FR2509614A1 - INTERFERON PRODUCTION - Google Patents

Abstract

PROCESS FOR THE PRODUCTION OF HUMAN INTERFERON OF FIBROBLAST B AND LEUCOCYTE A TYPES, CHARACTERIZED IN INCLUDING THE INTRODUCTION OF GENOMIC, HUMAN, DNA INTO AN APPROPRIATE PHAGE AND THE INFECTION OF A BACTERIA WITH SUCH A PHAGE RECOMBINANT , OR THE RECOVERY OF AN APPROPRIATE DNA SEGMENT FROM THIS PHAGE, INTRODUCTION OF THIS SEGMENT INTO THE EXPRESSION PLASMID OF A BACTERIA, CULTURE OF IT, AND RECOVERY OF THE INTERFERON AS SOON.

Description

The present invention relates to the production in large quantities

  of human INF interferons, fibroblast (È) or leukocyte (<i) type, using appropriate bacteria into which a human genomic DNA fragment has been introduced. Interferon, and in particular human interferon, is becoming increasingly important in many

  Its production is rather difficult and

  in this area are of great importance.

this and value.

  Expression of mammalian genes in bacteria such as E. coli is usually impossible because of the

  presence of intermediate sequences, interrupting the

  There is, however, a number of genes that do not have introns. Recently, Nagata et al (Nature 287, 401-408, 1980) have shown that genes for human leukocyte interferon are lacking. also of introns They could detect low rates

  of expression of these genes in E Coli.

  Human diploid fibroblasts, induced by double-stranded RNA

  filament, contain at least two m RNA encoding interferon (Weissenbach et al PNAS 77, 7152-7156,

  1980). One of them, 0.9 kb (11 S) long, corresponds to the

  of the main interferon IFN 1 produced by these

  lules, whose amino acid sequence is partially

  terminated (Knight et al. Science 207, 325-326, 1980) Cloning via AD Nc of this IFN Pl AR Nm, using plasmid p BR 322 from E Coli as vector, was achieved (Goeddel et al. Nucleic Acid Res 8, 4057-4075, 1980) The nucleotide sequence of these DNA clones corresponds to that which is

  determined for the protein and, after construction of

  Expression mediates, IFN- / 31 AD Nc is recognized to direct the synthesis of human interferon in E coli The applicant has isolated genomic fragments carrying IFN-1 gene (Mory et al., Eur J Biochem 120, 197202, 1981) and IFN-c genes <These genes are devoid of introns, and can

  to be accordingly directly expressed in E Coli.

  The new method of producing fibroblast (") and leukocyte-type (>) human interferon comprises the introduction of human genomic DNA into a

  appropriate phage, followed by either contamination of

  appropriate series by this recombinant phage, or else the

  recovery from this phage of a DNA segment con-

  the introduction of this segment into the plasmid of

  pressure of the appropriate bacterium, culture of these bacteria

  and recovery of interferon.

  The invention relates to a method, in which a specific fragment of human genomic DNA, extracted from

  human blood cells, is cloned into a bacillary vector

  serophage, and used directly to program the synthesis of IFN in bacteria, which are cultured for the production of interferon During this process, there is no need for painful preparation and cloning of complementary DNA (AD Nc) Total length, from Arim According to the invention, a suitable portion of DNA is taken directly from a readily available human cell, as a white blood cell, and is cloned into a vector. phage The IFN gene needed is not

  interrupted by introns, he is able to

  directly metabolize IFN in bacteria A strong

  IFN activity is produced by the infection of

  as E Coli, by a recombinant> -phage,

  closing a human DNA inserted into the IF Np 1 gene or the gene

  IFN-c O (IFN is collected and purified from the bacterial lysate

  resulting from this contamination It has been found that

  phages were appropriate vectors for the introduc-

  fragmentation of human DNA in bacteria; X Charon -

  4 A is particularly appropriate and leads to a strong

  The invention also relates to the introduction of

  of these cloned fragments of human genomic DNA into plasmid expression vectors, which are provided with

  strong initiators This process can be carried out with different

  IFN- genes "as well as with different genes

  IFN-1 ', provided that they are free of introns.

  In one embodiment, the DNA from

  from a human adult is fragmented by partial digestion

  Eco RI, and is cloned into Charon 4A. The clone thus obtained, designated Clone C 15, with a human DNA insertion of the order of 17 kb, is identified as containing

  a gene for fibroblast IFN-1 interferon

  restriction shows that this gene, located on a frag-

  1.8 kb Eco RI, is not interrupted by introns.

  This human genomic DNA fragment is capable of directing the synthesis of human active IFN Ai in E coli and other suitable bacteria. High IFN activity can be harvested from phage lysates, which

  certain conditions, may reach 107 U / liter.

  sats are advantageously subjected to chromatography,

  preferably on "Cibacron Blue Sepharose" or on co-

  similar types, and the interferon thus obtained has the same immunological properties and the same specificity of

  species that IFN produced from human fibroblasts.

  Similar results are obtained with IFN-α, the

  The gene is identified in clone 18-3 containing a fragment

13 kb human genome.

