CS273181B2 - Method of escherichia coli cells production that are capable to produce mature human leucocytic interferon - Google Patents

Abstract

A method of production of Escherichia coli cells capable of producing mature human leukocytic interferon with a partial amino acid sequence Cys-Ala-Trp-Glu-Val-Val-Arg-Ala-Glu-Ile-Met-Arg-Ser with 165 to 166 amino acids and with the amino acid Asp, Glu or Val in position 114 or, if methionine is linked to the N-terminal of the first amino acid of the abovementioned interferon, with 166 to 167 amino acids and with the amino acid Asp, Glu or Val in position 115, based according to the invention on the fact that a) E. coli cells are incubated and treated with calcium chloride DNA by an expressive vector selected from the group of the plasmid pLeIF A trp 25 DNA, the plasmid pLeIF B trp 7 DNA, the plasmid pLeIF F trp 1 DNA, the plasmid pLeIF C trp 35 DNA, the plasmid pLeIF D trp 11 DNA, the plasmid pLeIF H DNA, the plasmid pLeIF I trp 1 DNA and the plasmid pLeIF J trp 1 DNA in a ligating buffer solution for 30 minutes b) selection of the transformed E. coli cells occurs on a solid layer of agar containing tetracycline. Specifically, the E. coli strain K-12 No 294 ATCC 31446 is used.

Description

The invention relates to a process for the production of E. coli cells capable of producing mature human leukocyte interferon 8 with a partial amino acid sequence of Cys-Ala-Trp-Glu-Val-Val-Arg-Ala-Glu-Ile-Mst-Arg-Ser of 165 to 166 amino acids and amino acid A9β, Glu or Val at position 114 or, if the methionine is attached to the N-terminus of the first amino acid of the above interferon, with 166 to 167 amino acids and a amino acid Aep; Glu or Val at position 115.

The essence of the process according to the invention is that

a) incubating E. coli cells treated with calcium chloride with an ONA expression vector selected from the group consisting of plasmid pLelF A trp 25 DNA; pLelF B trp 7 DNA plasmid, pLIFF trp 1 DNA plasmid, pLelF C trp 35 DNA plasmid ^ pLelF D trp 11 DNA plasmid; plasmid pLelF H DNA; plasmid pLelf I trp 1 DNA and plasmid pLelF 0 trp 1 DNA in ligation buffer for 30 minutes, and

b) selecting transformed E-coli cells on a solid layer of tatracycline-containing agar.

Human leukocyte interferon (LelF) was first discovered and prepared in the form of very crude precipitates by Issacs and Lndemann (Proc. R. Soc. B 147, 258-267 (1957) and U.S. Pat. No. 3,699,222). Since then, efforts have been made to clean and characterize this material. These efforts have led to the preparation of relatively homogeneous leukocyte interferons derived from normal or leukemic donor leukocytes (German Patent Publication No. 2,947,134). These intarferons form a group of proteins known to have the ability to confer resistance to viruses on their target cells. Interferon may also act to inhibit cell proliferation and modulate the immune response. These properties of interferons have inspired their clinical use as therapeutic agents for the treatment of viral infections and malignancies.

Leukocyte interferons have been purified to a substantially homogeneous state / see Rubinatein et al., 'Why. Nati. Acad. Sci. USA 76: 640-644 (1979); Zaon and coworkers; ibid. 76? 5601-5605 (1979)]; and their reported molecular weights range from about 17,500 to about 21,000. The specific activity of these preparations is remarkably high; 2 x 10 to 1 x 10 units per mg protein, and the yields of cell culture methods are discouragingly low. Yet advances in working methods for determining protein sequences have allowed the determination of partial amino acid sequences in these intarfarons (see Zoon et al., Science 207, 527 (1980)) Lava et al. Why. Nati. Acad. Sci. USA 77, 5102-5104 (1980)]. The clarification of glycosylation of vigorous leukocyte interferons is not yet complete, but it is now apparent that differences in glycosylation between members of the group do not in themselves explain the spectrum of observed molecular weights. In addition, leukocyte intarferons clearly differ in amino acid composition and order, and the amino acid identity is in some cases less than 80%.

