (1978)]. These oligonucleotides were treated with T4 polynucleotide kinase in 50 yl of a reaction 203661 mixture (0.2 yg oligonucleotide, 10 mM MgCl , 10 mM 32 mercaptoethanol, 5 0 yCi y- P-ATP, and 3 units T4 polynucleotide kinase in 50 mM Tris HC1 (pH 8.0), at 37°C for an hour. These oligonucleotides thus labeled with 32 P at the 5'-end were used as probes and annealed with the DNA on the above-mentioned nitrocellulose filter by the method of Lawn et al [Nucleic Acids Res., 9, 6103 (1981)]. Four colonies reactive to the above two oligonucleotide probes were isolated using autoradiography.

Plasmid DNAs were isolated from the bacterial cells of each of these colonies by the method of Birnboim and Doly [Nucleic Acids Res. 1_, 1513 (1979)]. The inserts in the plasmid DNAs were excised with the PstI restriction enzyme. From among the isolated plasmids, the one containing the longest cDNA insert was chosen and named "pHIT3709." The restriction enzyme map of this plasmid is shown in Fig. 1.

The primary structure (base sequence) of the cDNA sequence inserted in the pHIT3709 plasmid was then determined by the dideoxynucleotide synthetic chain termination method and by the Maxam-Gilbert method.

Said primary structure was as shown in Fig. 2.

This primary structure is in agreement with that of IFI cDNA as reported by Gray et al. [Nature, 295, 503-508 (1982) ] except that the former differs from the latter in one codon; namely the former's codon for the No. 140 amino acid is CGA, while the latter1 s is CAA for Gin. The protein determined by this base sequence presumably consists of 166 amino acids whose synthesis is initiated from the No. 30 nucleotide, namely the ATG codon, which is the signal for the start of protein synthesis. The first 20 amino acids probably constitute a signal peptide. The amino acid sequence, too, is different from the IFI reported by Gray et al. with regard to the No. 140 amino acid (Arg in place of Gin). 2 03 6 6 Prom the above-mentioned primary structure, it is clear that this plasmid has the entire coding region for the IFI protein. This fact permits making hosts such as Escherichia coli produce immune interferon by transfering 5 the DNA sequence inserted in this plasmid to another expression plasmid.

Example 2 The insert in the pHIT3709 plasmid as obtained in Example 1 was excised from the No. 100 nucleotide thereof 10 with the BstNI restriction enzyme (cf. Fig. 2 and Fig. 3) and, after filling in of the sticky ends with DNA polymerase I large fragment (also called Klenow's DNA polymerase I), ligated, with the use of T4 DNA ligase, with the oligonucleotide adapter: 15 CGATAATGTGTTACTGCC TATTACACAATGACGG which was chemically synthesized by the above-mentioned triester method and contains the protein synthesis start codon ATG.

A second plasmid (ptrp601) containing pBR322 and the tryptophan promoter portion of Escherichia coli (i.e., the promoter- and operator-containing DNA fragment), containing 276 base pairs, was constructed as the expression plasmid, using the method of G. N. Bennett et 25 al. [J. Mol. Biol. 121, 113 (1978)].

This plasmid ptrp601 was cut with the Clal restriction enzyme, and the IFI gene with the above-mentioned adapter ligated thereto was inserted into the plasmid vector downstream from the tryptophan promoter, using T4 30 DNA ligase. An IFI expression plasmid pHIT-trp301 was thus constructed (Fig. 3).

A bacterial strain containing this plasmid pHIT* trp3 01 was obtained by transforming Escherichia coli 2 94 with that plasmid by the method of Cohen et al. supra. 35 Example 3 The bacterial strain containing the IFI expression .. ,-^TT ^ ■■ ' yr-i VT"' 1 ' 1 ^ ^ 203661 plasmid as obtained in Example 2 is grown in M9 medium ''■"containing 0; 5% glucose and 0.5% casamino acids at .37 °C ■ • for 6 hours. The cells are then collected and suspended in 0.15 M NaCl solution in 0.02 M phosphate buffer (pH 6.8) 5 and lysed by ultrasonic treatment. Centrafugation gives a supernatant. The interferon activity of this supernatant can be measured by assay on WISH cells in accordance with the method mentioned in Example l(i) supra, and described by Vf. E. Stewart, supra.

