CH664761A5 - METHOD FOR PRODUCING INTERFERON FROM HUMAN GENES. - Google Patents

Description

DESCRIPTION

The present invention relates to the production of interferon (IFN). In particular, it relates to the production of large amounts of human interferons of the fibroblast (ßi) or leucocyte (ac) type by suitable bacteria into which a fragment of human genome DNA has been introduced. Human DNA, which is taken directly from blood cells, is cloned into a suitable phage. High interferon production can be obtained in bacteria infected by this recombinant phage, and the interferon can be obtained and purified from the bacterial lysates resulting from this infection. The genomic DNA fragment is also expressed in bacteria into which it has been introduced via a plasmid expression vector.

Interferon and especially human interferon is becoming increasingly important in many areas. Its manufacture is rather cumbersome, and improvements in the process for its manufacture are of great importance and value.

The expression of mammalian genes in bacteria such as E. coli is usually prevented by the presence of intervening sequences that disrupt the coding sequence of the gene. However, there are a number of genes that do not have introns. Recently, Nagata et al. (Nature 287, 401-8, 1980) that human leucocyte interferon a genes also have no introns. They were able to detect low levels of expression of these genes in E. coli

Human diploid fibroblasts which have been induced by double-stranded RNA contain at least two mRNA codes for interferon (Weissenbach et al., PNAS 77, 7152-7156, 1980). An mRNA, 0.9 kb long (IIS) corresponds to the main type of interferon IFN-β, which is generated by these cells, the amino acid sequence of which has been partially determined (Knight et al., Science 207, 325-6, 1980). Cloning via cDNA of this IFN-ßrmRNA using E. coli plasmid pBR322 as a vector was carried out (Goeddel et al., Nucleic Acid Res., 8, 4057-4075, 1980). The nucleotide sequence of this DNA clo-

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ne corresponds to that determined for the protein, and after construction of expression plasmids, it was shown that IFN-ßrcDNA directs the synthesis of human interferon in E. coli. The inventors of the present invention have previously isolated human genome fragments which carry the IFN-ßr gene and the IFN-ac gene (Mory et al., Eur. J. Biochem. 120, 197-202, 1981). These genes have no introns and can therefore be expressed directly in E. coli.

The methods according to the invention are defined in claims 1, 2, 8 and 9.

The invention relates to a method in which a specific human genomic DNA fragment, e.g. from human blood cells, cloned into a bacteriophatic vector and used directly to program the synthesis of IFN in bacteria that are grown and result in the production of interferon. This method therefore does not require lengthy preparation and cloning of full-length complementary DNA (cDNA) from mRNA. According to the present invention, a relevant DNA fragment is obtained directly from an easily accessible human cell, e.g. taken from a white blood cell and cloned into a phage vector.

The required IFN gene is not interrupted by introns and is therefore able to direct IFN synthesis in bacteria. A high IFN activity was generated by infection of suitable bacteria, such as E. coli, by a recombinant phage which contains a human DNA insert with the IFN-ßr gene or IFN-ac gene. The IFN was obtained from bacterial lysate obtained from such an infection and purified. Phages have proven to be suitable vectors for introducing human DNA fragments into bacteria; the X-Cha-ron 4A phage is particularly suitable and leads to high IFN generation. The invention further includes the introduction of such cloned human genomic DNA fragments into plasmid expression vectors which can be provided with strong promoters. The method can be carried out with various IFN-a as well as IFN-ßi genes, provided that they do not contain introns.

In one embodiment, DNA from an adult was fragmented by partial Eco RI digestion and cloned in X-Charon 4A. The clone thus obtained, designated as clone C15, with an inclusion of human DNA of approximately 17 kb, was identified as containing a gene for the fibroblast interferon IFN-ß]. Restriction location showed that this gene, which was located on a 1.8 kb Eco-RI fragment,

is not interrupted by introns. This human genomic DNA fragment is capable of directing the synthesis of active human IFN-ßj in E. coli and other suitable bacteria. A high IFN activity can be obtained from phage lysates. Under certain conditions, an activity of the order of 107 U / liter was obtained from phage lysates. The lysates are advantageously subjected to chromatography, preferably on Ci-bacron-blue-Sepharose or similar columns, and the interferon thus obtained has the same immunological properties and species specificity as IFN, which was generated from human fibroblasts. Similar results were obtained with IFN-ac, whose gene was identified in clone-18-3, which contained a 13 kb human genome fragment.

In a further method, the human genomic DNA fragment containing the IFN-ßr or IFN-ac gene is inserted into a plasmid, e.g. Plasmid pBR322,

which contains the E. coli recA promoter. Cultures of E. coli, transformed by this recombinant plasmid, generate large amounts of IFN-ß] or IFN-ac when the recA promoter is introduced by nalidixic acid. The IFN-ßr genome fragment is then cut near the codon for the first methionine of the mature IFN-ß] and bound to the ribosomal binding site of the E. coli lac pro-motor. This lac promoter is modified in such a way that it contains the RNA polymerase binding site of the tryptophan promoter. With this hybrid tryp-lac promoter, the genome IFN-ßrDNA is capable of producing 108 U / liter IFN-ß] in E. coli, mini cell strain P679-54. Similarly satisfactory yields of IFN-ctj are obtained when the IFN-ac gene is appropriately linked to the tryp-lac promoter. In all cases, the IFN activity is obtained from the bacterial cells by lysis with Lysozyme and 30% propylene glycol and then, as with the phage lysates, purified on a hydrophobic chromatographic base. For IFN-ßi, cibacron blue sepharose is a preferred base. Elution can be carried out with substances such as ethylene glycol or preferably propylene glycol. Further purification of the IFN-ß] or IFN-ac can be carried out by immunoaffinity chromatography on monoclonal antibody columns or by another known method.