While isolation of interferons from donor leukocytes provided sufficient material. For partial characterization and limited clinical trials and homogeneous leukocytic interferon, this source is completely insufficient to obtain interferon in the amounts required for its wide-ranging clinical trial and for its extensive use for prophylactic and / or therapeutic purposes. Indeed, recent clinical trials using intarferones derived from human leukocytes to test their antineoplastic and antiviral effects have in fact been limited to crude preparations (less than 1% purity) and the long time required to produce sufficient quantities together with unrealistic pricing have critically delayed research on front.

The advent of ONA recombination technology has enabled controlled microbial production of an enormous array of useful polypeptides. Currently, bacteria modified with this technology are already available! the production of such polypeptide products such as CS 273 181 B2 tetatin, A and B chains of human insulin and human growth hormone (see Itakura et al., Science 198; 1056-1063 (1977); Goeddel et al., Nature 281, 544548 (1979)]. More recently, recombinant DNA technology has been used for bacterial production of proinsulin and thymosin alpha 1, and many authors report that they found DNA encoded for human leukocyte interferon and that the resulting proteins had leukocyte interferon activity (Nagata et al. Nature 284, 316-320 (1980)). Mantel et al., Gene 10;

1-10 (1980); Taniguchl et al., Nature 285, 547-549 (1980)].

A powerful feature in DNA recombination technology is a plasmid, a chromosome-free loop of double-stranded DNA found in bacteria and other microbes, often in multiple copies in a single bacterial cell. The information encoded in the plasmid DNA incorporates the information needed to reproduce the plasmids into the daughter cells (the so-called replicon) and usually one or more selection characteristics, such as antibiotic resistance bacteria, which allows the host cell clones containing the plasmid that It is the object of our interest that they can be recognized, selected and preferably grown in a selective growth environment. The usefulness of bacterial plasmids lies in the fact that they can be cleaved specifically by one or other restriction endonuclease or restriction enzyme, each of which recognizes a different cleavage site on the plasmid DNA.

After cleavage, foreign (heterologous) genes or gene fragments can be introduced into the plasmids either by direct attachment at the cleavage site or by attachment at the reconstructed ends adjacent to the 8 cleavage site. DNA recombination takes place outside the cells, but the resulting recombinant plasmid can be introduced into bacterial cells by a method known as transformation, and large quantities of recombinant plasmids containing a heterologous gene can be produced by culturing the transformant obtained. In addition, depending on where the gene is suitably introduced with respect to portions of the plasmids (which have driven the transcription and translation of the encoded DNA information), the expression obtained capable of being used for actual production of the polypeptide sequence; for which the built-in gene is encoded; this procedure is referred to in the description of the invention as expression.

Expression is initiated in a region known as the promoter; which is recognized and to which RNA polymerase is attached. In some cases; as in the case of the tryptophan (trp) promoter; which is preferably used in the practice of the method of the invention, the promoter regions are overlapped by operator regions and a combined promoter-operator is formed. Operators are DNA sequences that are recognized by so-called reporter proteins that serve to regulate the frequency of transcription initiation at a specific promoter. Polymaraza ae moves along the DNA strand; transcribes the information contained in the DNA coding strand and its 5 'end to the 3' end of the ribonucleic acid information (m-RNA), and this information is immediately translated into a polypeptide sequence having the amino acid sequence for which the DNA is encoded. Each amino acid is encoded by a single nucleotide breaker, or koden, for which the term structural gene is used in the present disclosure to denote that portion of DNA; wherein the encoded amino acid sequence of the expressed peptide product is encoded. After the RNA polymarase binds to the promoter, it first transcribes the nucleotides by encoding the ribosome binding milling, then initiates translation or the start signal (usually the ATG codon, which becomes the AUG in the resulting m-RNA), and then translates the nucleotide ksdon within itself structural gene. At the end of the structural gene, so-called stop codons are transcribed, and the polymerase may be pleased to generate an additional m-RNA sequence which, due to the presence of the stop signal, remains untranslated by riboeomas. The ribosomes bind to the binding site prepared for m-RNA, in bacteria usually after the m-RNA is produced, and produce the encoded polypeptides themselves, beginning with the translation start signal and ending with ehor above the stop signal. The desired product is Jan when the ribosome binding site coding sequences are positioned correctly relative to the AUG initiation codon and all remaining codons follow the initiation codon in the correct phase. The resulting product can be obtained by lysing a host cell and separating it from other bacterial proteins by a suitable purification method.