Example 4 The bacterial strain containing the IFI expression plasmid, pHIT*trp301, as obtained in Example 2 was grown in M9 medium containing 0.5% glucose and 0.5% casamino acids at 37°C for 6 hours under shaking.

Indole acrylic acid was added to a final concentration of 2 0 yg/ml and incubation was further continued at 37°C for 3 hours under shaking.

I A 10 ml sample was taken, the cells were collected by centrifugation and resuspended in 0.1 ml of 50 mM 20 Tris (pH 7.4) containing 10 percent sucrose, and the suspension was guick-frozen in a dry ice/ethanol bath.

Then, the cells were thawed at 20°C, and transferred to an ice bath. To the cell suspension NaCl was added to a concentration of 100 mM, EDTA to 10 mM, spermidine to 25 20 mM, and lysozyme to 200 pg/ml. The mixture was kept on ice for 45 minutes, then incubated at 37 °C for 2 minutes. Lysis was apparent by the increase in viscosity of the suspension. Cell debris was removed by centrifugation and the supernatant was assayed for IFI anti-viral activity on 30 WISH cells. This experiment resulted in a yield of 1280 units of IFI activity per ml of cell extract. 'sj' The following illustrative embodiments illustrate the expression of IFI utilizing the >. P, promoter system ±j together with the IFI coding DNA segment of the present invention. 203661 Example 5 In order to isolate a 350 base pair .(bp) fragment .. containing the A p promoter, 250 pg of X cI857 Sam7 Jj "— — DNA (Miles Laboratories) was digested with restriction 5 endonucleases BamHl and Bgl II, and the products were separated on agarose gel by electrophoresis. A 1200 bp fragment containing the P^ promoter was isolated from the gel (see Fig. 4).

This 1200 bp fragment was then digested completely 10 with Hpal and partially digested with Hinf I and a 350 bp fragment was isolated from a 5% polyacrylamide gel. Digestion with Hpal eliminated 2 Hinfl partial fragments of about 350 bp that would have otherwise contaminated the desired fragment.

This 350 bp fragment contains the promoter and the operator sites (O^].' ^L2' ^L3^ *"° X cl repressor binds. The Hinfl site, which occurs between the Shine—Dalgarno (SD) seguence of the X N-gene and the initiating codon for the N gene (see Fig. 4), 20 allows for the construction of hybrid ribosome-binding sites for the purpose of expressing foreign genes in E. coli. This Hinfl site, may also be converted to an EcoRl site.

The 350 bp Bgl II - Hinfl fragment was cloned into 25 plasmid pRCl which had been digested with EcoRl and Bgl II. pRCl is a derivative of pBR322 that contains a Bgl II site adjacent to the EcoRl site (see Fig. 5). Figure 5 shows the sequences of the joining terminal and indicates how the EcoRl terminus of the vector can join to the 30 Hinfl terminus of the fragment, thus reconstituting the EcoRl site. (The circled A in Figure 5 was filled-in in vivo.) The resulting recombinant plasmid was designated pRC14.

Using the same techniques, a number of other expres-35 sion vectors using the P promoter were also constructed (Figs. 6, 7, 8, and 9). pRCl5 contains, in addition to/^- .'-Cv 203661 the 350 bp Bgl II - Hinfl fragment described above, a 55 base pair fragment) (Hinf [-Mbol]) that contains the SD sequence of the x *int gene (see Figs. 6 and 7). pRC21 and pRC22 are analogous to pRC14 and pRC15, respectively. The principle difference between these plasmids is the orientation of the inserted PL - containing fragment, and thus the direction of transcription (see Fig. 8). pRC23 was constructed by ligating synthetic oligonucleotides containing a "consensus" RBS (Scherer, et al., Nucleic Acids Research, 8y 3895 (1980) to a 250 bp Bgl II - Hae III fragment containing the PL promoter, and inserting the ligation product into pRC2, which is a derivative of pBR322 that contains a Bgl II site adjacent to the EcoRl sfte (these sites in pRCl are transposed in pRC2), as shown in Figure 9.

