GB2103222A - Production of interferon - Google Patents

Abstract

A process for the production of human interferon of the fibroblast type ( beta ) and of the leukocyte type ( alpha ), which comprises introducing human genomic DNA into a suitable phage and either infecting a suitable bacteria by such phage recombinant, or recovering from such phage a suitable DNA segment, introducing same into the expression plasmid of a suitable bacterium, cultivating such bacteria and recovering the interferon is described.

Description

SPECIFICATION Production of interferon The invention relates to the production of interferon (IFN). More specifically it relates to the production of large amounts of human interferons of the fibroblast (ss) or leucocyte (a) types by suitable bacteria, into which a fragment of human genomic DNA was introduced. Human DNA taken directly from blood cells is cloned in a suitable .a.-phage. High interferon production can be obtained in bacteria infected by this recombinant A-phage and the interferon can be recovered and purified from the bacterial lysates resulting from this infection. The genomic DNA is also expressed in bacteria into which it has been introduced via a plasmid expression vector.

BACKGROUND OF THE INVENTION Interferon, and especially human interferon is attaining an increasing importance in a number of fields. Its production is rather cumbersome and improvements in the processes of its production are of great importance and value.

Expression of mammalian genes in bacteria such as E. coli is usually prevented by the presence of intervening sequences interrupting the coding sequence of the gene. There are, however, a certain number of genes that do not have introns. Recently, Nagata et al. (1980) (Nature 287,401-8) showed that human leucocyte interferon-a genes also lack introns. They could detect low levels of expression of these genes in E. coli.

Human diploid fibroblasts, induced by double-stranded RNA, contain at least two mRNAs coding for interferon (Weissenbach et al., 1980, PNAS 77,7152-7156). One mRNA, 0.9 kb long (1 1 S) corresponds to the major interferon species IFN-p1 produced by these cells, whose amino acid sequence was partially determined (Knight et al., 1980, Science 207, 325-6). Cloning via cDNA of this IFN-P, mRNA, using E. coli plasmid pBR322 as vector, has been achieved (Goeddel et al., 1980, Nucleic Acid Res., 8, 4057-4075). The nucleotide sequence of these DNA clones corresponds to that determined for the protein and, after construction of expression plasmids, the IFN-P, cDNA was shown to direct the synthesis of human interferon in E. coli. We have isolated human genomic fragments carrying the lFN-p1 gene (Mory et a/., 1981 Eur. J. Biochem. 120, 197-202), and the IFN-ac gene. These genes lack introns and can be therefore directly expressed in E. coli.

Summary of the Invention The invention relates to a process wherein a specific fragment of human genomic DNA, extracted from human blood cells is cloned in a bacteriophage vector, and used directly to program the synthesis of IFN in bacteria, which are cultivated resulting in the production of interferon. In this method, there is, therefore, no need for the lengthy preparation and cloning of full-length complementary DNA (cDNA) from mRNA.-According to the present invention, a suitable piece of DNA is taken directly from a readily available human cell, such as white blood cell, and cloned in a A-phage vector. The required IFN gene is not interrupted by introns and is thus capable to direct IFN synthesis in bacteria.High IFN activity was produced by infecting suitable bacteria, such as E. coli by a recombinant A-phage containing a human DNA insert with the IFN-pX gene or IFN-cgc gene. IFN was recovered and purified from bacterial lysate resulting from such an infection. Phages were found to be suitable vectors for introducing human DNA fragments into bacteria; A Charon 4A phage was especially suitable, and led to high IFN production. The invention further relates to the introduction of such cloned human genomic DNA fragments into plasmid expression vestors, which have been provided with strong promotors. The process can be carried out with different IFN-a as well as IFN-p genes, provided that these lack introns.

In one embodiment, DNA from a human adult was fragmented by partial Eco Rl digestion and cloned in A Charon 4A. The thus obtained clone, designated as Clone C15, with a human DNA insert of about 1 7 kb, was identified as containing a gene for the fibroblast interferon IFN-P,. Restriction mapping showed that this gene located on a 1.8 kb Eco RI fragment, is not interrupted by introns. This human genomic DNA fragment is able to direct the synthesis of active human IFN-p, in E. coli and in other suitable bacteria. A high IFN activity can be recovered from phage lysates.Under certain conditions an activity of the order of 107 U/liter was recovered from phage lysates. The lysates are advantageously subjected to chromatography, preferably on Cibacron Blue Sepharose or on similar columns, and the thus obtained interferon has the same, immunological properties and species specificity as IFN produced from human fibroblasts. Similar results were obtained with lFN-a, whose gene was identified in clone 18-3 containing a 13 kb human genomic fragment.

In a further embodiment the human genomic DNA fragment containing the IFN9, or IFN-cgc gene were introduced into a plasmid such as plasmid pBR322 containing the recA promotor of E. coli.