CS 273 181 B2

The inventors realized that applying recombinant DNA technology (i.e., inserting interferon genes into microbial expression carriers and expressing them under the control of microbial gene regulatory elements) would be the most effective way to provide large amounts of lysucocyte interferon which, although not glycosylated as in the case of a human leukocyte product, it has been used clinically in the treatment of a wide range of viral and neoplastic diseases.

According to the invention, the first leukocyte gene can be prepared by the following sequence of operations:

1) Using homogeneous purified human leukocyte interpharon partial amino acid sequences, sets of synthetic DNA test samples are constructed whose codons in total represent all possible nucleotide combinations capable of encoding said partial amino acid sequences.

2) Bacterial colony flasks containing complementary DNA (cDNA) from induced mRNA are prepared. Other induced radiolabeled mRNAs hybridize to plasmid oDNA from this flask. Hybridizing mRNA is eluted and tested for its ability to transplant to interferon using an oocyte assay. Plasmid ONAs from colonies found to induce interferon activity in this method are further tested for their ability to hybridize to test samples prepared as described above and

1).

3) In parallel to the procedure described above in section 2), oDNA derived from induced mRNA in plasmids is used to create an independent bank of transformed colonies. The test samples of part 1) were used to count 3ynthesis of radiolabeled single stranded cDNA (s) and used as hybridization test samples. Synthetic assay samples hybridize to the induced mRNA template and extend reverse transcription to produce induced, radiolabeled cDNA. Colony bank clones that hybridize to the radiolabeled cDNA obtained as described above can be tested to confirm the presence of the full length interferon coding gene. Any putative partial-length gene fragment obtained in the above sections may be used as a test sample for the full-length interferon gene.

1) and 2).

4) Adjust the full-length interferon gene / obtained by the above procedure, using synthetic DNA; to remove any leader sequence that could prevent microbial expression of the mature polypeptide and to provide suitable placement in an expression-capable carrier relative to the start initiation signals and to the ribosome binding milling of the microbial promoter. The expression of the interferon obtained is purified to a degree allowing confirmation of its character and determination of its activity.

5) The interferon gene fragment prepared by ehor as described above is used as such for testing; via hybridization, * for the preparation of other partially homologous leukocyte species of interferon.

The above-described application of ONA recombinant technology techniques has resulted in microbial production, in high yields and high purity, of a group of homologous laukocyte interferons (non-glycolysed) in the form of mature polypaptides, not accompanied by substantially corresponding sequencing or any portion thereof. These interferons can be directly expressed, isolated and purified to a degree of purity suitable for their use in the treatment of viral and malignant diseases in animals and humans. The microbial expression of the products of this group so far has been shown to be effective in in vitro testing, and in the first tests of this kind also in in vivo testing, including the first mature leukocyte interferon produced in a microbial fashion.