It can be seen from the above that human genomic DNA fragments can be used to produce human IFN in E. coli and other suitable guest bacteria in high yield.

An important advantage of using this invention is that genes from healthy and sick individuals can be compared for their ability to generate IFN, making it possible to examine genetic defects in IFN generation.

Materials and methods

Production of human gene library

High molecular weight DNA was prepared from peripheral blood cells of an adult with β-thalassemia. Aliquots (40 (ig) of DNÄ were digested separately with 100, 200 or 300 units of Eco-RI for 30 minutes, then heated to 65 ° C for 10 minutes and applied together on a 10-40% sucrose gradient as described. DNA Fragments between 10 and 20 kb were bound to X-Cha-ron 4A-DNÄ arms using T4 DNA ligase as described by Mory et al. (Mol. Biol. Reports 6, 203-208, 1980), which were isolated by Eco Ri-Digestion according to Maniatis et al. (Cell 15, 687-701, 1978).

In vitro packing was carried out as described by Hohn and Murray (PNAS 74, 3259-3263, 1977) by mixing several 8 (μ aliquots of the ligation reaction (0.5 (ig vector DNA and 0.125 ig human DNA) with 50 (ü extracts of E. coli BHB2688 [N205 recA "(X imm 434 clts b2 red 3 Eam4 Sam7) / À] and BHB2690 [N205 recA ~ (X imm 434 clts b2 red 3 Dam 15 Sam7) / A,]. Pro ng of recombinant DNA were obtained 3 x 105 pfu, compared to 108 pfu / (ig intact A. DNA. A total of 1.8 x 106 recombinant phages were in 6 ml of 10 mM Tris-HCl pH 7.5, 10 mM MgS04, 0.01% gelatin diluted After adding 0.1 ml of chloroform and removing the fragments by microfuge centrifugation, the phages were increased 105-fold by coating on E. coli DP50 (Leder et al., Science 196 , 175/177, 1977) with 4 x 103 pfu / 15 cm plate, 2 x 104 pfu on 15 cm plates were inoculated for screening and double nitrocellulose filters were lifted off successively, as disclosed by Benton et al. (Science 196, 180-182 , 1977). This work was done under P3 conditions.

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Preparation of JFN-ßrcDNA sample

Poly-A "from human foreskin fibroblast SFll cultures, which were passed for 4 hours through 100 μg / ml ml poly (rl): (rC), 50 μl cycloheximide (with 2 (μg / ml actinomycin-D during the last Hour) was generated and the two tips of interferon mRNA were fractionated by sucrose gradient centrifugation as described in detail by Weissenbach et al. (PNAS 77, 7152-7156, 1980), Weissenbach et al. (Eur. J. Biochem. 98, 1-8, 1979. The IIS mRNA (1.5 (ig) was used to convert RNA-cDNA hybrids with reverse transcriptase from avian (avian) myeloblastosis virus (J. Beard) and Oli -go (dT) as described by Weissenbach et al. (PNAS 77, 7152-7156, 1980) However, the RNA-cDNA hybrids were cloned directly according to Zain et al. (Cell 16, 851-861, 1979) using dCTP as the end of the hybrid and dG-terminated, Pst I-cleaved pBR322 as vector (Chang et al, Nature 275, 617-624, 1978). A total of 12,500 tetr amps transformants from E. coli MM294 were obtained as detailed by Stenlund et al. (Gene 10, 47-52, 1980). The colony hybridization was carried out using 32P-c DNA samples which were transcribed either from IIS mRNA (6 (ig) from induced SF 11 cells or from Total Poly A + RNA (3.7 (ig) from non-induced cells The cDNA synthesis was initiated with a synthetic complementary primer (see Narang et al., Methods Enzymol. 68, 90-98, 1979), namely 5'GA-GATCTTCAGTTTC 3 ', which is the Bgl Ii seat of the IFN- With 32P-5 'end-labeled primer (Hough-ton et al., Nucl. Ac. Res. 8, 1913-1931, 1980), an expected 640 nucleotide long cDNA was only observed when induced RNA was used as a template To prepare these samples, 20 pMoles of primer were reacted with each RNA in 4 (il 0.4 M KCl at 30 C for 60 minutes, then in 25 (il with 200 (iCi 32P-cc-dATP and dCTP (both 400 Ci mmol), 0.5 mM dGTP and TTP, 50 mM Tris-HCl pH 8.3, 4 mM MgCl2, 5 mM dithiotreitol, 50 (ig / ml actinomycin-D, 4 mM pyrophosphate and 30 units of reverese transcriptase , w Incubated at 37 C for 2 hours. After addition of 25% E. coli DNA, the reaction mixture was treated with 0.3 N NaOH for 60 minutes at 70 C, then neutralized and purified by Sephadex G-50 in 50 mM TrisHCl pH 8, 0.1 M NaCl , 10 mM ED-TA, 0.5% dodecyl sulfate filtered. 5 x 106 cpm cDNA were obtained from each RNA. Colonies grown on nitrocellulose filters were fixed and DNA denatured as described by Thayer (Anali. Biochem. 98, 60-63, 1979). Each filter was washed with 10 ml 6XSET (SET = 150 mM NaCl. 2 mM EDTA, 30 mM Tris-HCl pH 8) 10 x Denhardt's solution (Denhardt, Biochem. Biophys. Res. Comm. 23, 641-646, 1966), 0.1% sodium pyrophosphate, 0.1% dodecyl sulfate. 50 (ig / ml denatured E. coli DNA prehybridized for 4 hours at 67 C. The filters were at 67 C for 14 to 18 hours in the same solution with 0.5 x 106 cpm filter of one of the two 32P cDNA samples, 10 (ig / ml poly-A and 2.5 (ig / ml poly-C. The filters were washed for 1 hour at 67 ° C in the prehybridization solution, then three times for 30 minutes at 67 ° C with 3XSET, 0, 1% pyrophosphate, 0.1% dodecyl sulfate, then 60 minutes at 25 C with 4XSET, see Mania-tis (Cell 15, 687-701, 1978) The filters were dried and at -70 C Agfa Curix for 1 to 2 days X-ray films exposed with an intensifying screen.