Cultures of E. coli transformed by these recombinant plasmids produced large amounts of IFN-P or IFN-a respectively when the recA promotor was induced by nalidixic acid. The IFN-p1 genomic fragment was then cut next to the codon for the first methionine of the mature IFN-/31 and ligated to the ribosomal binding site of the E. coil lac promotor. This lac pro motor was modified so as to contain the RNA polymerase binding site of the tryptophan promotor.With this hydrid tryp-lac promotor, the genolic IFN DNA was able to produce 1 08U/liter lFN-k1 in E. coli minicell strain P679--54. Similarly satisfactory yields of IFN-c were obtained when the IFN-c gene was appropriately linked to the tryp-lac promotor.

in all cases the IFN activity was recovered from the bacterial cell by lysis with lysozyme and 30% propylene glycol, and was then purified as for the phage lysates on a hydrophobic chromatography support. For IFN-,BI a preferred support is Cibacron Blue Sepharose. Elution can be effected by substances such as ethylene glycol or preferably propylene glycol. Further purification of lFN-p or can be effected by immunoaffinity chromatography on monoclonal antibody columns or by other conventional techniques.

It is, therefore, clear that human genomic DNA fragments can be used for high yield production of human IFN in E. coli and other suitable host bacteria. As set out above, the process according to the present invention comprises of introducing a fragment of human genomic DNA into a suitable vector such as a phage, infecting a culture of bacteria with such phages resulting in the lysis of such bacteria, and recovering the resulting interferon content of the lysate.The invention, relates to the novel phages obtained by the introduction of human genomic DNA and especially to recombinant phages of the #- type such as clone C15, resulting in IFN-/32 and 18.3, resulting in lFN-a. The invention further relates to a process for the production of interferon by introducing human genomic DNA into a suitable phage, recovering from said phage a suitable DNA section, introducing same into the expression of plasmid of suitable bacteria, cultivating same and recovering the desired interferon. Other and further objects of the invention will become apparent from the following detailed description which is to be construed in a non-limitative sense.

An important advantage of this invention is that genes from healthy or diseased individuals can be compared for their ability to produce IFN, thereby allowing us to study genetic defects in IFN production.

MATERIALS AND METHODS Preparation ofhuman gene library: High molecular weight DNA was prepared from peripheral blood cells of an adult with ,!3- thalassemia. DNA aliquots (40 g) were separately digested 30 min with 100, 200 or 300 units Eco RI, then heated 1 0 min to 650C and applied together on a 10-40% sucrose gradient as described. DNA fragments between 10 and 20 kb were ligated with T4 DNA ligase as described by Mory eft at (1980, Moi. Biol.Reports 6, 203-208) to A Charon 4A DNA arms prepared by Eco RI digestion according to Maniatis et al. (1978, Cell 15, 687-701). ln vitro packaging was done as described by Hohn and Murray (1977, PNAS 74,3259-3263) by mixing several 8,ul aliquots of the ligation reaction (0.5,ug vector DNA and 0.125, g human DNA) with 50 ul extracts of E. coli BHB2688 [N205 recA- (R imm 434 clts b2 red 3 Eam4 Sam7)/A] and BHB2690 [N205 recA-Aimm 434 clts b2 red 3 Dam 15 Sam 7)/A].Per g recombinant DNA, 3 x 105 pfu were obtained, compared to 1 06 pfu/,bg intact A DNA. A total of 1.8 x 106 recombinant phages were diluted in 6 ml 10 mM Tris-HCI pH 7.5, 10 mM MgSO4, 0.01% gelatine. After adding 0.1 ml chloroform and removing debris by microfuge centrifugation, the phages were amplified 105-fold by plating an E. coli DP50 (Leder et al., 1977, Science 196, 1 75-177) at 4 x 103 pfu/1 5 cm plate. For screening, 2 x 104 pfu were seeded on 1 5 cm plates and duplicate nitrocellulose filters lifted sequentially as set out by Benton et al., (1977, Science 196, 1 80-182). This work was done under P3 containment conditions.