Pl - expression vectors were also constructed containing the Immune Interferon (IFI) gene from plasmid pHIT3709, from Example 1, supra, as shown in the following examples.

Example 6 A 900 bp BstNl fragment was isolated from pHIT3709 which contains 430 bp of coding sequence for IFI and 470 bp of the 3'-noncoding region. Not present on this fragment are the sequences forthe "signal" portion of IFI and the codons for the first 3 amino acids of the presumed mature protein. To restore the three missing codons and to provide an initiating Met codon, synthetic oligonucleotides were prepared and ligated to the 900 bp fragment (see Fig. 10). The synthetic segment converted both of the BstNl termini to EcoRl termini. The resulting fragment was cloned into vector pRC22 which had been restricted with EcoRl. Orientation of the insert was determined by restriction analysis with Bgl I which cuts within the 3'-noncoding region.

The pRC22 vector containing the IFI gene (denoted pRC22/IFI-900) was seguenced across the promoter-gene junction to assure that all ligation steps occurred as expected. (See Fig. 10).

The test for expression of the IFI gene, strain RRI (pRK248£lts, pRC22/IFI-900) was grown in M9-glucose medi 2 03 6 61 at 30°C to 3-4 x 10** cells/ml, then induced at 42°C for one hour. Prior to induction, additional glucose and casamino acids were added to 1.0 and 0.5 percent, respectively. A 10 ml sample was taken, the cells were 5 collected by centrifugation and resuspended in 0.1 ml of 50 mM Tris (pH 7.4), 10 percent sucrose, and the suspension was quick-frozen in a dry ice/ethanol bath.

The cells were thawed at 20°C, then transferred to an ice bath. NaCl was added to 100 mM, EDTA to 10 mM, spermidine to 20 mM, and lysozyme to 200 ug/ml. The mixture was kept on ice for 45 minutes, then incubated at 37°C for 2 minutes. Cell debris was removed by centri-fugation and the supernatant was assayed for IFI antiviral activity on WISH cells. This initial experiment 15 resulted in a yield of 1280 units of IFI activity per ml of cell extract.

In order to determine the kinetics of induction, the above procedure was repeated. One sample was kept at 30°C as a control, and other samples were taken after 20 induction at 42°C for 30, 60, 90, 120, and 180 minutes.

The samples were processed as described above. The results, shown in Table 2, indicate that at 30°C no activity is produced and that following induction at 42°C the amount of activity detected reaches a maximum at 25 around 90 minutes, then gradually declines.

Table 2 Induction conditions IFI Strain Temperature (°C)-Time Activity, unit/ml RR1(pRK248cIts, pRC22/IFI—900) 30° 0 " 42° - 30'* 120 " 42° - 60 1 120 " 42° - 90' 320 42° - 120' 160 " 42° - 180' 40 *' = minutes 203661 Example 7 pRC22/IFI-900 DNA was further restricted with EcoRl and the 900 bp fragment containing the IFI gene was isolated and inserted into pRC23 which had been restricted with EcoRl (see Fig. 11). The resulting construction, pRC23/IFI-900, contained a RBS significantly different from that in pRC22/IFI (compare Figs. 10 and 11). To test for expression of the IFI gene, strain RRI (pRK24 8cIts, prC23/IFI-900) was grown in M9-glucose media at 30°C to g 3-4 x 10 cells/ml, then induced at 42°C for one hour.