The term mature leukocyte interferon, as used in the description of the present invention, refers to a microbially (e.g. bacterially) produced glycolyl-free intarfarone molecule. The mature leukocyte interferon in the method of the invention without CS 273 181 B2 is expressed directly, starting with the tlanslation initiation signal (ATG), before the first amino acid codon of the natural product. Thus, a mature polypeptide may contain as the first amino acid in its sequence methionine (for which it encodes ATGs without substantially altering its nature. On the other hand, the microbial host may process the translation product so that the initial methionine is omitted. other than the conventional leader protein, said conjugate being specifically digested in an intra- or extracellular environment (see British Patent No. 2,007,676 A.) Finally, the mature interferon can be produced in conjunction with a microbial signal peptide that transports the conjugate to the cellular the walls where the signal peptide is cleaved to exclude the mature polypeptide. The expression of mature leukocyte interferon refers to bacterial or other microbial production of interferon molecules lacking glycosyl groups or amino acid presequences, which are directly involved in mRNA translation of the human leukocyte interferon genome.

The specific leukocyte interferon proteins disclosed herein are defined by the determined DNA gene (see Figures 3 and 8) and the fallen deduced amino acid sequence (see Figures 4 and 9). It is understood that, for these specific interferons, although they can indeed all be included in the group of leucooytic interferon proteins, there are natural allelic variations that occur from case to case. These variations can be demonstrated by a difference (or differences) in the total amino acid sequence, or by omission; subtitling, by insertion; by inverting or adding an amino acid (or amino acids) in said sequence. For each leukocyte interferon protein referred to in the present invention / designated by LelP A to LelF 0, said allelic variations are included within the scope of said labels or of the terms defining these interferons.

The preferred method of the invention is explained in more detail in the following detailed description and in the accompanying figures 1 to 9.

In Figure 1, two amino acid sequences are shown; which are common to all species of interferons isolated from human leukocytes and purified to a homogeneous state (designated T1 to T-15). All possible mRNA sequences encoding these peptides are shown as corresponding DNA sequences. The letters A, T / G, 0 and U refer to nucleotides containing base adenine; thymine, guanine, ¹ cytoein and uracin. Breed N refers to any of said nucleotides A; G, C and U. The polynucleotides are shown as follows; to read from the 5 * (left) to 3 '(right) direction, and where a double-stranded (dv) ONA is shown, it in turn reads from below or from the non-coding strand.

Figure 2 An autoradiogram showing hybridization of potential LelF 32 plaemids with synthetic P-labeled deoxyoligenuclaotides is shown.

Figure 3: Nucleotide sequences (coding strands) of eight gene fragments; which have been isolated As possible candidates for use in axprasion of leukocyte interferons, and which are indicated by the letters A to H. The translation initiation ATG codons and termination triplets of each LelF are underlined. Stop codons or thermirectional triplets are followed by non-translated regions at the 3 * position. The full length incorporated LelF A gene has omitted one codon that was found in the other drawn genes as shown in the third A line of Figure 3. At position 5 *, the untranslated regions precede the leader sequences. Fragment E lacks the full-length leader sequence after isolation, but the entire gan for the putative mature LwIF E is included. Fragment G lacks the entire coding sequence after isolation.

Figure 4 shows a comparison of eight LalF protein sequences determined by nucleotide sequences. For amino acids The one-letter abbreviations used by the IUPAC-IUB Biochemical Nomenclature Committee are used: Aalanine; C »cysteine: D» aspartic acid: E »glutamic acid F phenylalanine; G glycine; H and histidine;

I isoleucine: K lysine: L lauci .; M methionine: N aeparagine: P-proline: Q-glutamine: R-arginine, S-serine, T-threonine V-valine: W = tryptophan: and Y-tyrosine.

CS 273 181 B2

The numbers indicate the location of the amino acids (S ee refers to the signal peptide). A dash at position 44 of the 165 amino acid sequence of interferon LelF A is introduced to align said LelF A sequence with a sequence of 166 amino acids of other LelF A. The LelF E sequence was determined by neglecting the extranucleotide (position 187 in Figure 3) in its coding sequence. areas. Asterisks indicate the termination codons that are in phase. Amino acids common to LelF (with the exception of LelF E pseudogen) are also evident. Underlined residues are amino acids that are also present in human fibroblast interferon.