Preparation of IFN-, 1 cDNA probe: Poly A+ from human foreskin fibroblasts FS11 cultures induced 4 hours by 100 g/ml.poly(rl):(rC), 50 yg/ml cycloheximide (with 2,tzg/ml actinomycin D for the last hour) was prepared, and the two peaks of interferon mRNA were fractionated by sucrose gradient centrifugation as detailed, Weissenbach et ai., (1980 PNAS 77, 7152-7156; Weissenbach et al., 1979, Eur.J.Biochem. 98 1-8). The 11S mRNA (1.5 y9) was used to prepare RNA-cDNA hybrids with reverse transcriptase from avian myeloblastosis virus (J.Beard) and oligo(dT) as before Weissenbach et al., (1980, PNAS 77, 7152-7156).The RNA--cDNA hybrids were directly cloned according to Zain et al., (1979, Cell 16 851-861), but using dCTP for tailing the hybrids and dG-tailed, Pst I-cleaved pBR322 as vector (Chang et al., 1978, Nature 275, 617-624). A total of 12,500 tetr amps transformants of E. coli MM294 were obtained as outlined by Stenlund et al., (1980 Gene 10, 47-52). Colony hybridization was done with 32P-cDNA probes transcribed either from 11S mRNA (6 g) of induced FS1 1 cells or from total poly A±RNA (3.7 yg) of non-induced cells.The cDNA synthesis was initiated with a synthetic complementary primer, (see No rang et al., 1979, Methods Enzymol, 68, 90-98), 5'GAGATCTTCAGTTTC 3', which includes the Bgl II site of the IFN-p1 sequence. With aZP5' end labeled -- (Houghton et al., 1980, Nucl. Ac Res. 8, 1913-1931) primer, an expected 640 nucleotideslong cDNA was observed only when induced RNA was used as template.To prepare the probes, 20 pmoles primer were annealed with each RNA in 4 l 0.4 M KCI at 300C for 60 min, then incubated in 25 l with 200 Ci each of 32P-ar-dATP and dCTP (both 400 Ci/mmol), 0.5 mM each of dGTP and TTP, 50 mM Tris-HCl pH 8.3, 4 mM MgCl2, 5 mM dithiothreitol, 50 /lg/ml actinomycin D, 4 mM pyrophosphate and 30 units reverse transcriptase for 2 hrs at 370C. After adding 25 ug E. coli DNA, the reaction was treated by 0.3 N NaOH 60 min at 700C, neutralized and filtered through Sephadex G-50 mM Tris-HCI pH 8, 0.1 M NaCI, 10 mM EDTA, 0,5% dodecyl sulfate. From each RNA, 5 x 106 cpm cDNA was obtained. Colonies grown on nitrocellulose filters were fixed, and DNA denatured as described by Thayer (1979 Anal. Biochem. 98 60-63). Each filter was prehybridized with 10 ml 6XSET (SET = 150 mM NaCI, 2 mM EDTA, 30 mM Tris-HCI pH 8) 10 x Denhardt's solution (Denhardt 1966, Biochem.Biophys.Res.Comm. 23, 641--646), 0.1% Na pyrophosphate, 0.1% dodecylsulfate, 50 yg/ml denatured E. coli DNA for 4 hrs at 67 C. Filters were hybridized at 67 OC for 14-1 8 hrs in the same solution with 0.5 x 106 cpm/filter of either of the two 32P-cDNA probes,10 yg/ml poly A and 2.5 ,ug/ml poly C.Filters were washed for 1 hr at 670C in prehybridization solution, then 3 times more for 30 min at 67CC with 3XSET, 0.1% pyrophosphate, 0.1% dodecylsulfate, then 60 min at 25 OC with 4XSET, see Maniatis (1978, Cell 15, 687-701). Filters were dried and exposed to Agfa Curie X-ray films with intensifying screens for 1-2 days at -700C.

Out of 3,500 colonies screened, 1 4 hybridized preferentially to primered cDNA from induced mRNA, while 130 hybridized also to primered cDNA of non-induced mRNA. DNA (0.3, g) of plasmids from the first group was hybridized on filters (Kafatos et al., 1979, Nucl. Ac. Res. 7, 1 541-1 552) with the 5'-end 32P-labeled primer itself (0.3 pmoles, 4 x 106 cpm) in 6XSSC, 10 x Denhardt's solution at 350C for 21 h, followed by washing with 6XSSC (900 mM NaCI, 90 mM Na citrate) at 350C. One clone 1-6-5 was strongly positive and DNA sequencing (see Maxim et al., 1 980, Methods Enzymol. 65, 499-560) showed that it contained about 370 nucleotides from the 3'-side of the IFN-pt sequence.

There were screened another 50,000 clones prepared as previously by introducing dC-tailed ds-cDNA from sucrose gradient purified induced mRNA in the Pst I site of pBR322: the proportion of IFN-, clones were found to be 0.16% (80 clones) as compared to 0.65% IFN-p2 clones.

The clone lFN-1 1-6-5 described above and prepared from FS-1 1 mRNA was used to screen the human library.

Isolation of the genomic clone: IFN-fi1 clone 15 Plasmid DNA from IFN-p1 cDNA clones was labeled by nick-translation, according to Rigby et al., (1977, J.Mol. Biol 113,237-251) to 2 x 108 cpm/yg DNA and hybridized at 106 cpm/filter to the human gene library. Preparation of filters, DNA denaturation and hybridization procedures were as described above. Phages from plaques giving positive hybridization, were replated at a density of 1-2 x 102 pfu on 9 cm plates and rescreened. This procedure was repeated 3 times until more than 95% of the plaques on the plate hybridized to IFN-P, cDNA. Phage clone Cl 5 was isolated from one such plaque.

Phages were grown in liquid cultures as described by Blattner et al., (1977, Science 196, 1 61-169). E. coli DP50 were grown overnight in 1% tryptone, 0.5% NaCI, 0.5% yeast extract, 0.2% maltose, 0.25% MgSO4,0.01 diaminopimelic acid, 0.004% thymidine. About 0.3 ml of the culture was mixed for preadsorption with 0.3 ml of 10 mM MgSO4, 10 mM CaCI2 and 107 phages for 20 min at 37 C.The cultures were then diluted into a 2 liter flask with 500 ml of prewarmed medium containing 1% NZ amine, 0.5% yeast extract, 0.5% NaCI, 0.1% casamino acids, 0.25% MgSO4, 0.01% diaminopimelic acid, 0.004% thymidine, and incubated with good aeration for 1 5-18 hrs at 370C.