Of 3500 colonies examined, 14 hybridized preferentially to primed cDNA from induced mRNA, while 130 also hybridized to primered cDNA from non-induced mRNA. DNA (0.3 (ig) plasmids from the first group were hybridized on filters (Kafatos et al.,

Nucl. Ac. Res. 7.1541-1552, 1979) with the 5'-end-32P-labeled primer itself (0.3 pMoles, 4 x 106 cpm) in 6XSSC, 10 x Denhardt's solution at 35 C for 21 hours, followed by washed with 6XSSC (900mM NaCl 90mM sodium citrate) at 35C. Clone 1-6-5 was highly positive and DNA sequencing (see Maxam et al., Methods Enzymol. 65, 499-560, 1980) showed that it had approximately 370 nucleotides from the 3 'side of the IFN-ßr sequence contained. A further 50,000 clones, produced as before by introducing dC-terminated ds cDNA from sucrose gradient purified purified mRNA in the Pst I seat of pBR322 were examined: the ratio of IFN-β, clones was 0.16% (80 Clones) compared to 0.65% IFN-β2 clones.

The above-described clone IFN-β, 1-6-5, which was made from SF-11 mRNA, was used to examine the human library.

Isolation of the genomic clone IFN-ßrClon 15

Plasmid DNA from IFN-ß, cDNA clones was labeled by nick translation, according to Rigby et al., (J. Mol. Biol. 113, 237-351, 1977) to 2 x 108 cpm / ig DNA and hybridized at 106 cpm / filter to the human gene library. Filter preparation, DNA denaturation and hybridization procedures were the same as described above. Phages from «plaques», which gave positive hybridization, were resettled at a density of 1 to 2 x 102 pfu on 9 cm plates and examined again. This procedure was repeated three times until more than 95% of the "plaques" on the plate became IFN-ß! cDNA hybridized. The phage clone C15 was isolated on such a "plaque".

Phages were grown in liquid cultures as described by Blattner et al. (Science 196, 161-169, 1977). E. coli DP50 were grown overnight in 1% tryptone, 0.5% NaCl, 0.5% yeast extract, 0.2% maltose, 0.25% MgS04, 0.01% diaminopimelic acid, 0.004% thymidine. About 0.3 ml of the culture was mixed with 0.3 ml of 10 mM MgSO4, 10 mM CaCl: and 107 phages for 20 minutes at 37 C for pre-adsorption. The cultures were then placed in a 2 liter flask with 500 ml preheated medium containing 1% NZ amine, 0.5% yeast extract, 0.5% NaCl, 0.1% casamino acids, 0.25% MgSO4, 0.01% Diaminopimelic acid, containing 0.004% thymidine, diluted and incubated at 37 C with good ventilation for 15 to 18 hours. After the lysis was completed, the cell debris was removed by centrifugation in the cold for 15 minutes at 7000 rpm in a Sorvall GSA rotor. From this cleared lysate, the phages were precipitated with 7% polyethylene glycol 6000 and purified by two successive CsCl gradients. The phage C15-DNA was phenol-purified and its structure was analyzed by restriction enzyme digestion, horizontal agarose gel electrophoresis in 20 mM Tris base, 10 mM sodium acetate, 1 mM EDTA with 0.5 (ig / ml ethidium bromide), transferred to nitrocellulose filter (Southern, J. Mol. Biol. 98, 503-517, 1975) and hybridized to translated IFN-β, cDNA as above Eco RI fragments from C15 DNA were Eco RI-seated from pBR322 for accurate restriction mapping Plasmid DNA subcloned using the Bolivar method introduced (Methods Enzymol, 68, 245-267, 1979).