Figure 5 is a map of the restriction endonucleases of eight types (A to H) of cDNA cloned by LelF interferons. Hybridized plasmids were constructed using the dC: dG end-residue method (Goeddel DV et al., Nature 287 / 411-416 (1980)), whereby cDNA inserts were excised using a rheumetric endonuclease Five. homopolymsrical dCidG terminal residues.

The location of PvuII is also indicated in the figure; EcoRI and BglII cleavage sites. The shaded regions of the figure indicate the coding sequences of mature LelF; shaded regions indicate sequences encoding a signal peptide; the free regions represent the 3 ^r and 5 'non-coding sequences.

Figure 6 schematically depicts the construction of the gene encoding the direct microbial synthesis of mature LelF A. 3a and cleavage sites and residues formed (Pst I / etc.) are indicated. Abbreviation b.p. indicates “a pair of bases.

Figure 7 is a schematic representation of a reetric map of two gene fragments used to express mature leukocyte interferon LsIF B. The codon sequences indicated are end-strand coding sequences obtained by digestion with the restriction enzyme Sau3a in the two cases.

Figures 8 and 9 show the DNA and amino acid sequences (when referring to the corresponding one-letter amino acid abbreviations as described above for Figure 4) of the five LelF proteins / including type I and 3. The asterisk in Figure 9 indicates the termination codon of the adjacent DNA sequence and the delimiter indicates a further or gap in the sequence.

A. Used micro-organisms

The method of the invention comprises the use of two microorganisms: E. coli x 1776 (as described in U.S. Pat. No. 4,190,495) and E. coli K-12 strain 294 (A / thi ', har' / hsmj zakon terminus). as described in British Patent No. 2,055,382 A. Each of these microorganisms is deposited with the American Type Culture Collection (ATCO) under the designation ATCC Nos. 31537 and 31446. All work with recombinant DNA has been carried out in accordance with binding National Health Authority guidelines (NIH) in the United States.

The method of the invention in its most preferred embodiment is described using E. coli microorganisms, including not only strains of E. coli x 1775 and E. coli K-12 strain 294 mentioned above, but also other known strains of E. coli such as E. coli B / or other microbial strains, many of which are stored and available in known collection institutes / such as the American Type Culture Collection; see also German Patent Publication No. 2,644,432. Other microorganisms include, for example, Basilli strains, such as Eacillus subtilis / and other enterobacterial microorganisms, including Salmonella typhimuriura and Serrata marcescens strains which use plasmids and are capable of replication, and expressing heterologous gene sequences contained therein.

As a host organism for the preparation of interferon proteins, the expression of genes encoded therein, under the control of a yeast promoter (preferably yeasts) such as Sacharomyces cereviae.

B. Source and purification of leukocyte interferon raRNA (LsIF mRNA)

LelF mRNA can be obtained from human leukocytes (usually from leukocytes suffering from chronic myeloid leukemia), and these leukocytes can be induced to produce interferon by Sendai or Newcastle disease virus, as described, for example, in U.S. Pat. No. 2,781,118 B2 cim file 2,947,134. a source, also used in this work, is a cell line designated KG-1 obtained from patients with acute mysloid leukemia. This cell line, described by HP Koeffler and DW Gold in Science 200, 1153 (1978), readily grows in culture medium containing RPMI (Rosewell Park Memorial Institute) 1640 plus 10% FCS (fetal calf serum) inactivated by heating, 25 mM buffer HEPES (N-2-hydroxyethyl-piperazine-N ^- 2-ethanesulfonic acid) and 50 µg / ml gentamicin; and subculture to one to three divided twice a week. Cells from the previous culture medium are frozen with 10% OMSO (dimethylsulfoxide). KG-1 cells are deposited with the American Type Culture Collection under the designation ATCC No. CRL 8031.