After lysis was complete, the cell debris were removed by centrifugation in the cold 15 min at 7,000 rpm in a Sorvall GSA rotor. From this clarified lysate the phages were precipitated with 7% polyethylene glycol 6000 and purified by two successive CsCI gradients. Phage C15 DNA was phenol-purified and its structure analyzed by restriction enzyme digestions, horizontal agarose gel electrophoresis in 20 mM tris base, 10 mM Na acetate, 1 mM EDTA with 0.5,ug/ml ethidium bromide, transferred to nitrocellulose filters (Southern, 1975, J. Mol. B ol. B 98,503-517) and hybridization to nick-translated IFN-,B1 cDNA as above.Eco RI fragments of Cl 5 DNA were subcloned in the Eco RI site of pBR322 for precise restriction mapping of the plasmid DNA according to the established prc,cedure, see Bolivar (1979, Methods Enzymol. 68 245-267).

Assay of interferon activity in phage lysates: Clarified phage IFN-pI C15 lysates, prepared as above were dialyzed against phosphate buffered saline (PBS) for 7 hrs. A 10 ml aliquot was ioaded on a 0.3 ml column of Cibacron Blue-Sepharose CL6B (Pharmacia Fine Chem.). The column was washed with 10-15 ml of 1 MNaCI, 0.02 M Na Phosphate buffer pH 7.2, and then with 10 ml of 50% propylene glycol in the wash solution. Fractions (0.5 ml) were coilected and stored at 40C; in some cases 0.1% human serum albumin was added for stabilization.

Interferon activity was assayed, as described by Weissenbach et al., (1979, Eur. J. Biochem. 98, 1-8) by diluting each fraction serially in a 95-well micropiate. Each well received 2-3 x 104 FS1 1 human fibroblasts in 0.1 ml Minimum Essential Medium (Gibco), 5% fetal calf serum (FCS), 0.5% Gentamycin.

After 18 hrs at 370C, medium was removed and vesicular stomatitis virus (VSV) was added at 1 pfu/cell in medium with 2% FCS. Inhibition of the cytopathic effect was recorded 30 to 40 hrs after infection, in comparison to IFN-ss NIH standard G023-902-527. The antiviral activity was also measured by the reduction of 3H-uridine incorporation after VSV infection as described previously (Weissenbach et al., 1979, Eur. J. Biochem. 98, 1-8). Anti-serum to IFN-ss was obtained from rabbits and assayed as before (Weissenbach et al., 1979, Eur. J. Biochem. 98, 1-8). For the neutralization test, 0.1 ml anti-serum (titering 104 U/ml) or non-immune serum was mixed with 0.2 ml of a 50% suspension of protein A Sepharose beads (Pharmacia Fine Chem.) in PBS, for 1 h at 370C. The beads were washed twice with PBS and 0.1 ml of bacterial interferon (100 U/ml) were added. After 2 hrs at 37 C, the beads were centrifuged and the supernatants assayed for inhibition of 3H-uridine incorporation in VSV infected cells.

Isolation of genomic clone: IFN-a, clone 18-3 Two short single-stranded oligonucleotides, chemically synthesized as above, were used to screen directly the human gene library. The oligonucleotides are complimentary to common sequency of IFN- genes and had the sequence 5'CTCTGACAACCTCCC 3' and 5'CCTTCTGGAACTG 3'. The oligonucleotides were used after 5' labeling, as above, either directly or as primers for cDNA synthesis on RNA from Sendai-virus induced human leukemic cells. The 32P-oligonucleotides or primed cDNAs were hybridized to a total of 500,000 A recombinant phages, as demonstrated above. Approximately 5 x 105 cpm were used per 9 cm nitrocellulose filter. Hybridizations were done in plastic sealed cooking pouches in minimal volumes of liquid.Hybridization with the 1 5 base primers were performed as follows: first, prehybridization in 6xSSC, 10 x Denhardts at 370C for 4 hrs; then, hybridization in 6xSSC, 10 x Denhardts at 370C for 18 hrs, and washes, 3-4 times, 30 min each in 6xSSC at 370C or until no more counts were found in wash. For hybridization with the 1 3 base primer, hybridization and washing temperatures were dropped to 300C. A total of 1 6 phages gave positive hybridization and were further analyzed by restriction mapping and DNA sequencing.Clone 18-3 was demonstrated to carry 3 IFN-a genes, one of which has the sequence identical to the cDNA clone a0 of Goeddel et al., (1981, Nature, 290, 20--25). Clone 18-3 was purified as described for clone C15 above. Expression of clone 1 8-3 in A bacteriophage and after insertion into expression plasmid vector is described in the Results.

RESULTS 1. Structure of genomic IFN-P, C15 clone: This phage clone was isolated from a library of human DNA which had been partially digested with Eco RI and cloned in the arms of;l Charon 4A. The clone hybridized strongly to two different IFN-j31 cDNA probes, independently prepared frorn mRNA fractions of two strains of human fibroblasts. Digestion of the phage DNA by Eco RI showed, in addition to the two A arms, four fragments of 12, 2.6, 1.84 and 0.6 kilobases (Fig. 1 A). Transfer to nitrocellulose by the method of Southern (1975, J. Mol.Biol. 98 503-517), and hybridization with nick-translated DNA from the lFN-1 1-6-5 cDNA clone indicated that the 1.84 kb fragment contains the IFN-p, gene. This Eco RI fragment was recloned in the Eco RI site of pBR322; Restriction analysis of this 1.84 kb subclone with Bgl II, Pvu II, Pst I, Hinc II and Tag I, and hybridization with partial and full-length IFN-P, cDNA probes, allowed to conclude that the coding sequence for IFN-p, preprotein (Hinc II site) starts about 350 nucleotides from the Eco RI site (Fig. 1 A).