  In another embodiment, the fragment

  Human genomic DNA containing the IFN-1 or IFN-qc gene is introduced into a plasmid such as a plasmid.

  p BR 322 containing the rec A promoter of E Coli The cul-

  E coli transformed by these recombinant plasmids.

  of large amounts, respectively, of IFN-A 1 or IFN-α, when the rec A promoter is induced by

  nalidixic acid The IFN-Ai genomic fragment is

  subsequently cut near the codon for the first methionine of mature IFN-1, and is bound to the ribosomal binding site of

  E coli lake promoter This lake initiator is modified

  in order to enclose the binding site of the po-

  lymerase of the tryptophan promoter With this hybrid initiator

  tryp-lac flange, genomic IFN- / 1 DNA is capable of producing 108 U / liter of IFN-i 1 in E coli P 679-54 minicell strain Similarly satisfactory yields of IFN-α are obtained when the IFN c gene is appropriately bound to the tryp-lac initiator. In all cases,

  IFN activity is recovered from the bac / cell

  by lysis using lysozyme and propylene glycol at 30%, and is then purified, as far as

  phage lysates, on a hydrophobic support of chroma-

  tography: for IFN-j 1 the preferred medium is Cibacron

  Blue Sepharose Elution can be achieved by sub-

  stances such as ethylene glycol, or better, propylene glycol.

  Further purification of IFN-1 or IFN-c may be performed by immunoaffinity chromatography on monoclonal antibody columns, or by other conventional techniques. It is therefore clear that fragments

  human genomic DNA can be used for the production of

  high yield of human IFN, in E coli and other appropriate bacteria As mentioned above

  the method according to the present invention consists in introducing

  a fragment of human genomic DNA in a vector

  such as a phage, to contaminate a culture of bac-

  with such phages, resulting in the lysis of

  these bacteria, and to collect the resulting interferon,

  The invention relates to novel phages, obtained by the introduction of human genomic DNA, and in particular to recombinant phages of the -> type as shown in FIG.

  clone C 15, giving IFN 2 and 18.3 giving IFN oc The invention

  further relates to a process for the production of

  terferon, by introducing human genomic DNA into an appropriate phage, recovering from this phage a section of suitable DNA, introducing it into the plasmid expression of appropriate bacteria, culture

  of these bacteria and recovery of the desired interferon.

  Other objects of the invention will appear from

  the following detailed description, not limiting.

  A great advantage of this invention lies in the fact that the genes of healthy or sick individuals can be compared for their ability to produce IFN, which allows the study of genetic deficiencies.

in the production of IFN.

  To begin, we study substances and

the processes used.

  Preparation of a collection of human genes High molecular weight DNA is prepared from

  peripheral blood cells of an adult, with j-thalas-

  Sow portions (40 / g) of DNA are digested separately for 30 minutes with 100, 200 or 300 Eco RI units, then are heated for 10 minutes at 65 C, and are applied.

  together on a 10 to 40% gradient of sucrose, as de-

  crit The DNA fragments between 10 and 20 kb are linked with

  T 4 DNA ligase, as described by Mory et al (Mol Biol.

  Reports 6, 203-208, 1980) to the branches of> Charon 4 A DNA

  prepared by Eco RI digestion according to Maniatis et al (Cell.

687-701, 1978).

  In vitro conditioning is done as described by Hohn

  and Murray (PNAS 74, 3259-3263, 1977) by mixing several

  8 μl aliquots of the binding reaction (0.5 μg of DNA vector and 0.125 μg of human DNA) with 50 μl of extracts of E Coli BHB 2688 / N 205 rec A - ( 434 c Its b 2 red 3 Eam 4 Sam 7) /> 1 l and BHB 2690% N 205 rec A (Limm 434 c Its b 2 red 3 Dam 15 Sam 7) / 7 By gag of recombinant DNA, one

  obtains 3 x 105 pfu, compared with 8 pfu / g intact DNA.

  A total of 1.8 × 10 6 recombinant phages are diluted in 6 ml of Tris-HCl 10 mM, pH 7.5, 10 mM of SO 4 Mg, 0.01% of

  gelatin After addition of 0.1 ml of chloroform and

  microfuge centrifuge debris, the phasing

  are developed 10 times by deposit on E Coli DP 50 (Le-

  der et al., Science 196, 175-177, 1977) at 4 x 103 pfu / 15 cm

  of plaque For screening 2 x 104 pfu are

  placed on 15 cm plates, and nitrocellulose double filters are raised in follow-up as indicated by Benton et al (Science 196, 180182, 1977), This work

  is carried out under P 3 restraint conditions.

  Test Preparation of IFN-A 1 AD Nc Poly A from foreskin fibroblast F 511 cultures induced for 4 hours per 100 μg / ml of poly (r I): (r C), 501 g / ml of cycloheximide (with 2 μg / ml Actinomycin D for the last hour) are prepared, and both

  peaks of AR Nm interferon are fractionated by centrifugation

  Sucrose gradient procedure, as detailed in Weissenbach et al (PNAS 77, 7152-7156, 1980, and Eur J Biochem 98, 1-8, 1979) 11 S m RNA (1.5 μg) is used to prepare RNA-AD Nc hybrids with reverse transcriptase from avian myeloblactosis virus (J Beard) and oligo (d T), as above, Weissenbach et al (PNAS, 77, 7152-7156, 1980) These RNA-AD Nc hybrids are directly cloned according to Zain et al (Cell 16, 851-861, 1979), but using CTP for tailing hybrids, and ds I, split Pst I-split BR 322 as vector (Chang et al Nature 275 , 617-624, 1978) A total of 12,500 tetr S is obtained

  transformants of E Coli MM 294, as indicated by Sten

  lund et al (Gene, 10, 47-52 1980) The hybridization of

  lonie is made with tests of AD Nc to 32 P transcripts

  either from 11 S AR Nm (6 / g) of F 511 cells or from

  Non-induced cell poly A ± RNA (3.7 / Ag) extraction Synthesis of AD Nc is initiated with a synthetic, complementary primer (see Narang et al, Methods Enzymols, 68, -98, 1979). 'GAGATCTTCAGTTTC 3', which includes the Bgl site

  II of the IFN-b1 sequence With a primer marked to

  32-5 '(Houghton et al., Nul Ac Res 8, 1913-1931, P-5 minus' Houghton et al., Nucl Ac Res 8, 1913-1931.