Investigation of interferon activity in phage lysates

Purified phages IFN-β, C15 lysates, prepared as above, were dialyzed against phosphate buffered saline (PBS) for 7 hours. A 10 ml portion was loaded onto a 0.3 ml column of Cibacron Blue Sepharose CL6B (Pharmacia Fine Chem.). The pillar was

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washed with 10 to 15 ml of 1 M NaCl, 0.02 M sodium phosphate buffer pH 7.2 and then with 10 ml of 50% propylene glycol in the washing solution. Fractions (0.5 ml) were collected and stored at 4 3C; in some cases 0.1% human serum albumin was added for stabilization. Interferon activity was examined as described by Weissenbach et al. (Eur. J. Biochem. 98, 1-8, 1979) by diluting each fraction in series in a 95-well microplate. Each well received 2 to 3 x IO4 SF11 human fibroblasts in 0.1 ml "Minimum Essential Medium" (Gibco), 5% fetal calf serum (FCS), 0.5% gentamycin. After 18 hours at 37 ° C., the medium was removed and vesicular stomatitis viruses (VSV) were added in an amount of 1 pfu / cell in medium with 2% FCS. Inhibition of cytopathic effects was recorded 30 to 40 hours after infection. Compared to IFN-NIH standard G023-902-527. Antiviral activity was further measured by reducing 3H uridine incorporation after VSV infection as previously described (Weissenbach et al., Eur. J. Biochem. 98, 1-8, 1979). An antiserum to IFN-ß was obtained from rabbits and examined as previously described (Weissenbach et al., Eur. J. Biochem. 98, 1-8, 1979). For the neutralization test, 0.1 ml of antiserum (with a titer of 104 U / ml) or nonimmunosum was mixed with 0.2 ml of a 50% suspension of Protein A Sepharose beads (Pharmacia Fine Chem.) In PBS for 1 hour mixed at 37 ° C. The beads were washed twice with PBS and 0.1 ml bacterial interferon (100 U / ml) was added. After 2 hours at 37 C, the beads were centrifuged and the supernatant liquid examined for the inhibition of 3H uridine incorporation into VSV-infected cells.

Isolation of genomic clone: IFN-a c clone 18-3

Two short single-stranded oligonucleotides, chemically synthesized as above, were used to directly screen the human gene library. The oligonucleotides are complementary to ordinary sequences of IFN-a genes and had the sequence 5'OTCTGACAACCTCCC 3 'and 5'CCTTCTGGAA CTG 3'. The oligonucleotides were used after 5 'labeling as above, either directly or as primers for cDNA synthesis on RNA as Sendai virus-induced human leukemia cells. The 32P-01igonucleotides or primed cDNAs were hybridized to a total of 500,000 X recombinant phages as shown above. About 5 x 105 cpm were used per 9 cm nitrocellulose filter. The hybridizations were carried out in minimal volumes of liquid in sealed plastic cooking bags. The hybridization with the 15 base primers was carried out as follows:

First pre-hybridization in 6xSSC, 10 x Denhardts at 37 C for 4 hours; then hybridization in 6xSSC, 10 x Denhardts at 37 C for 18 hours and three to four washes, 30 minutes each in 6xSSC at 37 ° C or until no more counting in the washing liquid could take place. For the hybridization with the 13 base primer, the hybridization and washing temperatures were reduced to 30 ° C. A total of 16 phages resulted in positive hybridization and was further analyzed by restriction map ping and DNA sequences. Clone 18-3 has been shown to carry three IFN-a genes, one of which has an identical sequence to that of Goeddel et al. CDNA clon-ac. (Nature 290, 20-25, 1981). Clone 18-3 was purified as described for clone C15 above. The expression of clone 18-3 X, bacteriophage and after introduction into the expression plasmid vector is described in the results.

Resultale

1. Structure of genome IFN-βrCJ5 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 /.-charon 4A. The clone hybridized strongly to two different IFN-ß, cDNA samples, which had been prepared independently from mRNa fractions from two strains of human fibroblasts. In addition to the two X arms, the digestion of the phage DNA by Eco RI showed four fragments of 12, 2.6, 1.84 and 0.6 kilobases (FIG. 1A). The transfer of nitrocellulose by the method of Southern (J. Mol. Biol. 98, 503-517, 1975) and hybridization with translated DNA on the IFN-ß, 1-6-6 cDNA clone showed that the 1, 84 kb fragment containing the IFN-β, gene. This Eco RI fragment was recloned into the Eco RI seat of PBR322. Restriction analysis of this 1.84 kb subclone with Bgl II, Pvu II, Pst I, Hinc II and Taq I and hybridization with partial and full length IFN-ß, cDNA samples allowed the conclusion that the coding sequence for IFN- β, preprotein (Hinc Ii seat) begins about 350 nucleotides from the Eco RI seat (Fig. 1A). The distances between the different restriction seats in the genomic DNA are the same as in the cDNA sequence within the experimental error limits (Table I). No unexpected restriction seats were found within the IFN-ß, coding region, indicating the absence of any detectable interference sequence.