The distances between the various restriction sites in the genomic DNA are the same as in the cDNA sequence within experimental error (Table 1). No unexpected restriction sites were found within the IFN-,B, coding region, indicating the absence of any sizeable intervening sequence.

Partial Eco RI digests of Cl 5 DNA showed that the 0.6 kb Eco RI fragment is located between the 1.84 and 2.6 kg Eco RI fragments. A Bam HI site was found in the 2.6 kb, but not in the other Eco RI fragments of the human DNA insert. The 1.84 kb Eco RI fragment, that hybridizes to iFN-p, cDNA, was found on a 9.6 kb Bam HI fragment of Cl 5 DNA. As shown in Figure 1 , this fragment can originate only from the Bam HI site of the A right arm (5.1 kb from Eco RI), leaving 4.5 kb for the Eco Rl-Bam HI segment of the human insert, and indicating that the 1.84 kb Eco RI fragment must be located next to A right arm. The gene orientation (Fig. 1A) was determined as follows.When C15 DNA was cut with Pvu II and hybridized to the full-length or to the 3'-half of IFN-p, cDNA, a 0.66 kb fragment hybridizing only to the 5'-end of IFN-P, was found. But, when the 1.84 kb Eco RI piece was cut with Pvu II, the 5'-end of íFN-p, was on a smaller 0.54 kb fragment. Since no Pvu II site was found in the 0.6 kb Eco RI piece of the human insert, the 0.66 kb Pvu II fragment must end in the A right arm, hence, the 5'-end of íFN-ss is closest to the A arm. The structure shown in Fig. 1 A is supported by several other restriction mapping experiments using Hind Ill, Sma I and Bgl II digestions.The coding sequence of the IFN-P, gene in clone Cl 5 was, therefore, inserted about 1,775 nucleotides away from the strong A P, promotor that controls leftwardtranscription (Williams et af., 1980, Genetic Engineering Viol.11, pp201--281, Plenum Corp.), and in the proper lefward orientation. This together with the the absence of detectable introns, prompted us to investigate if interferon could be produced in E. coli infected by the Cl 5 recombinant phage.

2. Recovery of interferon biological activity from lysates of E. coil infected by the IFN-,B CI 5 phage: Phages from clone Cl 5 were grown in 500 ml cultures of E. coli DP50 as decribed in Methods.

Ten ml of the clarified lysate was dialyzed and loaded onto a small Cibacron Blue-Sepharose column (0.3 ml) and the fractions were assayed for the inhibition of Vesicular Stomatitis virus (VSV) cytopathic effect, with an IFN-,5 standard as reference. In one experiment, where the phage titer in the lysate was 1.3 x 10'0 phages/ml, the interferon activity pattern shown in Fig. 2 was observed. A total of 7,500 units of IFN activity were recovered in the fractions eluted by 50% propylene glycol-1 M NaCI from the column. This would correspond to 0.75 x 106 units/liter lysate. In the crude lysate, however, the measurable activity was much lower suggesting that some material in the lysate prevented correct assay of IFN activity.In a reconstruction experiment, standard human IFN-P was added to a wild type A Charon 4A lysate and indeed the activity measured was only one tenth of the input. When this mixture was passed on the Blue-Sepharose column, 75% of the input activity was recovered. In a control run, lysate from wild type A Charon 4A phages (without adding 1 FN was analyzed, and no lFN activity was found (Fig. 2).

The chromatographic behavior of IFN from lysates of clone C15 may be variable. In an experiment where the phage titer was 3 x 10" pfu/ml, the IFN activity was high but was not retained on the Blue Sepharose column. In this experiment, the total activity recovered from the column amounted to 70-80,000 units IFN for an input of 10 ml lysate (7-8 x 106 units/liter). The effluent fractions were readsorbed on a second Blue-Sepharose column; this time the activity was retained on the column, but again eluted by washing the column just with PBS.Human IFN-p, should normally be eluted from Blue Sepharose only by hydrophobic solvents (Knight et a/., 1980, Science 207, 525-526). Therefore, standard human IFN-p was mixed with the same phage lysate and applied to the column: 30% of the activity was not retained and recovery of activity was only 25%. This suggests that components of this lysate probably destabilized the interaction of IFN-p, in particular that produced by the bacteria, with Blue-Sepharose. Even though interferon was not retained on the column, over 90% of the protein of the lysate was removed from the actiye fractions which had a specific activity of 4-5 x 105 U/mg protein.

This partial purification was essential to remove the chemicals that masked the activity in the crude lysates. In one experiment, clone Cl 5 1 FN activity was also recovered after chromatography of the lysate on carboxymethyl-Sepharose (not shown).

3. Properties of interferon produced in E. coliinfectedby clone C 15 The interferon activity recovered after the chromatographic step, reduced 3H-uridine incorporation in VSV-infected human cells, treated by actinomycin D (Weissenbach, 1979, Eur.J.Biochem. 98, 1-8).

The titration curve obtained for the bacterial IFDB- was similar to that for standard human IFN-/3 (Fig.