  1980), 1980), a desired AD Nc, 640 nm long, is observed.

  cleotides, only when using induced RNA as a template To prepare the assays, 20 # of primer are annealed to each RNA in 4/1 KC 1 0.4 M, at 30 C for 60 minutes, then put to incubate in 25 d with p Ci each of d CTP and "-d ATP at 32 p (both 400

  Ci / mmole), 0.5 m M each of d GTP and TTP, 50 m M of Tris-

  HCl pH 8.3, 5 mM dithiothreitol, 50 / g / ml actinomycin

  D, 4 mM pyrophosphate and 30 transcription units.

  reverse phase, for 2 hours at 37 ° C. After addition of 4 g of E coli DNA, the medium is treated with 0.3N NaOH for 60 minutes at 70 ° C., it is neutralized and filtered on Sephadex G-50. in 50 mM Tris-HCl pH 8, 0.1M NaCl, 10mM EDTA, 0.5% dodecyl sulfate.

  from each RNA, 5 x 106 cpm of AD Nc The colonies, cul-

  Nitrated on nitrocellulose filters, are fixed, and the DNA is denatured as described by Thayer (Anal Biochem 98, 60-63, 1979) Each filter is prehybridized with 10 ml

  of 6 XSET (SET = 150 mM NaCl, 2 mM EDTA, 30 mM Tris).

  HCl p H 8) 10 x Denhardt's solution (Denhardt 1966, Biochem Biophys Res Comm 23, 641-646), 0.1% Na pyrophosphate,

  0.1% dodecyl sulfate, 50 μg / ml E-modified DNA

  These filters are hybridized at 67 ° C. for 1418 hours in the same solution with 0.5 × 10 6 cpm / filter of one of the two tests of AD Nc at 32 μ, 10 μg / ml. The filters are rinsed for 1 hour at 67.degree. C. in the prehybridization solution and then 3 more times for 30 minutes at 67.degree.

  with 3 XSET, 0.1% pyrophosphate, 0.1% dodecahydrate

  cyle, then for 60 minutes at 25 C with 4 XSET (see Mania-

  tis, 1978, Cell 15, 687-701) The filters are dried and exposed to Agfa Curix ray + films for 1 to 2 hours.

  days at -70 C with reinforcement screens.

  Among 3,500 colonies examined, 14 hybridized with

  of primed AD Nc from the induced AR Nm, while

  130 have hybridized to AD Nc primed with AR Nm no

  The DNA (0.3 μg) of the plasmids from the first group was hybridized on filters (Kafatos et al. 1979, Nucl Ac Res 7, 1541-1552) with the 5 'end of the labeled antibody. 32 p (0.3 + oles, 4 × 10 6 cpm) in 6 XSSC, Denhardt's solution, at 35 ° C. for 21 hours, then rinsed with 6 XSSC (900 mM NaCl, 90 mM citrate). Na) at 35 ° C. An I-6-5 clone is highly positive and the succession of DNA (see Maxam et al, 1980, Methods Enzymol 65, 499-560) shows that it contains about 370

  nucleotides from the -3 'side of the IFN-A 1 sequence.

  A further 50,000 clones prepared as above were examined by introduction of C -equents ds-AD Nc from the purified sucrose gradient purified N N AR into the Pst I site of p BR 322: the proportion of clone IFN-A 1 is 0.16% (80

  clones) for 0.65% of IFN-1 2 clones.

  The clone IFN-1 I-6-5, described above and prepared from the AR Nm FS-11, is used for sorting the collection.

human.

  Isolation of genomic clone: IFN- / 1 clone Plasmid DNA clones from IFN 1, AD Nc are labeled as "notch translation", according to Rigby et al (1977, J. Mol Biol 113, 237-251 ) at 2 x 108 cpm g of

  DNA and hybridized at 106 cpm / filter with the gene from the

  The preparation of filters, the operating modes

  The DNA modification and hybridization methods are as described above. Phage from plates giving positive hybridizations are redeposited at a density of 1 to 2 × 102 pfu on 9 cm plates and re-examined. The procedure is repeated 3 times, until more than 1% of the plates hybridize to IFN 1 AD Nc. Clone phage

  is isolated from one of these plates.

  Phage are grown in liquid cultures, as

  described by Blattner et al. (1977, Science, 196, 161-169).

  E Coli DP 50 are grown overnight in 1% tryptic

  tone, 0.5% yeast extract, 0.2% maltose, 0.25%

  504 mg, 0.01% diaminopimelic acid, 0.004% thymidine.