Partial Eco RI digest of C15 DNA showed that the 0.6 kb Eco RI fragment lies between the 1.84 and 2.6 kb Eco RI fragments. A Bam HI seat was found in the 2.6 kb, but none in the other Eco RI fragments of the human DNA insert. The 1.84 kb Eco Ri fragment, which hybridizes to IFN-ß, cDNA, was found on a 9.6 kb Bam HI fragment of C15 DNA. As shown in Figure 1, this fragment can only originate from the Bam HI seat of the right arm (5.1 kb from Eco RI), 4.5 kb for the Eco RI-Bam HI segment of the human Be left under influence and indicate that the 1.84 kb Eco RI fragment must be adjacent to the X-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-ß, cDNA, a 0.66 kb fragment was found which was only attached to the 5 'end of IFN-ß, hybridized However, when the 1.84 kb Eco RI piece was cut with Pvu II, the 5 'end of IFN-ß was on a smaller 0.54 kb fragment. Since no Pvu Ii seat could be found in the 0.6 kb Eco RI piece of human inclusion, the 0.66 kb Pvu II fragment must end in the X-right arm and therefore is the 5 'end of IFN-ß , closest to the right arm. The structure shown in Fig. 1A is supported by various other "restriction map ping" attempts using Hind III, Sma I and Bgl II digestions. The coding sequence of the IFN-β, gene in clone C15 was therefore introduced approximately 1.775 nucleotides away from the strong À.PL promoter, which regulates the left-hand transcription (Williams et al. Genetic Engineering, Vol. II, pages 201- 281, 1980, Plenum Corp.), and in the right leftward orientation. This, together with the absence of detectable introns, led to the investigation as to whether interferon could be generated in E. coli which are infected with the C15 recombinant phage.

2. Obtaining biological interference activity from lysates of E. coli infected by the IFN-ß / C 15 phage:

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Phages from clone C15 were grown in 500 ml cultures of E. coli DP50. as described above in the methods. 10 ml of the clarified lysate was dialyzed and loaded onto a small Cibacron Blue Sepharose column (0.3 ml), after which the fractions were examined for the inhibition of the cytopathic effect of vesicular stromatitis virus (VSV) with an IFN -ß standard for reference. In an experiment in which the phage titer in the lysate was 1.3 x 10 ″ phage ml, the interferon activity pattern, which is shown in FIG. 2, was observed. A total of 7500 units of IFN activity was determined on the Fractions obtained which had been eluted from the column with 50% propylene glycol - 1 M NaCl. This would correspond to 0.75 x 106 units of liter of lysate. However, in the crude lysate the measurable activity was significantly lower, which suggests that certain materials in the lysate prevented correct determination of IFN activity In a reconstruction attempt, standard human IFN-ß was added to a wild-type A. Charon 4A lysate, and the activity measured was actually only a tenth of the addition the blue Sepharose column was passed, 75% of the original activity was recovered and in a control experiment the wild type /. -charon 4A phage lysate (without the addition of IFN) was analyzed and no IFN act was found ivity found (Figure 2).

The chromatographic behavior of IFN from lysates of clone C15 can vary. In an experiment in which 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 obtained from the column was 70 -80,000 units of IFN using 10 ml of lysate (7 to 8 x 106 units / liter) The effluent was readsorbed onto a second blue Sepharose column, this time the activity was retained on the column but again by washing the column eluted only with PBS. Human IFN-ß, should normally be eluted from blue-Sepharose only with hydrophobic solvents (Knight et al., Science 207, 525 -526, 1980). Standard human IFN-ß was therefore made with the same phage -Lysate mixed and placed on the column: 30% of the activity was not retained and the recovery of the activity was only 25%, suggesting that components of this lysate are likely to interact with IFN-ß, in particular e the one that is produced by bacteria is destabilized with blue Sepharose. Although interferon was not retained on the column, over 90% of the protein of the lvsate was removed from the active fractions, which had a specific activity of 4 to 5 x 105 U mg protein. This partial purification was essential to remove the chemicals which had marked the activity in the crude lysates. In one experiment, the clone C15 IFN activity was also obtained after the lysate had been chromatographed on carboxymethyl Sepharose (not shown).

3. Properties of interferon generated in E. coli infected by clone C15.

The interferon activity obtained after the chromatography step reduced 3H-uridine incorporation in VSV-infected human cells which were treated with actinomycin-D (Weissenbach, Eur. J. Biochem. 98, 1-8, 1979). The titration curve obtained for the bacterial IFN-ß was similar to that for standard human IFN-ß (Fig. 3). To investigate the immunological properties of bacterial IFN, the partially purified product was treated with rabbit anti-IFN-ß-Se-

rum (inactive on IFN-a) or mixed with rabbit nonimmune serum which had been pre-bound to Protein A-Sepharose. After centrifugation, the supernatant was assayed for IFN activity: anti-IFN-ß serum removed any interferon activity while activity remained when nonimmune serum was used (Fig. 3). The bacterial IFN (20 U / ml) was neutralized in a similar manner when it was only mixed with anti-IFN-ß (20 U / ml) on the cell culture and examined by inhibiting the cytopathic effect VSV.

The species specificity of the bacterial interferon was examined by comparing different cell types. As human IFN-ß, the product of E. coli infected with clone C15 showed less than 10% activity on monkey kidney cells BSC-1 and bovine MDBK cells compared to human cells. No detectable antiviral activity was observed on mouse L or A9 cells (Table II). The same results were obtained with 6000 U / ml of the bacterial IFN generated by clone C15.

The present invention thus shows that the IFN-ßr gene, which was isolated directly from partial Eco RI fragments of human genomic DNA by cloning in X-Charon 4A, can be expressed and to accumulate significant amounts of IFN activity in the Culture lysates. The activity generated is clearly fibroblast interferon (ß), as demonstrated by its immunological properties and species specificity. The activity is determined by the bacterial culture only in response to the infection by the IFN-ß | C15 phage clone generated. The IFN activity generated by clone 15 in E. coli is similar to that of human IFN-ß in its chromatographic properties on Cibacron blue Sepharose. The chromatographic step is essential to obtain high activity (up to 7 x 106 U liters) from the bacterial lysate. Interferons thus provide the first examples of biologically active proteins that are produced by bacteria under the control of a piece of human DNA that was taken directly from an individual's genome.