3). To test the immunological properties of the bacterial IFN, the partially purified product was mixed with rabbit anti IFN-P serum (inactive on lFN-a) or with rabbit non-immune serum which had been prebound to protein A-Sepharose. After centrifugation, the supernatant was assayed for lFN activity: anti IFN-p serum removed all the interferon activity, while the activity remained when non-immune serum was used (Fig. 3). The bacterial IFN (20 U/ml) was similarly neutralized when just mixed with anti IFN-p (20 U/ml) on the cell culture, and assayed by inhibition of VSV cytopathic effect.

The species-specificity of the bacterial interferon was assayed by comparison of various cell types.

As human IFN-p, the product of clone Cl 5-infected E. coli, showed less than 10% activity on monkey kidney cell BSC-1 and Bovine MDBK cells as compared to human cells. There was no detectable anviral activity on mouse L or A9 cells (Table 2). The same results were obtained with 6,000 U/ml of the bacterial IFN produced by clone C15.

The present invention demonstrates, therefore, that the IFN-p1 gene, isolated directly from partial eco RI fragments of human genomic DNA by cloning in A Charon 4A, can be expressed and leads to the accumulation of significant amounts of IFN activity in the culture lysates. The activity produced is clearly fibroblast interferon (p) as shown by its immunological properties and its species-specificity. The activity is produced by the bacterial culture only in response to infection by the IFN-p1 C15 phage clone. The lFN activity produced by clone 1 5 in E. coli, resembles human IFN-p in its chromatographic properties on Cibacron Blue Sepharose. The chromatographic step is essential to recover high activity (up to 7 x 106 U/liter) from the bacterial lysate.Interferons provide therefore the first examples of biologically active proteins that are produced by bacteria under the direction of a piece of human DNA taken directly from the genome of one individual.

4. Genomic /FN-cgc 18-3 clone This clone was identified by direct hybridization of short, oligonucleotides, synthesized chemically to be complementary to sequences in IFN-a. From a total of 0.5 x 106 genomic, 16 clones were positive in hybridization. The Eco RI fragments hybridizing to the oligonucleotides were sequenced and clone 18-3 was shown as in Fig. 1 B, to contain a 2 kb Eco RI fragment 18-33 (insert of Fig. 1 B) in which the gene for lFN-a0 is located. This gene is, clearly, the source of the mRNA which gave rise to the IFN-a, clone reported by Goeddel petal., (1981, Nature 290,20-25). Clone 5-1 represents an overlapping DNA fragment.Together, clone 5-1 and clone 18-3 carries, in addition to lFN-c, two other IFN-a genes designated a-,, and Cg-c4 (Fig. 1 B).

5. Expression of human IFN genomic fragment under recA promotor control The recA promotor is considered to be one of the strongest E. coli promotors. Normally, it is tightly repressed by the product of the lex gene, even when present on a multicopy plasmid such as pBR322. It is induced, in the presence of damaged DNA, to cleave its own repressor and undergoes induction to very high levels (Sancar and Rupp, 1979, PNAS 76, 3144-3148). The standard inducers for recA are nalidixic acid (which blocks DNA synthesis) or mitomycin C (which cross-links DNA).

A plasmid, pDR1 453, containing the cloned recA gene was used to isolate the promotor on a Taql-BamHi fragment of about 1 kilobase. This was ligated into pBR322 that had been opened up with Clal and Baml to construct plasmid RAP-1 The Taqi-Clai ligation restores the C/al site in this case, allowing the plasmid to be uniquely opened in the third codon of the recA gene. RAP-2 was constructed by cutting RAP-1 with Eco RI and Clay, filling in the sticky ends and religating which restores the RI site. When RAP-2 is opened at the RI site, foreign DNA can be inserted at the 3rd codon of the recA protein.

The RAP-1 vector was used for indirect expression of interferon activity by genomic fragments from the human genomic library. The IFN-P, gene and severl isolated IFN-a genes of the a0a subclass, produced strong interferon activity (as determined by the standard antiviral assay) when inserted into this plasmid. The RI fragments of DNA on which these genes reside (Fig. 1 A and B) were subcloned into the RI site of the RAP-i plasmid. The plasmid was placed in a recA+ host, the bacteria grown and induced with nalidixic acid, the cells harvested, lysed by lysozyme plus 30% propylene glycol and the extract assayed for antiviral activity.The level of activity is clearly dependent upon induction by nalidixic acid, the optimum being at 50 ,ug/ml (Table 3). Interestingly, in some cases, the genomic fragment produces activity in both possible orientations relative to the recA promotor. The IFN-ss1 and IFN-a genes which showed activity were all located on DNA Eco RI fragments of 1.8-2 kilobases, so that the distance from the promotor to the ATG start codon of the interferon gene is on the order of hundreds of nucleotides.

These experments prove that the promotor is functioning as expected, and, they allow quick determinations of which interferon-like genes from the gene library can be active.