Prior to induction, additional glucose and casamino acids were added to 1.0 and 0.5 percent, respectively. A 10 ml sample was taken, the cells were collected by centrifugation and resuspended in 0.1 ml of 50 mM Tris (pH 7.4), 10 percent sucrose, and the suspension was quick-frozen in a dry ice/ethanol bath. The cells were thawed at 2 0°C, then transferred to an ice bath. NaCl was added to 100 mM, EDTA to 10 mM, spermidine to 20 mM, and lysozyme to 1200 yg/ml. The mixture was kept on ice for 45 minutes, then incubated at 37°C for 2 minutes. Cells debris was removed by centrifugation and the supernatant was assayed to IFI anti-viral activity on WISH cells. pRC2 3/IFI when used to transform E. coli strain RRI, produced about four times more activity than pRC22/IFI, as shown in Table 3.

Table 3 Induction IFI Activity Strain conditions units/ml RRI(pRK248cIts, pRC22/IFI-900) 42° - 90'* 320 RRI (pRK24 8cIts, pRC23/IFI-900) 42° - 90* 1280 RRI(pRK24 8cIts, pRC231/IFI-900) 42° - 90' 640 * i _ = minutes Example 8 pRC23/IFI was further restricted with EcoRl under conditions that resulted in only one of two sites being cut. The resulting molecules were then treated with Pol I 203661 "Klenow" to fill-in the EcoRl termini. The blunt-ends were ligated together with T^ DNA ligase and the DN£. was • used to transform RRI (pRl»248clts) . Transformants were screened for the loss of the EcoRl site at the beginning of the IFI gene and two positives were obtained. One of these, denoted pRC231/IFI-900, was used to transform E. coli strain RRI to express IFI. To test for expression of the IFI gene, this strain RRI PpRK248clts, pRC231/IFI-900) g was grown in M9-glucose media at 30°C to 3-4 x 10 cells/ ml, then induced at 42°C for one hour. Prior to induction, additional glucose and casamino acids were added to 1.0 and 0.5 percent, respectively. A 10 ml sample was taken, the cells were collected by centrifugation and resuspended in 0.1 ml of 50 mM.Tris (pH 7.4), 10 percent sucrose, and the suspension was guick-frozen in a dry ice/ethanol bath. The cells were thawed at 20°C, then transferred to an ice bath. NaCl vas added to 100 mM, EDTA to 10 mM, Spermidine to 20 mM, and lysozyme to 200 yg/ml. The mixture was kept on ice for 45 minutes, then incubated at 37°C for 2 minutes. Cell debris was removed by centrifugation and the 'supernatant was assayed for IFI anti-viral activity on WISH cells. The results are shown in Table 3.

The modification of pRC23/IFI described was designed to extend the linker region from 6 to 10 bp. In the case of IFI, a distance of 6 bp appears to be betterthan Iff-bp for expression of IFI.

Human immune interferon obtained in accordance with the present invention may be purified by a procedure such as the following: All steps are preferably carried out at low temperature, preferably below room temperature, and more preferably at about 4CC. The IFI-containinc materiel, e.g. the supernatant fluid of Example 4, can be sequentially filtered through a 3 micron and then an 0.22 mic Example 9 filter. 2 03661 The next purification step utilizes a high performance weak cation-exchange liquid chromatography column. CM 200 resin, 10 m (Separation Industries) is packed into a 25 cm * 0.46 cm I.D. column, and prepared for use by cleaning with 2 M KC1 and re-equilibrating with 24 mM phosphate buffer, pH 7.5, or with distilled, deionized water (which should be used throughout this procedure) the filtrate from the 0.22 microfilter is diluted with an equal volume of water and pumped at 1.5 ml/min. onto the CM column. The column is then washed with 25 mM potassium phosphate, pH 7.5, until protein in the column effluent is down to background levels, as determined by automatic monitoring of the column with fluorescamine. The column is then eluted at 0.5 ml/ min with a gradient of increasing KC1 (0 to 1 M) in 25 mM potassium phosphate buffer, pH 7.5.