  About 0.3 ml of this culture is mixed to prepare for

  adsorption with 0.3 ml of Mg 504 10 mM, 10 mM of CaCl and

7 2

  107 phages, for 20 minutes at 37 ° C. These cultures are then diluted in a 2-liter flask with 500 ml of

  preheated medium containing 1% NZ amine, 0.5% ex-

  yeast trait, 0.5% NaCl, 0.1% casamino acids, 0.25% MgSO 4, 0.01% diaminopimelic acid, 0.004% thymidine, and all is Incubate under good aeration for 15 to 18 hours at 37 ° C. After complete lysis, the cell debris is removed by centrifugation in the cold for 15 minutes, at 7000 rpm in the Sorvall GSA rotor. The phages are precipitated from this lysate clarified using polyethylene glycol 6000 at 7% and they are purified by two successive gradients of

  Cs C1 Phage DNA C is phenol-purified and its structure

  Ture is analyzed by restriction enzyme digestions, horizontal agarose gel electrophoresis in 20 mM tris base, 10 mM Na acetate, 1 mM EDTA with 0.5 / g /

  ml of ethidium bromide and then transferred to

  very nitrocellulose (Southern, 1975, J Mol Biol 98, 503-517) and hybridized with translational IFN 1 AD Nc

  As above Eco RI fragments of C15 DNA are underdeveloped.

  cloned into the Eco RI site of p BR 322 for precise restriction labeling of the plasmid DNA, according to the established procedure (see Bolivar, 1979, Methods Enzymol 68,

245-267).

  Interferon Activity Assay in Phage Lysates Clarified IFN A 1 C 15 phage lysates, prepared as

  above, are dialyzed against a buffered saline solution

  Phosphate born (PBS) for 7 hours A 10 ml portion is loaded into a 0.3 ml column of Cibacron Blue

  Sepharose CL 6 B (Pharmacia Fine Chem) The column is rinsed

  10-15 ml of 1M NaCl, phosphate buffered saline,

  dium 0.02 M p H 7.2 and then with 10 ml of propylene gly-

  col in the rinsing solution Fractions (0.5 ml) are collected and stored at 4 ° C; in some cases, 0.1% of human serum albumin is added to stabilize. The interferon activity is tested, as described by Weissenbach et al (1979, Eur J Biochem 98, 1-8), by dilution of each fraction in series in a 95-well microplate Each housing receives 2-3 x 104 human fibroblasts F 511 in 0.1 ml of Minimal Essential Medium (Gibco),% fetal calf serum (FCS), 0.5% of Gentamine After 18 hours at 37 C, the medium is removed and

  vesicular stomatitis virus (VSV) at 1 pfu / cel-

  lule in medium at 2% FCS Inhibition of effect

  cytopathic is recorded 30 to 40 hours after the infection.

  compared to the standard NIH IFN G 023-902-527.

  Antiviral activity is also measured by the reduction

  incorporation of 3 H-uridine after contamination

  by VSV as previously described (Weissenbach et al.

  1979, Eur J Biochem 98, 1-8) Anti-serum against IFN-g

  is obtained from rabbits and is tried as

  (Weissenbach et al. 1979, Eur J Biochem 98, 1-8) For the neutralization test 0.1 ml of anti-serum (titrating 104 U / ml) or non-immune serum is mixed with

  0.2 ml of a suspension of 50% Sepha

  The beads are rinsed twice with PBS, and 0.1 ml of bacterial interferon (100 U / ml) is added.

  2 hours at 37 ° C, the beads are centrifuged and the

  the supernatant is tried as an inhibitor of the incor-

  poration of H-uridine in cells contaminated with VSV. Isolation of the genomic clone IFN c clone 18-3

  Two short oligonucleotides with a single filament, chemical-

  synthesized as above, are used to directly sort the human collection These oligonucleotides are complementary to the usual sequence of IFN-α genes and have the sequence 5 'CTCTGACAACCTCCC 3' and 'CCTTCTGGAACTG 3' These oligonucleotides are used after marking in 5 ', as above, either directly,

  or as primer for synthase of AD Nc on cellular RNA

  human leukemia induced from Sendal virus. The 32 Poligo nucleotides or AD Nc primed are hybridized for a total of 500 000 recombinant Xphages, as demonstrated above. Approximately x 10 5 cpm per 9 cm nitrocellulose filter is used. Hybri -

  dations are made in sealed cooking bags.

  plastic materials in minimum volumes of

  The hybridization with the basic primers 15 is

  read as follows: first prehybridization in 6 XSSC, x Denhardts at 37 C for 4 hours; then, hybridization

  in 6 XSSC, 10 x Denhardts at 37 C for 18 hours, and rinse with

  3-4 times, 30 minutes each, in 6 XSSC at 37 ° C or until no more counts are found in the rinsing. For hybridization with the base primers 13, the hybridization temperatures and rinses are lowered to 30 C A total

  of 16 phages give a positive hybridization and are

  The clone 18-3 is shown to carry 3 IFN-1 genes, one of which has a sequence identical to that of the clone.

  oc NDA of Goeddel et al (1981, Nature, 290, 20-25).

  Clone 18-3 is purified as described for clone 15 below.

  above Expression of clone 18-3 in a bacteriophage and after insertion into an expression plasmid vector,

  is described in the results below.