KG-1 cells are induced to produce leucooytic interferon mRNA by Sendaisko virus or Newcastle disease as described by Rubinstein and coworkers / see Proc. Nati. Acad. Sci. USA 76; 640-644 (1979)]. The es cells are isolated 5 hours after induction and RNA is prepared by the procedure using guanidine thiocyanate with guanidine hydrochloride (see Chirgwin et al., Biochemiatry 18, 5294-5299 (1979)). In the same way, RNA is isolated from uninduced cells. Using oligodeoxythymidine (dT) -cellulose chromatography and ultracentrifugation with eacharose gradient to obtain a 12S poly (A) mRNA fraction (as described by Gresn et al., Arch.). Biochem. Biophys. 172, 74-89, (1976) and Okimura et al., Arch. Biochem. Biophys. 188; 98-104 (1978)]. The mRNA thus obtained has an interferon activity of 8000 to 10,000 units per microgram in an oocyte assay using a Xenopua Laevis frog / see Cavalieri et al., Proc. Nati. Acad. Sci USA 74 / 3287-3291 (1977)].

C. Preparation of colony flasks containing LelF cONA sequences

Using 5 / µg of mRNA with standard procedures, prepare! double-stranded cONA / see Wickens et al., 0. Biol. Chem. 253 / 2483-2495 (1978) and Goeddel et al. Nature 281, 544-548 (1979). The obtained cDNA was ee fractionated by electrophoresis on a 6% polyacrylamide gel (PAGE); and 230 ng of material having a size ranging from 500 to 1300 b.p. is isolated by electroelution. For a portion of this cONA (100 ng), deoxycytidine residues (dC) were finally ligated as described by Chang and co-workers in Nature 275, 617-624 (1978) / product ee analyzed with 470 plasmid pBR322, to which the Pet I restriction mills are bound desoxyguanidine residues (dG) (see Bolivar et al., Gene 2, 95-113 (1977)) and the plasmid thus modified is used to transform E. coli x 1776. About 130 tetracycline-resistant and ampicillin-sensitive transformants are obtained. per ng of cDNA.

In a second similar experiment, approximately 1000 E. coli K-12 strain 294 (tetracycline resistant and ampicillin sensitive) / per ng ng cDNA were obtained, in which case cDNA fractionation by molecular size and electroelution yielded a material ranging from 600 up to 1300 bp · which ee will use to bind the terminal dC.

O. Preparation of synthetic oligonucleotides and their use

Knowledge of the aminokyeeline sequences of various tryptic fragments of human leukocyte tare interferon allows to design synthetic deeoxyoligonucleotides complementary to different regions of LelF mRNA. Two tryptic peptides T1 and T13 were selected; since they have an amino acid sequence requiring only 12 or 4 undecamer eynthesis (to explain all possible coding sequences (see Figure 1)). They synthesize 3Θ4 sets of test deoxyoligonucleotides for each sequence, each containing either three (T-1A, B, C, 10) or One (T-13A / B / C, O) oligonucleotide. Said 11 base complementary deoxyoligonucleotides are synthesized chemically using the phosphfortriester method (see Grea et al., Proc.). Nati. Acad. Sci. USA 75 / 5765-5769 (1978)]. In the T-13 series four individual samples are prepared, in the T-1 series twelve samples in four groups of three samples as shown in Figure 1.

four individual test samples in the T-13 series and twelve T-1 test samples at 7

CS 273 181 B2 prepared in four groups of three primers in each ee will use radiolabeled single stranded cDNA for information synthesis for use in hybridization assays.

Buď either 12S RNA from KG-1 cells induced by Sendai virus (8000 units of IF activity per µg) or total poly (A) mRNA from induced leukocytes (activity less than 10 units per µg) was used as exemplary mRNA. From the aa primers, prepare labeled cDNA