4. Genomic IFN-a, 18-3 clone

This clone was identified by direct hybridization on short oligonucleotides that were chemically synthesized to be complementary to sequences in IFN-a. Out of a total of 0.5 x 106 genomes, 16 clones were positive in the hybridization. The Eco RI fragments which hybridized to the oligonucleotides were sequenced and it was found that clone 18-3 (as in FIG. 1B) contains a 2 kb Eco RI fragment 18-33 (inclusion of FIG. 1B), in which the gene for IFN-ac is contained. This gene is clearly the source of the mRNA, which resulted in the IFN-ac clone, which Goeddel et al. (Nature 290, 20-25. 1981). Clone 5-1 represents an overlapping DNA fragment. In addition to IFN-ac, clone 5-1 and clone 18-3 together carry two further IFN-a genes, which are designated as at.2 and a-c4 (FIG 1B).

5. Expression of human IFN genomic fragment under recA promoter regulation

The recA promoter is considered one of the strongest E. coli promoters. Usually it is pushed back tightly by the product of the lex gene even when it is present on a multicopy plasmid such as pBR322. In the presence of damaged DNA, it is induced to cleave its own repressor and experiences very high levels of induction (Sancar and Rupp, PNAS 76,

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3144 3148, 1979). The standard inducers for recA are nalidixic acid (which blocks DNA synthesis or mitomycin-C (which crosslinks DNA).

A plasmid, pDRI453, containing the clonized recA gene was used to isolate the promoter on a Taql-BamHI fragment of approximately 1 kilobase. This was bound in pBR322 which had been digested with Clal and BamI to form plasmid RAP-1. In this case, the Tagl-CIal ligation restores the Clal seat, which enables the plasmid to be opened only in the third codon of the recA gene. RAP-2 was constructed by cutting RAP-1 with Eco Ri and Clal, filling in the blunt ends and reconnecting, thereby restoring the RI seat. When RAP-2 is opened at the RI seat, foreign DNA can be introduced at the third codon of the recA protein.

The RAP-1 vector was used for the indirect expression of interferon activity by genomic fragments from the human genomic library. The IFN-ßi gene and various isolated ac subclass IFN-u genes produced strong interferon activity (as determined by standard antiviral assays) when introduced into this plasmid. The Ri fragments of DNA on which these genes are located (Figures 1A and 1B) were subcloned into the RI site of the RAP-1 plasmid. The plasmid was placed in a reeA "host, the bacteria grown and induced with nalidixic acid, the cells harvested, lysed by lysozyme plus 30% propylene glycol and the extract examined for its antiviral activity. The level of activity clearly depends on the induction by nalidixic acid, the optimum being 50 ml (Table III). Interestingly, in some cases the genomic fragment generates activity in both possible directions with respect to the recA promoter. The IFN-ßr and IFN-a genes, which Showed activity, were all on DNA Eco RI fragments of 1.8 to 2 kilobases, so that the distance from the promoter to the ATG start codon of the interferon gene is on the order of hundreds of nucleotides.

These experiments show that the promoter is working as expected and allow quick determinations of which interferon-like genes from the gene library can be active.

6. High expression of IFN-β, gene under the regulation of the hybrid trip-kic promoter

The plasmid PLJ3 (Johnsrud, PNAS, 75, 5314-5318, 1978) contains two lac promoters, each 95 base pairs long, separated by 95 unrelated nucleotides. This 285 nucleotide fragment is inserted into the Eco RI seat of the plasmid PMB9. These 95 base pair long lac sequences contain the operator and motor sequence and stop immediately before the ATG of the β-galactosidase. A coding DNA sequence with an ATG initiator inserted at the correct distance from the ribosomal binding site of the β-gaiactosidase gene should be experimented correctly in E. coli cells. The 285 base pair Eco RI fragment was recloned in pBR322 at the Eco RI seat; the recombinants were screened by growing the cells on plates containing 40 µg ml X-gal and 15 µg / ml tetracycline. Only positive blue colonies were collected and the DNA was purified by CsCl gradient centrifugation.

Two directions of the lac promoters were expected; against the ampicillin gene or against the tetracycline gene of pBR322. Since we are interested in having only the Eco RI seat that lies between the lac-ribosomal binding site and the pBR322 sequence, this seat was protected by binding RNA polymerase to the plasmid DNA while the distal seat was closed Eco RI restriction was open, filled with the Klenow fragment from DNA polymerase and bound again. After transformation of MM294 E. coli cells, plasmids were isolated and the distance between the Eco RI seat and the Bam Hi seat of pBR322 was measured. The plasmids containing a 375 beta-pair fragment were those in which the lac promoters were directed against the tetracycline gene, while those containing a 670 beta-pair fragment (375 ± 295 bp) were the lac promoters against the ampicillin gene contained directed.