6. High expression oflFN-P, gene under the control of tryp-lac hybrid promotor The plasmid PLJ3 (Johnsrud, PNAS, 1978, 75, 5314--53181 contains two lac promotors each 95 base pair long separated by 95 non-related nucleotides. This 285 nucleotides fragment is inserted in the Eco RI site of the plasmid PMB9. The 95 base pair long lac sequences contain the operator and promotor sequence and stops just before the ATG of the p-galactosidase. A coding DNA sequence with an ATG initiator, inserted at the proper distance from the ribosomal binding site of the p-galactosidase gene, should be correctly expressed in E. coli cells.The 285 base pairs Eco RI fragment was recloned into pBR322 at the Eco RI site; the recombinants were screened by growing the cells on plates containing 40 Mg/ml X-gal and 1 5 LLg/ml tetracycline. Only positive blue colonies were pooled and the DNA purified by CsCI gradient centrifugation.

Two orientations of the lac promotors are expected; towards the ampicillin gene or towards the tetracycline gene of pBR322. Since we are interested in having only the Eco RI site, located between the lac ribosomal binding site and the pBR322 sequence, this site was protected by binding RNA polymerase to the plasmid DNA, while the distal site was open by Eco RI restriction, filled in with the Klenow fragment of DNA polymerase and religated. After transformation of MM294 E. coli cells, plasmids were isolated and the distance between the Eco RI site and the Bam HI site of pBR322 was measured.The plasmids containing a 375 base pair fragment were those in which the lac promotors are toward the tetracycline gene, while those containing a 670 base pair fragment (375 + 295 b.p. had the lac promotors towards the ampicillin gene.

From the Eco RI 1.84 kb subclone of the IFN-p1 genomic fragment we cut a Hincll--Bgll fragment containing the preinterferon sequence. The mature IFN-p1 protein contains at its NH2-terminal end, a methionine which can be used as an initiator codon in E. coli. There are two Alul sites close to this ATG codon separated by 13 nucleotides, one Alul site is 8 nucleotides in front of the ATG, while the other is 2 nucleotides after the ATG. A partialAlul digestion was performed on the Hincll-Bglll fragment and fragments around 510 base pairs were isolated from agarose gels. The vector containing the lac promotors towards the tetracycline gene was restricted with Eco RI, the site fillied-in by DNA polymerase and re-cut with BamHI.Upon ligation of the AlulBglll fragments to be filled-in Eco RI BamHI vector, the Eco RI site is restored. To be able to differentiate between the clones containing the ATG codon (13 nucleotides longer) the clones obtained were restricted with Eco RI and Pst1; the clones containing the expected 151 base pairs fragments were sequenced. The Eco RI site was then reopened and the distance between the ribosomal binding site and ATG shortened by Bal 31 digestion, and religation. E. colt cells transformed by these plasmids were screened for IFN-,l31 production.

One clone, L-1 1, was found to produce about 3 x 106 Liter of 1 FN-ss. The lac promotor region in this clone was cut with HpaHI, i.e. 1 7 nucleotides before the start of the mRNA, To cut out the IFN-,8 gene, the enzyme Sau3a was used which cuts the DNA just 3 nucleotides beyond the termination codon of IFN-/31. This Hpall-Sau3a lac-lFN-,B1 fragment was introduced between the Clal and Hindlll sites of a tryp promotor plasmid prepared as follows.A 1 80 nucleotide long Taql--Taql fragments from -21 to -201 before the start of the tryp mRNA, was cut out from the tryp plasmid pEP 121-221, and cloned in the Clal site of pBR322. We selected the orientation in which the tryp promotorfragment is oriented clockwise. This operation restors a Clal site at position -21 of the try promotor. After ligation to the lac-FIN-,B1 fragment a hybrid promotor tryp-lac is formed before the IFN9, gene. This plasmid TL-1 1, was used to transform E. coli MM294 or E. coli minicell strain p679-54. These bacteria produce 1 08 U/liter of lFN-,31 under fermentor culture to an OD650 of 1 0, demonstrating that the IFN-P, genomic DNA fragment is very active (Fig. 4).

TABLE 1 Restriction sites in IFN-p1 cDNA and gene DISTANCES BETWEEN SITES Restriction Calculated from Measured from sites ,B1 cDNA sequence* ss1 genomic DNA** nucleoddes nucheotides Taq l-Pvu 11 255 250 HjncllPvull 11 204 210 Hinc Il-BglII 570 570 Pvu II-Bgl II 366 360 Pst I-Bgl II 363 360 * From Taniguchi et al., 1980 Gene 10 (11-15).

** Measured from 1.84 kb Eco RI fragment.

The Hinc II and Taq I site taken into consideration are those closest to 5'-end of gene.

TABLE 2: Species-specificity of bacterial interferon produced by genomic IFN-p1 C15 clone INTERFERON TITER, U/ml Inferferon Source Human Monkey Bovine Mouse Mouse FS11 BSC-1 MDBK L A9 cells cells cells cells cells 1. Human FS11 cells 1,500 32 32 < 4 < 4 2. E. coli infected by C15 clone 1,000 32 32 < 4 < 4 The bacterial IFN was used after chromatography of the Cl 5 lysate on Cibacron Blue-Sepharose as in Fig. 2.