  1 Structure of the Genomic Clone C IFN-i 1.

  This phage clone is isolated from a collection of human DNA that has been partially digested with Eco RI and cloned

  in the branches of X Charon 4 A The clone hybridizes strongly

  in 2 different tests of IFN-3 1 AD Nc, prepared independently

  with fractions of AR Nm from 2 strains of fi-

  human broblasts The digestion of phage DNA by Eco RI shows, in addition to the two X branches, four fragments

  of 12, 2.6, 1.84 and 0.6 kilobases (Fig. 1A).

  nitrocellulose by the method of Southern (1975 J.Mol Biol 98, 503-517), and hybridization with DNA of notch translation, from clone IFN-141 I-6-5 AD Nc, indicates that the 1.84 kb fragment contains the IFN A gene. This Eco RI fragment is recloned in the Eco RI site of p BR 322; restriction analysis of this 1.84 kb subclone with Bgl II, Pvu II, Pst I, Hinc II and Taq I, and hybridization with total or partial length trials of IFN-1 1 AD Nc, conclude that the sequence

  coding for IFN-1 preprotein (Hinc II site)

  mence to about 35 Sucleotides from the Eco RI site

  (Fig 1 A) The distances between the different sites of

  triction in genomic DNA are the same as in the sequence of AD Nc in the limit of the experimental error

  (Table 1) There are no unintentional restriction sites

  within the confines of the IFN-A 1 coding region, this

  which indicates the absence of any intermediate net sequence.

  Partial Eco RI digests of C-DNA show that the 0.6 kb Eco RI fragment is located between the 1.84 and 2.6 kb fragments. A Bam HI site is found in 2.6 kb, but not

  in the other Eco RI fragments of the human DNA insert.

  The 1.84 kb EcoRI fragment, which hybridizes to AD Nc of IFN-31, is on a Bam HI 9.6 kb fragment of C-DNA. As shown in FIG. from the Bam HI site of the right branch> (5.1 kb of Eco RI), leaving 4.5 kb for the Eco RI-Bam HI segment of the insert

  showing that the 1.84 kb EcoRI fragment must be

  The orientation of the gene (Fig. 1A) is determined as follows. When the C-DNA is cut with Pvu II and hybridizes with the total length or half-length at 3 'of the AD Nc of IFN e 1, only a 0.66 kb fragment hybridizing with the 5 'end of IFN- / i is found But when the 1.84 kb Eco RI portion is cut with Pvu II the 5 'end of IFN 1 is on a smaller 0.54 kb fragment. Since no Pvu II site is found in the 0.6 kb Eco RI portion of the human spacer, the 0.66 fragment kb-Pvu II must terminate the right branch 'and, consequently, the 5' end of IFN-il, is the closest to the right branch X The indicated structure

  in Figure 1A is supported by several other

  none of these restriction markers, using diges-

  Hind III, Sma I and Bgl II The coding sequence

  IFN / 1 in clone C 15 is therefore

  to the 1775 nucleotides from the strong PL initiator that controls the transcription to the left (Williams et al., 1980, Genetic Engineering Vol. Ipp 201-281, Plenum Corp) and in a suitable left orientation.

  together with the absence of detectable introns,

  cline the plaintiff to look for interferon could

  to be produced in E coli contaminated with recombinant phage

from C 15.

  2 Recovery of the biological activity of interferon from

  IFN-41 Phage-Contaminated E Coli ELISA Lysates Phage from clone C were cultured in 500 ml cultures of E Coli DP 50, as described above in the 10 ml procedures. of clarified lysate are dialyzed and loaded onto a small column of Cibacron Blue Sepharose (0.3 ml) and the fractions are tested for their inhibitory effect on the cytopathic effect of VSV, with

  as a reference to an INF- / standard During an experiment

  tation, in which the phage titer in the lysate is 1.3 x 10 10 phages / ml, the type of interferon activity shown in Figure 2 is observed in the fractions eluted from the column propylene glycol 50% -NaCl 1 M, a total of 7,500 activity units

  This corresponds to 0.75 x 106 units / liter of lysate.

  However, in this crude lysate, the measurable activity is much lower, suggesting that in this lysate, some substance prevents a correct test of IFN activity. During a reconstruction experiment, IFN- / standardized human is added to a wild-type X Charon 4 A lysate, and indeed the measured activity represents only one-tenth of the intake. When this mixture is passed over the Blue Sepharose column, 75 % of the

* tivity introduced In a control operation, one analyzes

  lysis of phage lysate \ Charon 4 A wild type (without addition of IFN), and no IFN activity was observed

(Figure 2).

  The chromatographic behavior of IFNs from clone C lysates may vary. In one experiment, in which the phage titer is 3 × 11 pfu / ml,

  IFN activity is strong but is not retained on the co-

  Blue Sepharose In this experiment, the ac-

  total activity, recovered from the column, amounts to 70-80 000 units of IFN, for an introduction of 10 ml of lysate (7-8 x 106 units / liter). The effluent fractions are

  re-adsorbed on a second column of Blue Sepharose: this

  Once the activity is retained on the column, but again

  calf is eluted by rinsing the column with PBS IFN-I 1

  should normally be eluted from Blue Sepharose, only

  Therefore, normalized IFN- / human is mixed with this same phage lysate and the whole is applied to the column: 30% of activity is not

  retained, and the recovery of activity is only 25%.

  This suggests that constituents of this lysate probably destabilize the interaction of IFN- /, especially

  which is produced by bacteria, with Blue Sepharose.

  Even if interferon is not retained on the column, more

  90% of the lysate protein is removed from the active fractions.

  which have a specific activity of 4-5 x 105 U / ml of

  This partial purification is essential to remove the chemicals which conceal the activity in the crude lysates. During an experiment, the IFN activity of clone C is also recovered after chromatography of the lysate on carboxymethyl sepharose (not shown). .