A HincII-BglII fragment containing the pre-interferon sequence was cut from the Eco RI 1.84 kb subclone of the IFN-β genomic fragment. The mature IFN-ßr protein contains at its NH2 end a methionine which can be used as an initiator codon in E. coli. There are two Alul seats close to this ATG codon, separated by 13 nucleotides, one Alul seat is 8 nucleotides before the ATG, while the other is 2 nucleotides after the ATG. Partial Alul digestion was performed on the HinclI-ßglll fragment, and fragments of approximately 510 base pairs were isolated from agarose gels. The. Vector containing the lac promoters against the tetracycline gene was restricted with Eco RI, the seat was filled with DNA polymerase and cut again with BamHI. After connecting the AluI-BgHI fragments to the filled Eco Ri-BamHI vector, the Eco RI sites were restored. In order to be able to distinguish between the clones which contain the ATG codon (13 nucleotides longer), the clones obtained were restricted with Eco RI and PstI; the clones containing the expected 151 base pair fragments were sequenced. The Eco RI seat was then opened again and the distance between the ribosomal binding site and the ATG was shortened by Bai 31 digestion, and closed again. E. coli lines transformed by these plasmids were selected for IFN-ß generation.

A clone L-II was found to produce approximately 3 x IO6 U / liter IFN-ß. The lac promoter region in this code was cut with HpaHI, i.e. 17 nucleotides before the start of the mRNA. To cut the IFN-ßrGen, the enzyme Sau3a was used, which cuts the DNA exactly 3 nucleotides behind the end codon of IFN-ß]. This HpaII-Sau3a-lac-IFN-ßr fragment was inserted between the Clal and Hindill seats of a tryp promoter plasmid, which was prepared as follows. A 180 nucleotide Taql-Taql fragment from -21 to -201 before the start of the tryp mRNA was excised from the tryp plasmid pEP 121-221 and cloned into the clal seat of pBR322. The direction was chosen in which the tryp promoter fragment is currently oriented. This operation restores a clal seat in position - 21 of the try promoter. After binding to the lac-IFN-ßr fragment, a hybrid promoter tryp-lac is formed in front of the IFN-ßr gene. This plasmid TL-11 was used to convert E. coli MM294 or E. coli mini cell strain p679-54. These bacteria produce 108 U / liter IFN-ß, under fermenter culture to an OD650 of 10, which shows that the IFN-ß, genomic DNA fragment is very active (Fig. 4).

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

664 761

8th

Table /

Restriction seat in IFN-ß-cDNA and gene

Restriction seats

Distance between seats calculated from

ß, cDNA sequence *

measured from ß, aenomic DNA **

Taq I - Pvu II Hinc II - Pvu II Hinc II - Bel II Pvu II - Bgl II Pst I - Bal II

Nucleotides

255

204

570

366

363

Nucleotides

250

210

570

360

360

* From Taniguchi et al. (Gen 10, 11-15. 1980)

** measured from 1.84 kb Eco RI fragment

The Hinc II and Taq 1 seats considered are the closest to the 5 end of the gene.

Table II

Species specificity of bacterial interferon generated by genomic IFN-ßi CI5 clone

interferon

Interferon titer.

U ml

source

human

monkey

Beef

mouse

mouse

FS-1I-

BSC-1-

MDKB-

L cells

-A9 cells

Cells

Cells

Cells

1. Human

1,500

32

32

<4

<4

SF11 cells

2. E. coli infected

1.00

32

32

<4

<4

by C15 clone

The bacterial IFN was determined after chromatography of the C15 lysate on Cibacron blue Sepharose. as used in Fig. 2.

Table III

IFN-a and ß activities generated by genomic fragments in the recA plasmid RAP-1

Try clon

conditions

IFN activity U ml

1. RAP-1 (1833) - + nalidic acid 500 IFN-a,

RAP-1 (1833) -

IFN-ac - nalidic acid

2. RAP-1 (1833) - correct

IFN-a,

RAP-1 (631) -IFN-ß,

RAP-1 (631) -IFN-ß,

Clone RAP-1

Clone RAP-1

Orientation correct orientation opposite orientation

<32

1000

3000

750

(1833) contains the Eco RI fragment with IFN-atGen (Fig. 1B).

(631) contains the Eco RI fragment with IFN-β | gene (Fig. 1A)

Fragments are introduced at the beginning of the recA gene. The orientation is given in relation to the recA promoter orientation.

Legends for the characters:

Fig. IA: Schematic structure of IFN-ßrClon CI5 DNA

(a) Schematic map of X-Charon 4A. The two 50 arms are shown in solid lines.

(b) Structure of the human DNA insert in clone C15, the blackened area shows the Eco RI fragment which hybridizes to IFN-ß, cDNA.

(c) Enlargement of the blackened fragment area from 55 (b), shown as a double line without blackening. The single line is part of the right arm. PL is the rear I promoter.

(d) Position of IFN-ß, mRNA. The upper scale is for (a) and (b). The lower scale for (c) and (d). For single

60 th see text.

Fig. IB: Schematic structure of IFN-a .- clone 18-3.

The restriction map of the inclusions of two ^ -Charon 4A clones is shown. The IFN-ac gene is identified by the 65 arrow Alph-c *. Two other IFN-a genes are present near IFN-ac. The inclusions show the Eco RI 2 kb fragment 18-33, which contains the IFN-ac genes and was recloned into the recA plasmid, as in the text

9

664 761

executed. The symbols for restriction enzyme seat are indicated.