The active fractions from the CM column are pooled and concentrated on a prewashed CF 25 filter cone (Amicon Corp.). The concentrated solution (0.5 ml or less) is then injected into a TSK 25 0 silica gel permeation column (30 x 0.75 cm) (Bio-Rad Laboratories), which has been pre-equilibrated, and eluted with 0.3 M KC1, 25 mM potassium phosphate, pH 7.5 at 24 ml/hr. Fractions are collected at 1 minute intervals. Interferon activity will correspond to a peak of protein. Homogeneous immune interferon may be obtained in one or more fractions at this stage, depending on the starting material and the efficiency of the earlier steps. If necessary, impure fractions can be reconcentrated and re-chromatographed on the TSK or CM column to achieve homogeneity.

The present invention has made it possible to produce human immune interferon or an equivalent thereto in an economical manner. Said interferon is useful as an antiviral or antitumor agent.

Claims (4)

203641 _ - 24 - WHAT-WWE CLAIM IS- What . i n-rl a i mci3. a v-.

1. contains a base sequence' of the formula: (5') tgt tac tgc cag gac cca tat gta aaa gaa gca gaa' aac ctt aag aaa tat ttt aat gca ggt cat tca gat gta gcg gat aat gga act ctt ttc tta ggc att ttg aag aat tgg aaa gag gag agt gac aga aaa ata atg cag agc caa att gtc tcc ttt tac ttc aaa ctt ttt aaa aac ttt aaa gat gac cag agc atc caa aag agt gtg gag acc atc aag gaa gac atg aat gtc aag ttt ttc aat agc aac aaa aag aaa cga gat gac TTC GAA AAG ctg ACT AAT TAT tcg GTA ACT GAC TTG AAT gtc CAA CGC AAA GCA ATA CAT GAA CTC ATC CAA GTG ATG GCT GAA CTG tcg CCA gca gct AAA ACA GGG AAG cga AAA AGG agt cag atg ctg TTT CGA GGT CGA AGA GCA TCC cag -X (3' ) wherein X is TAA, TGA or TAG.

2. A DNA according to Claim 1, wherein it has at the 5'-end thereof the sequence (5') ATG AAA TAT ACA AGT TAT ATC TTG GCT TTT CAG CTC TGC ATC GTT TTG GGT TCT CTT GGC (3').

3. A DNA according to Claim 1, wherein it has ATG at the 5'—end thereof. .

4. A DNA according to any of Claims 1-3, wherein it for the polypeptide : Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin He Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe-ssssss^ Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe /^r b\ & G^SpH V O V* fy ■ - 25 - Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin .

5. A DNA according to any of Claims 1-3, wherein it codes for a polypeptide equivalent to the immune interferon polypeptide in immunological or biological activities.

6. A DNA according to Claim 1, 3 or 5, wherein it codes for the polypeptide: Met Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala lie His Glu Leu lie Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin .

7. A DNA according to Claim 1,2 or 5, wherein it codes the polypeptide: Met Lys Tyr Thr Ser Tyr He leu Ala Phe Gin Leu • o lie Val Leu Gly Ser Leu Gly Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys o * 2 1 FEB 1986 ** "^rroci 203661 . - 26 - { " 'h> V" ..' Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gin Arg Lys Ala lie His Glu leu He Gin Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin .

8. A DNA according to «uny of Claims 1-7, wherein it is linked at a site downstream from a promoter.

9. A method of producing a DNA containing a base sequence of the formula: (5 *) TGT TAC TGC CAG GAC CCA TAT GTA AAA GAA GCA GAA AAC CTT AAG AAA TAT TTT AAT GCA GGT CAT TCA GAT GTA GCG GAT AAT GGA ACT CTT TTC TTA GGC ATT TTG AAG AAT TGG AAA GAG GAG AGT GAC AGA AAA ATA ATG CAG AGC CAA ATT GTC TCC TTT TAC TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA AAG AGT GTG GAG ACC ATC AAG GAA GAC ATG AAT GTC AAG TTT TTC AAT AGC AAC AAA AAG AAA CGA GAT GAC TTC GAA AAG CTG ACT AAT TAT TCG GTA ACT GAC TTG AAT GTC CAA CGC AAA GCA ATA CAT GAA CTC ATC CAA GTG ATG GCT GAA CTG TCG CCA GCA GCT AAA ACA GGG AAG CGA AAA AGG AGT CAG ATG CTG TTT CGA GGT CGA AGA GCA TCC CAG -X (3*) wherein X is TAA, TGA or TAG, which comprises (a) growing human immune interferon-secreting cells, ^ (b) isolating from the cells a messenger RNA coding for human immune interferon, (c) Synthesizing a single-stranded complementary DNA ■with the use of said messenger SNA and reverse fry A transcriptase, (d) converting said complementary DNA to a double- stranded DNA, (e) inserting said double-stranded DNA into a plasmid, (f) transforming a host with the recombinant plasmid, (g) isolating a plasmid containing the required DNA from the culture of said host, (h) if desired, excising out the cloned DNA from the plasmid, and (i) if desired, connecting said cloned DNA at a site downstream from a promoter in a vehicle.

10. A method according to Claim 9, wherein the required for the polypeptide: Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Glu Thr lie Lys Glu Asp Met Asn Val Lys Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Arg Lys Ala lie His Glu Leu lie Gin Val Met Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Gin,. -LA» A method according to Claim 9, wherein the required DNA codes for a polypeptide equivalent to the immune interferon polypeptide in immunological or biological activities.

12. A method according to any. of Claims 9-11, wherein the required ' DNA forms a part of a recombinant DNA molecule. 0 2 1 FEB 1986* DNA codes Cys Asn Val Lys Gin Lys Val Phe Lys Gin Ala Lys Ser ■.p 203661 28

13. A host transformed with a recombinant DNA containing a base sequence of the formula: (5 •) TGT TAC TGC CAG GAC CCA TAT GTA AAA GAA GCA GAA AAC CTT AAG AAA TAT TTT AAT GCA GGT CAT TCA GAT GTA GCG GAT AAT GGA ACT CTT TTC TTA GGC ATT TTG AAG AAT TGG AAA GAG GAG AGT GAC AGA AAA ATA ATG CAG AGC CAA ATT GTC TCC TTT TAC TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA AAG AGT GTG GAG ACC ATC AAG GAA GAC ATG AAT GTC AAG TTT TTC AAT AGC AAC AAA AAG AAA CGA GAT GAC TTC GAA AAG CTG ACT AAT TAT TCG GTA ACT GAC TTG AAT GTC CAA CGC AAA GCA ATA CAT GAA CTC ATC CAA GTG ATG GCT GAA CTG TCG CCA GCA GCT AAA ACA GGG AAG CGA AAA AGG AGT CAG ATG CTG TTT CGA GGT CGA AGA GCA TCC CAG -X (3* ) wherein X is TAA, TGA or TAG.

14. A host according to Claim 13, wherein the DNA contains said base sequence linked at a site downstream from a promoter.