TABLE 3: lFN-a and ss activities produced by genomic fragments in the recA-plasmid RAP--1

IFN activity Exp. Cone Conditions U/ml 1. RAP-i (1833)-IFN-&alpha;c + nalidixic acid 500 RAP-i (1 833)-IFN- - nalidixic acid < 32 2. RAP-1 (1833)--IFN-&alpha;c right orientation 1000 RAP-1 ( 631 )-IFN-ss1 right orientation 3000 RAP-1 (631)-IFN-ss1 opposite orientation 750 Clone RAP-1 (1833) contains the Eco RI fragment with IFN-a, gene (Fig. 1 B).

Clone RAP--1 ( 631) contains the Eco RI fragment with IFN-ss, gene (Fig. 1 A).

Fragments are inserted at the beginning of the recA gene. Orientation is given with respect to recA promotor orientation.

Legends for Figures: Fig. 1A: Schematic structure oflFN-p1 clone C15 DNA (a): Schematic map of # Charon 4A. The two arms are shown in full line. b): Structure of the human DNA insert in clone Cl 5, the blackened area shows the Eco RI fragment which hybridizes to IFN-p1 cDNA. c): Enlargement of the blackened fragment from b, is shown as a double line. The single line is part of A right arm. PL is the A leftward promoter. d): Position of the IFN-p mRNA.

The upper scales is for a) and b). The lower scale for c) and d). For details see text.

Fig. 7B: Schematic structure oflFN-cY, clone 18-3 The restriction map of the inserts of two A Charon 4A clones is shown. The IFN-a, gene is designated by the arrow alpha-c*. Two other IFN-a genes are present near IFN-ac. The inserts shows the Eco Rl 2 fragment 18-33 which contains the IFN-a0genes and was recloned in the recA plasmid, as explained in the text. Symbols for restriction enzyme site are shown.

Fig. 2: Assay ofinterferon produced by genomic clone IFN-P, C15 on Blue-Sepharose Crude lysate of E. coli DP50 infected by phage C15 was fractionated as described in Methods and Materials and IFN-activity was assayed in each fraction. Protein concentration was measured in the same fractions A parallel chromatography and assay of lysate from A Charon 4A phage is shown (points indicated by squares).

Fig. 3: Titration of genomic clone IFN-p, C15 interferon A fraction of phage C15 IFN from the column in Fig. 2 (about 103 U/ml), was diluted 1:40 and then serially diluted in the microplate for assay of IFN by dilution of VSV RNA synthesis. Standard human fibroblast interferon 25 U/ml was assayed in parallel. Control without VSV and with VSV without IFN are shown. The two columns at the right show the residual activity of the phage C15 1 FN (at final dilution 1 :40) after adsorption on immobilized anti IFN-p or non-immune IgG, as described in Methods.

Fig. 4: Growth and IFN production in E coli containing plasmid TL 11 The TL1 1 piasmid contains a hybrid tryp-lac promotor and the coding sequence for mature human lFN-,31, originating from the human genomic fragment as described in the text. Bacterial cultures were grown in a 1 liter New Brunswick fermentor and at the indicated times, bacteria were lysed by Isozymepropylene glycol and IFN activity assayed on human cells challenged with VSV. The IFN activity is calculated per ml of bacterial culture.

Claims (14)

CLAIMS:

1. A process for the production of human interferon of the fibroblast type (ss) and of the leukocyte type (a), which comprises introducing human genomic DNA into a suitable phage and either infecting suitable bacteria by such phage recombinant, or recovering from such phage a suitable DNA segment, introducing same into the expression plasmid of a suitable bacterium, cultivating such bacteria and recovering the interferon.

2. A process according to claim 1 , wherein the interferon is of the (p) type, and the phage used is of the A type.

3. A process according to claim 1 or 2, wherein the phage is of A Charcn 4A resulting in a clone designated as Clone C15.

4. A process according to claim 1, wherein the interferon produced is of the (cr) type, the phage used is of the Atype.

5. A process according to claim 4 wherein the phage is A-Charon 4A resulting in Clone 1 8-3.

6. A process according to any of claims 1 to 5, wherein the phage used for the infection is of the A-type and the bacterium so infected is E. coli.

7. A process according to claim 6, wherein the phage is A-Charon 4A and the bacterium of the E.

coli DP-50.

8. A process according to claim 1, wherein a human DNA fraction is recovered from such phages, introduced into a plasmid expression vector of a suitable bacterium, and cultivating the bacteria to produce interferon.

9. A process according to claim 8, wherein the DNA is introduced into a plasmid provided with a strong pro motor.

10. A process according to claim 8, wherein the plasmid used is pBR322 and the gene introduced is the IFN-ss1 or IFN-xc gene.

11. A process according to claim 10 wherein the plasmid is pBR322 containing the recA promotor of F. coli.

12. A modification of the process of claim 8 or 10 wherein the human genomic DNA fragment containing the IFN-cu or IFN-P gene is removed from the phage or from the plasmid, cut next to the codon for the first methionine of the mature IFN ligated to the ribosomal binding site of E. coli lac promotor modified to contain the RNA polymerase binding site of the tryptophan promotor, resulting in a hybrid-tryp-lac promotor, reintroducing the plasmid into the bacterium and cultivating the bacteria to produce interferon.

13. A process for the production of human interferon of the fibroblast type, substantially as hereinbefore described and illustrated by the foregoing Examples.

14. Human interferon of the fibroblast type when produced by a process in accordance with any one of claims 1 to 13.