P using known Noyea reaction conditions / visas and co-workers. Why. Natl, Acad. Sci. USA 76, 1770-1774 ¹ (1979) /. Reactions aa are performed in 60 microliter buffers of 20 mM Tris-HCl (pH 8.3), 20 mM potassium chloride, 8 mM anhydrous magnesium chloride, and 30 mM β-mercaptoethanol. For each reaction, one µg of each primary is used (i.e., a total of 12 µg for the primary T1 series and a total of 4- / µg for the T-13 series), 2 µg induced 12S fraction of mRNA (or 10 / u9 non-induced poles). mRNA); 0.5 mM dATP; dCTP, * dTTP, 200 / UCi (c < ³² P) dCTP (Amersham supplier, 2000-3000 Ci / mmol) and 60 reverse transcriptase units (Bethesda Research Laboratories supplier). The desired product is separated from the incorporated marker material by gel filtration on a 10 ml column of Saphadex G-50 and the product ee is heated at 70 ° C for 30 minutes with 0.3 N sodium hydroxide solutions to decompose the RNA; and then neutralized with hydrochloric acid. Hybridisations aa were performed as described by Kafatos and co-workers in Nucleic Acids Ras. 7; 1541-1552 (1979)].

E. Identification of clones pL1 to pL30

The rapid plasmid isolation method described by Birnboimam and co-workers in Niclaic Acids Res is used to prepare 1 / Ug plasmid ONA from each of the 500 individual E. coli K-I2 transformants, ¹ strain 294 (see section C). 7j 1513-1523 (1979). Each DNA sample obtained was denatured and then applied in triplicate to a nitrocalulose filter according to the procedure described by Kafatos and coworkers (see above).

The above three sets of nitrocellulose filters containing 500 plasmid DC samples hybridize

a) and induced cONA containing a T-1 set of primaries;

b) and induced cONA containing T-13 primers, and

(c) uninduced cDNA prepared using both primer sets.

Clones are considered positive when hybridizing more strongly on one or both samples induced by cDNA than on the total non-induced sample. Thirty positive clones (pL1 to pL30) were selected from the initial 500 plasmid samples for further analysis.

F. Identification of clones pL31 to pL39. Isolation of plasmid Number 104 containing the LelF gene fragment

The transformants of E. coli x 1776 ae will be short-listed by the method of colony hybridization according to Grunstein and Hogness / see Proc. Nati. Acad. Sci. USA 72; 3961-3965 (1975) (using ³² P labeled induced mRNA as a test sample), see Lillsnhaug et al. Biochemistry 15; 1858-1965 (1976)]. Unlabeled mRNA from induced cells is mixed with the test sample at a ratio of 200: 1 to compete with the 3 induced mRNA present in the P-labeled preparation. Hybridization of the labeled mRNA occurs preferably with 8 colonies containing induced sequences. Three types of transformants are obtained:

1) 2 to 3% colonies hybridized to ³² P-mRNA very strongly;

2) 10% colonies hybridized significantly less than in group 1); and

3) a residue; that swears a dategatable hybridization signal,

Positive colonies (Groups 1 and 2) are tested for the presence of interferon-specific sequences by assay; which consists in hybridizing intarferon mRNA specifically to plaemid DNA. First, 60 affinity-positive colonies (from group 1) are cultured individually in 100 ml of M9 broth; supplemented with tatracycline (20 µg / ml), diaminopimylic acid (100 µg / ml), thymidine (20 µg / ml) and d-biotinam (1 µg / ml). M9 broth contains sodium hydrogen phosphate (6 g) in 1 liter, dihydrogen phosphate