15. A host according to Claim 13 or 14, wherein said host is Escherichia coli or Bacillus subtilis.

16. A polypeptide, encoded by a recombinant DNA molecule* of the formula: ^ Y—Cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Asn lieu Lys Lys Tyr Phe Asn Ala Gly Bis Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin Ser Gin lie Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Ser Val Glu Thr lie Lys Glu Asp Met Asn Val lys Phe Phe Asn Ser Asn Lys lys Lys Arg Asp Asp Phe Glu lys heu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Gin Arg lys Ala lie His Glu Leu He Gin Val Met • # ■ -• . ; ; ,vi ! ....... v"" ! ' :.'f \. " i _ "> ■. . r / 203661 - 29 - Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala Ser Gin wherein Y is (a) hydrogen, (b) Net or (c) Met Lys Tyr Thr Ser Tyr lie Leu Ala Phe Gin Leu Cys lie Val Leu-Gly Ser Leu Gly. ni7a mrfi ®etf°d °/J£r°*lcln? 3 encoded by a Recombinant DNA molecule, of the formula: Y-Cys Tyr Asn Leu Val Ala Lys Asn Gin Ser Lys Asn Val Glu Phe Asn Lys Leu Gin Arg Ala" Glu Lys Arg Ser Gin wherein Y is (a) hydrogen, (b) Met or (c) Met Lys Tyr Thr Ser Tyr lie Leu Ala- Phe Gin Leu Cys life Val Leu Gly Ser Leu Gly, which comprises growing a host transformed with a recombinant containing a base sequence of the a H 1 • • €5*) TGT TAC TGC CAG GAC CCA TAT GTA AAA GAA GCA GAA AAC CTT AAG AAA TAT TTT AAT GCA GGT CAT TCA GAT GTA GCG GAT AAT GGA ACT CTT TTC TTA GGC ATT TTG AAG AAT TGG AAA GAG GAG AGT GAC AGA AAA ATA ATG CAG AGC CAA ATT GTC TCC TTT TAC TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA AAG AGT GTG GAG ACC ATC AAG GAA GAC ATG AAT CTC AAG TTT TTC AAT AGC AAC AAA AAG AAA CGA GAT GAC TTC GAA AAG CTG ACT AAT TAT TCG GTA ACT CAC TTG AAT GTC CAA CGC AAA GCA ATA CAT CAA CTC ATC CAA GTG ATG Cys Gin Asp Pro Tyr Val Lys Glu Ala Glu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Asp Asn Gly Thr Leu Phe Leu Gly lie Leu Trp Lys Glu Glu Ser Asp Arg Lys lie Met Gin He Val Ser Phe Tyr Phe Lys Leu Phe Phe Lys Asp Asp Gin Ser He Gin Lys Ser Thr lie Lys Glu Asp Met Asn Val Lys Phe Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val Lys Ala lie Bis Glu Leu lie Gin Val Met Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Ser Gin Met Leu Phe Arg Gly Arg Arg Ala - 30 - 20366i GCT GAA CTG TCG CCA GCA GCT AAA ACA GGG AAG CGA AAA AGG AGT CAG ATG CTG TTT CGA GGT CGA AGA GCA TCC CAG -X (3') wherein X is TAA, TGA or TAG, accumulating said polypeptide in the culture broth and recovering the same.

18. A method according to Claim 17, wherein the grown host is lysed by subjecting the same to freezing, thawing and then lysozyme treatment in the presence of ethylenediamine tetraacetic acid, to recover the required polypeptide.

19. A sterile pharmaceutical composition, comprising a polypeptide, encoded by a recombinant DNA molecule, of the formula Glu Asp Leu Met Phe Ser Phe Glu Val Met Arg Ala Tyr Thr Ser Tyr lie Leu Ala Phe Gin Leu cys lie Val Leu Gly Ser Leu Gly, and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition according to Claim 19, wherein Y is hydrogen, and the pharmaceutically acceptable carrier is sterile water, normal saline.or human serum albumin. BALD WIN, SON A CAREY olfeQ attorneys for the applic.v ,' g y-cys Tyr Cys Gin Asp Pro Tyr Val Lys Glu Ala Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Val Ala Asp Asn Gly Thr Leu Phe Leu Gly lie Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys lie Gin Ser Gin He Val Ser Phe Tyr Phe Lys Leu Lys Asn Phe Lys Asp Asp Gin Ser lie Gin Lys Val Glu Thr lie Lys Glu Asp Met Asn val Lys Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Gin Arg Lys Ala lie His Glu Leu lie Gin Val Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Lys Arg •Ser Gin Met Leu Phe Arg Gly Arg Arg , Ser -Whorei-n Y Gin is I [a) hydrogen, (b) Met or (c) Met Lys 23 APR.