  3 Properties of interferon produced in E Coli contagion

mined by clone C 15

  Interferon activity, recovered after the chroma-

  tographic, reduces the incorporation of 3 H-uridine into

  human cells contaminated with VSV, treated with

  nomycin D (Weissenbach, 1979, Eur J Biochem 98, 1-8)

  The titration curve, obtained for bacterial IFN, is

  to that of normalized IFN- / human (Figure 3) A-

  to test the immunological properties of the bacterial IFN

  the partially purified product is mixed with anti-IFN-A rabbit serum (inactive on IFN-O) or with non-immune rabbit serum which has been previously bound to protein A-Sepharose. IFN activity of the supernatant: anti-IFN-A serum

  removes all the activity of interferon, while the

  remains when the non-immune serum is used

  3) The bacterial IFN (20 U / ml) is similarly neu-

  when mixed with anti-IFN-A (20 U / ml)

  on cell culture and tried by inhibiting the ef-

  VSV cytopathic fetus The species specificity of

  Bacterial feron is tested by comparison of different cell types Like human IFN-A, the product of E Coli

  contaminated with clone C 15, has less than 10% activity

  on BSC-1 cell of monkey kidney and bovine Bovine MDBK cells compared to human cells. There is no detectable antiviral activity on mouse L or A 9 cells (Table 2). Similar results are obtained with 6000 U / ml of bacterial IFN produced by the

clone C 15.

  The present invention demonstrates, therefore, that the

  IFN-1, isolated directly from fragments

  Eco RI sequences of human genomic DNA by cloning in X Charon 4A, can be expressed and leads to the accumulation of significant amounts of IFN activity in cultured lysates. The activity produced is obviously of 1 '.

  interferon (e) fibroblast, as indicated by its

  Immunological properties and species specificity

  is produced by bacterial culture only in response to

  is contaminated with phage clone IFN e 1 C 15.

  The IFN activity produced by clone 15 in E Coli,

  appears to human IFN-A in its chromatographic properties.

  on Cibacron Blue Sepharose The chromatographic stage

  It is essential to recover a high activity (up to 7 x 106 U / liter) from the bacterial lysate.

  interferons therefore provide the first examples

  of biologically active proteins to have been pro-

  picked by bacteria under the direction of a party

  of human DNA taken directly from the genome of a

dividual.

  4 Clone 18-3 genomic IFN 4 j This clone is identified by direct hybridization of short

  oligonucleotides, chemically synthesized to be

  Complementary sequences in IFN-0 From a total of 0.5 genomic O, 16 clones proved positive in

  hybridization The Eco RI fragments hybridizing with the o-

  ligonucleotides are sequenced and it is shown that clone 18-3 as in Figure 1 B, contains a 2 kb Eco fragment

  RI 18-33 (insert of FIG. 1B) in which is

  This gene is, clearly, the source of the AR Nm which gives rise to the clone of IFN-? c reported by

  Goeddel et al (1981, Nature 290, 20-25) Clone 5-1 refers to

  presents a fragment of DNA that overlaps with another

  apparently, clone 5-1 and clone 1 -3-carry in addition to IFN-c 'two other genes of IFN-Kdesigned by i-c 2 and

4 (FIG. 1B).

  c Genomic fragment expression of human IFN under the control of rec-initiator A. Rec-initiator is considered one of the strongest E-Coli initiators Normally, it is strongly

  braked by the gene product, even when it is pre-

  on a multicopy plasmid such as p BR 322 It is

  induced, in the presence of damaged DNA, to split its

  It has been shown to be at a very high level (Sancar and Rupp, 1979, PNAS 76, 3144-3148). The usual inducers for rec A are nalidixic acid (which blocks the synthesis of DNA) or mitomycin C (which reticle

the DNA) -

  A plasmid, DR 1453, containing the cloned rec A gene, is used to isolate the initiator on a Taq I-Bam HI fragment of approximately 1 kilobase. This is bound in p BR 322 which has

  was opened with Cla I and Bam I to construct the RAP-1 plasmid.

  In this case, the Taq I-Cla I link re-establishes the Cla I site, which allows the plasmid to be opened only in the

  third codon of the rec A gene RAP-2 is published by

  of RAP-1 with Eco RI and Cla I, filling to the extremes

  adhering and rebinding, which restores the RI site.

  When RAP-2 is opened at the RI site, foreign DNA can be inserted at the third codon of the rec A protein. The RAP-1 vector is used for the indirect expression of interferon activity by genomic fragments of the Human genomic library The IFN-1 gene and several isolated IFN- <subclass Xc genes produce high interferon activity (as determined by the assay.

  antiviral standard), when inserted into this

  mide The DNA RI fragments on which these genes reside

  (FIGS. 1A and 1B) are subcloned into the RI site of the plasmid

  mide RAP-1 This plasmid is placed in a host rec A +, the

  bacteria are cultured and induced with nalidic acid

  cells are harvested and lysed with lysozyme plus 30% propylene glycol, and the extract is tested for

  antiviral activity The activity rate is clearly dependent on

  nalidixic acid induction, the optimal value being at 50/4 g / ml. Interestingly, in some cases the genomic fragment produces activity.

  in two possible orientations relating to the initia-

  The IFN- / 1 and IFN-i genes that show activity are all located on DNA Eco RI fragments.

  1.8-2 kilobases, so that the distance of the i-

  initiator ATG start codon of the interferon gene is

  of the order of several hundred nucleotides.

  These experiments show that the initiator functions as expected and they make it possible to rapidly determine those genes, of the interferon type, of the "gene library",

which can be active.