Figure 2: Determination of interferon produced by the genomic clone IFN-β) C15 on blue Sepharose. Raw lysate from E. coli DP50 infected by phage C15 was fractionated as described in the Methods and Materials section, and the IFN Activity was determined in each fraction (• •). The protein concentration was measured in the same fractions (• ■ •). A parallel chromatography and determination of the lysate from X-Charon 4A phage is shown (□ - □).

Fig. 3: Titration of genomic clone IFN-ß; C15-Interfe-ron

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 to determine IFN by dilution of VSV RNA synthesis, (o-o ).

Standard human fibroblast interferon 25 U / ml was determined in parallel (9- - • #). Control without VSV (i i) and with VSV without IFN (ywm) are shown. The two columns on the right show the remaining activity of the s phages C15 IFN (at the final dilution 1:40) after adsorption on immobilized anti-IFN or non-immune IgG, as described in the Methods section.

Fig. 4: Growth and IFN generation in E. coli containing 10 plasmid TL11

The TL11 plasmid contains a hybrid Tripylac-Pro-motor and the coding sequence for mature human IFN-ßb from the human genomic fragment, as described in the text. Bacterial cultures were grown in a 15 liter New Brunswick fermentor and at the indicated time bacteria were lysed by lysozyme propylene glycol and IFN activity on human cells challenged with VSV was determined. IFN activity is calculated per milliliter of bacterial culture.

20th

C.

5 sheets of drawings

Claims (16)

664 761 2nd PATENT CLAIMS 1. A process for the preparation of human fibroblast interferon-ß), characterized in that a human genome DNA fragment, which contains the genetic information for human fibroblast interferon-ß, is introduced into a phage with bacteria infected phage obtained, the bacteria grown and the interferon wins. 2. Process for the production of human fibroblast interferon-ß |. characterized in that a human genome DNA fragment which contains the genetic information for human fibroblast interferon-ßi is introduced into a phage, from this phage a DNA fragment which contains the genetic information for human fibroblast interferon -ß, contains, wins, inserts the fragment thus obtained into a plasmid which, after introduction into a bacterium, is capable of effecting the synthesis of Hu-man fibroblast interferon-ßi, infecting bacteria with the plasmid obtained, which thus infected Bacteria grow and the interferon produced wins. 3. The method according to claim 2, characterized in that the phage is À-charon 4A, from which clone C 15 is obtained with the gene card according to FIG. 1A. 4. The method according to claim 1 or 2, characterized in that the infected bacterium is E. coli. 5. The method according to claim 2, characterized in that the human genome DNA fragment obtained from the phage, is introduced into a plasmid expression vector, which is provided with a strong promoter. which is capable of effecting the synthesis of human fibroblast interferon-ß in a bacterium. 6. The method according to claim 5, characterized in that the plasmid used is pBR322, which contains the recA promoter of E. coli, 7. The method according to claim 1 or 2, characterized in that the human genomic DNA fragment, which contains the IFN-ßrGen, is removed from the plasmid, cut close to the codon for the first methionine of the mature IFN and on the ribosomal binding site of the E. coli lac promoter, which is modified to contain the RNA polymerase binding site of the tryptophan promoter, thereby obtaining a hybrid tryp-lac promoter, the plasmid back into the bacterium is introduced, and the bacteria are grown to produce interferon. 8. Process for the preparation of human leukocyte interferon ac, characterized in that a human genome DNA fragment which contains the genetic information for human leukocyte interferon ac is introduced into an À phage with bacteria infected phage obtained, the bacteria grown and the interferon wins. 9. A process for the production of human leukocyte interferon ac, characterized in that a human genome DNA fragment which contains the genetic information for human leukocyte interferon ac is introduced into an agen phage from the latter Phage obtains a DNA fragment which contains the genetic information for Hu-man leukocyte interferon ac, which inserts the fragment thus obtained into a plasmid which, after introduction into a bacterium, is capable of synthesizing human leukocyte interferon after introduction into a bacterium -ac to cause bacteria to be infected with the plasmid obtained, to cultivate the bacteria thus infected and to generate the interferon produced. 10. The method according to claim 9, characterized. that the phage is À-charon 4A, from which clone 18-3 is obtained with the gene map according to FIG. 1B. 11. The method according to claim 8 or 9, characterized in that the infected bacterium is E. coli. 12. The method according to claim 2, characterized in that the human genome DNA fragment is obtained from the phage and is introduced into a plasmid expression vector which is provided with a strong promoter which is capable of synthesizing human To cause leukocyte interferon ac in a bacterium. 13. The method according to claim 12, characterized in that the plasmid used is pBR322, which contains the recA promoter of E. coli. 14. The method according to claim 8 or 9, characterized in that the human genomic DNA fragment, which contains the IFN-ac gene, is removed from the plasmid, is cut close to the codon for the first methionine of the mature IFN and is bound to the ribosomal binding site of the E. coli lac promoter, which is modified such that it contains the RNA polymerase binding site of the trypthophan promoter, whereby a hybrid tryp-lac promoter is obtained, the plasmid is recovered the bacterium is introduced and the bacteria are grown to produce interferon. 15. Human fibroblast interferon -ßh produced according to one of claims 1 to 7. 16. Human leukocyte interferon ac, produced according to one of the claims 8 to 14.