  6 Strong IFN 1 gene expression under the control of a tryp-lac hybrid initiator

  Plasmid PLJ3 (Johnsrud, PNAS, 1978, 75, 5314-5318) contains

  me two lake initiators, each 95 base pairs long, separated by 95 unrelated nucleotides This fragment of

  285 nucleotides is inserted in the Eco RI site of the plas-

  mide PMB 9 Lake sequences, 95 kars long,

  contain the operator and initiator sequence and stop

  just before the ATG of β-galactosidase A DNA coding sequence with an ATG initiator, remotely intercalated

  of the ribosomal binding site of the (-galac-

  tosidase, must be correctly expressed in cells

  The 285 base pair Eco RI fragment is

  cloned into BR 322 at the Eco RI site; recombinants are es-

  tried by culturing the cells on plates containing

  / qg / ml X-gal and 15 μg / ml tetracycline.

  positive blue copies are pooled and the DNA is purified by

  gradient centrifugation of Cs Cl.

  Two orientations of lake initiators are expected: to the ampicillin gene or to the BR 322 p tetracycline gene. As only the Eco RI site located between the lac ribosomal binding site and the BR 322 p sequence. is very interesting, this site is protected by binding of RNA polymerase with plasmid DNA, while the furthest site is opened by Eco RI restriction, is loaded

  with the Klenow fragment of DNA polymerase and is connected A-

  after transformation of E coli MM 294 cells, the plasmids

  are isolated and the distance between the Eco RI site and the Bam HI site of p BR 322 is measured Plasmids containing a fragment at 375 base pairs are those in which

  the lake initiators are towards the tetracycline gene, tan-

  say that those containing a 670 base pair fragment (375,295 bp) have lake-initiated initiators

to the ampicillin gene.

  From the 1.84 kb subclone of Eco RI of the genomic fragment

  of IFN (1), a Hinc II-Bgl II fragment containing

  Preinterferon sequencing The parent protein of IFN-1 i-

  contains at its terminal end -NH 2, a methionine which

  can be used as an initiator codon in E Coli.

  There are two Alu I sites near this ATG codon, separated by 13 nucleotides, one of these sites is 8 nucleotides before ATG, while the other is 2 nucleotides after

  ATG Partial digestion of Alu I is performed on the fragment

  Hinc II-Bgl II and fragments around 510

  basal res are isolated from agarose gels.

  containing the lake initiators to the gene of

  tracycline is reduced with Eco RI, the site is loaded with DNA polymerase and cut with Bam HI When binding Alu I-Bgl II fragments to the vector loaded with Eco RI-Bam HI, the Eco RI site is restored to be able to differentiate between

  clones containing the ATG codon (longer than 13 nucleotides)

  tides), the clones obtained are reduced with Eco RI and PstI; the clones containing the desired 151 base pair fragments are sequenced. The Eco RI site is then reopened and the distance between the ribosomal binding site and A Tg is

  shortened by Bal 31 digestion, and relinking

  E coli lules transformed by these plasmids are sorted

for the production of IFN / 1.

  It is found that a clone, L-11, produces about 3 x 10 6 U / l.

  The lac-initiating region in this clone is cleaved with Hpa HI, i.e., 17-nucleotides before the start of mRNA. To cut the IFN-1 gene, use is made of Sai 3a enzyme which cuts the DNA just 3 nucleotides beyond the IFN I 1 termination codon This Hpa IISau 3a-IFN-p, 1 fragment is introduced between the Cla I and Hind III sites. a tryp initiator plasmid, prepared as follows A 180 nucleotide long Taq I-Taq I fragment from -21 to -201 before the start of tryp m mRNA is cleaved from the tryp p plasmid EP 121-221, and is cloned in the Cla I site of p BR 322 We arrange for the initiator fragment

  of trypsis oriented in the sense of the needles of a

  This operation restores a ClaI site at the -21 position of the tryp initiator. After binding to the IFN-A 1-lac fragment, a tryp-lac hybrid initiator is formed before the IFN- / 11 This plasmid TL-11, is

  used to transform E Coli MM 294 or the minicell strain

  These bacteria produce 108 U / liter of IFN-A 1, under fermentor culture, at a OD 650 of

  , which demonstrates that the genomic DNA fragment of IFN-

f 1 is very active (Figure 4).

  See Table 1 on the next page.

TABLE 1

  IFN restriction sites - (& I AD Nc and restriction site gene

Taq I -

  Hinc II Hinc II Pvu II Pst I Pvu II I Pvu II Bgl II Bgl II Bgl II Distances Calculated from DNA-A * nucleotide sequences between sites

measured at

  genomic DNA firing ** nucleotides * According to Taniguchi et al 1980 Gene 10 (11-15) ** Measured from 1.84 kb Eco RI fragment The Hinc II and Taq I sites considered are those

  which are closest to the -5 'end of the gene.

TABLE 2:

  Specificity of species: bacterial interferon produced by genomic C 15 IFN-, 1 Source of interferon Title in interferon, U / ml Cellu Cellu Cellu cells in cells of mouse mice monkey vines L A 9

F 511 BSC-1 MDBK

  1 Human cells F 511 2 E Coli contaminated with clone C 15 1,000

32 32 <4 <4

32 32 <4 <4

  Bacterial IFN is used after lysate chromatography

  C 15 on Cibacron Blue-Sepharose as in Figure 2.

TABLE 3

  IFN-Ket activities -produced by genomic fragments in rec A RAP-1 plasmid Conditions Activity of IFN U / ml

RAP-1 (1833) -IFN-c + nalidic acid

xique

RAP-1 (1833) -IFN- "

kc

nalidic acid

  xic <32 2 RAP-1 (1833) -IFN-Oc right orientation RAP-1 (631) -IFN-1 right orientation

RAP-1 (631) -IFN-A 1

RAP-1 clone

oppo-

  (1833) contains the Eco RI fragment with the

IFN-ic gene (Fig 1 B).

  c Clone RAP-1 (631) contains the Eco RI fragment with the

IFN 1 gene (Fig 1 A) -

  The fragments are interspersed at the beginning of the rec A gene. The orientation is given in accordance with that of the A. clone Exp. 1. -

LEGENDS OF THE ANNEXES

  Figure 1A: Schematic structure of cloned C15 IFN-e1 DNA (a) Schematic diagram of "Charon 4A Both

  branches are shown in full line.

  (b) Structure of the human DNA spacer

  clone C 15; the blackened surface shows the frag-

  Eco RI that hybridizes with AD Nc of IFN-1.

  (c) magnification of the blackened fragment from (b ?, indicated as a double line The simple line is a part of the PL \ branch

is the initiator> to the left.

  (d) position of the mRNA of IFN 1 'The upper scale corresponds to (a) and (b) and the lower scale to (c) and (d) For the detail

see the text.

  Figure 1B: Schematic structure of IFN ac clone 18-3

  It represents the diagram of reduction of in-

  serts of the two clones of> Charon 4 A The IFN gene * <is designated by the arrow alpha-c * Two

  other IFN- <genes are present near IFN- <c.

  Inserts show Eco RI fragment 18-33

  2 kb, which contains the IFN <c genes and is

  cloned in the plasmid rec A, as explained in the text The symbols for the enzyme sites of

reduction are indicated.

  FIG. 2: Interferon test, produced by IFN 1 genomic clone C 15, on Blue Sepharose

  The crude lysate of E Coli DP 50 contaminated with phage

  C 15, is fractionated as described above in the section on methods and substances, and IFN activity is tested on each fraction. (e) The protein concentration is measured

  in the same fractions () A chromatogra-

  parallel analysis and a lysate assay from N Charon 4 A phage are also shown.

(Q).

  IFN 1 genomic clone interferon titration 1 A phage C 15 IFN fraction from the column of FIG. 2 (approximately 10 U / ml) is diluted 1:40 and then diluted series in

  the microplate for IFN testing by di-

  Synthesis of VSV RNA (o o) 25 U / ml standardized human fibroblast interferon are tested in parallel (λ).

  without VSV (C) and with VSV without IFN (V) are

  presented The 2 columns on the right show the ac-

  Residual activity of phage C 15 IFN (diluted

  final concentration of 1/40 th) after adsorption on

  anti-IFN-f or non-immune IgG, as de-

  written above in the section on processes.

  : Culture and production of IFN in E Coli

  Closure of Plasmid TL 11 Plasmid-TL 11 contains a tryp-lac hybrid initiator and the coding sequence for IFN-1

  mature human, derived from the human genomic fragment

  as described in the text Bacterial cultures are made in a fermenter

  of New Brunswich of 1 liter, and at

  the bacteria are lysed with lysozyme supplemented with propylene glycol, and the activity of

  IFN is tested on human cells

  by VSV This activity is calculated per ml

of bacterial culture -

Figure 3 Figure 4

Claims (2)

claims   1 Process for the production of human interferon   fibroblast (<) and leukocyte (oi, characterized in that it comprises the introduction of genomic DNA, human, into a suitable phage and the infection of a bacterium with such a phage recombinant or recovery of an appropriate segment of DNA of this phage, introducing this segment into the expression plasmid of a bacterium, culturing it, and recovering the interferon thus formed 2 Process according to claim 1, characterized in that the interferon is of the type (, the phage used being of type \>.   Process according to claim 1 or 2, characterized   rised in that the phage is \ Charon 4 A giving a clone designated C 15.   4 Process according to claim 1, characterized in that the product interferon being of type <, the phage is of the type Process according to claim 4, characterized in that   what the phage is the X Charon 4 A giving the clone 18-3.   Process according to one of Claims 1 to 5,   wherein the infected bacterium is E Coli.   7 Process according to claim 6, characterized in that the phage is> -Charon 4 A, the infected bacterium is the E Coli DP-50.   8 Process according to claim 1, characterized in that a fragment of genomic DNA, human, is recovered from the phage, it is introduced into an expression vector of   a plasmid of a suitable bacterium, which is then cultured   for the production of interferon.   Process according to claim 8, characterized in that the DNA is introduced into the plasmid with a strong promoter. * 1 process according to claim 8, characterized in that the plasmid used is p BR 322, while   the introduced gene is IFN-i or IFN c.   Process according to Claim 10, characterized in that the plasmid p BR 322 contains the rec A promoter of the E Coli.   The process of claim 8 or 10, wherein   in that the human genomic DNA fragment   holding the IFN-1 or -δ gene, is deleted from the phage or plasmid, codon-cut for the first methionine group of mature IFN, bound to the ribosomal attachment site of the promoter   Lake E Coli, modified to contain the site of   tryptophan promoter RNA polymerase stain,   tryp-lac hybrid promoter, and that the plasmid is reintroduced into the bacterium which is then cultured to production of interferon. 2 Q w