CS 273 181 B2 potassium (3 g), sodium chloride (0.5 g) and ammonium chloride (1 g); After sterilization in an autoclave, 1 ml of sterile 1M magnesium sulphate solution (bszvodó) and 10 ml of sterile 0.01 M calcium chloride chloride solution are added. Plasma DNA cultures are isolated by ten joints from ten of the six cohesive groups, as described by Clawsllam et al., Biochemistry 9, 4428-4440 (1970). Ten µg of plasmid DNA from each group was digested with HindIII, the mixture danatured and covalently bound to OBM-paper (diazobsnzyloxymethyl filter paper). One µg of purified mRNA from the induced cells hybridized to the digested DNA bound to DBM-paper. The hybridized mRNA is removed by washing, specifically the hybridized mRNA is eluted and assayed with Xenopus laevie oocytes. In this test, all six groups were negative. Of the 59 weakly positive colonies (Group 2), five groups of 10 colonies and one group of 9 colonies were formed, and from these groups plasmids were prepared as described above and evaluated as above. Of the six test groups, one (K 10) hybridized to interferon mRNA and showed levels significantly higher than baseline at each time of testing. To identify a specific interferon cDNA clone, prepare from 9 colonies of the K10 group. plasmid DNA and tested individually. Two of these plasmids (number 101 and number 104) bound Interfsron mRNA significantly above baseline levels. A unique Bgl II rss32 trick fragment containing 260 b.p. was isolated from plasmids number 104, labeled with a radioactive P as described by Taylor and co-workers in Biochim. Biophys. Acta 442, 324-330 (1976) and used as a test sample for independent selection on 400 transformants. coli 294 using the in situ screening method / see Grunstsin and Hogness, Proc. Nati. Sci. USA 72, 3961-3965 (1975)]. Nine colonies (pL31 to pL39) were identified that hybridized to a different degree in this assay.

The above tagged 260 bp fragment. was further used in independent screening for 4000 transformants of E. coli 294 in the same manner. 50 colonies were identified that hybridized with the test sample to different degrees in this assay. The box contained a LalF G fragment, one LsIF H fragment, and one contained a fragment designated as LelF H1, an apparent alanic variant of interferon LelF H. The hybrid plasmids obtained were designated as pLsIF H ', and so on.

G. Isolation and sequence determination of the first LsIF gene and full-length chain

Of all 39 potential LsIF cONA clones, ss prepare plasmid ONAs, which are again subjected to another selection with the same 260 b.p. DNA sample, using the hybridization procedure described by Kafatos and coworkers (see above). Three plasmids (pL 4, pL 31, pL 34) gave very strong hybridization signals in this screening, four (pL 13, pL 30, pL 32, pL 36) hybridized slightly and three (pL 6, pL 8, pL 14) hybridized with the test sample weakly.

The above 39 potential LelF cDNA recombinant plasmids were also subjected to selective testing using synthetic radiolabeled P-labeled (groups of individual T-1 primers or individual 7-13 primers) directly as hybridization assay samples. The hybridization conditions were chosen so that perfect pairing of the base could be achieved for dsgging of the hybridization signals (see Wallacs et al., Nuclsic Acids Rss). 6, 3543-3557 (1979)]. Thus, plasmid DNA was prepared from 39 clones by standard lysate procedure (see Clevs11 et al., Supra), which were purified by Biorad Agarose A-50 agarose column chromatography. Samples (3 µg) of each preparation were linsarized with Eco RI, denatured with alkaline and applied to 3 separate nitrocellulose filters in a batch

1.5 µg per Jdd application (according to Kafatoss and co-workers, see above). Individual synthetic dsoxyoligonucleotide primers and pools of fused primers were phosphorylated with (P) ATP as follows: 50 pmol oligonucleotide and 100 pmol labeled

CS 273 181 82 (β) -ATP (supplied by Nsw England Nuclear, activity 2500 Ci / mmol) was added to 30 microliter buffer consisting of 50 mM Tris-HCl, 10 mM anhydrous magnesium chloride, and 15 mM / mercaptoethanol. Two units of polynucleotide kinase T 4 were added to the mixture and after 30 minutes heating at 37 ° C the resulting P-labeled primers were purified by chromatography on 10 ml Ssphadex®G-50 columns. Hybridizations were then performed using 10 ⁶ cpm primed T-130 or 3 x 10 ⁶ cpm fused primers T-10. Hybridizations were performed at 15 ° C for 14 hours in 6 x SSC (1 x SSC β 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.2) and 10 x Danhardt's solution (0.2% bovine). serumalbumin, 0.2% polyvinylpyrrolidone, 0.2% Ficoll), as described by Wallace and coworkers (cited above). The filters were washed for 5 minutes (3 Cr) at 0 ° C in 6 x SSC, dried and exposed to X-ray sensitive film. The results obtained with P-labeled T-13C fused primers and T-1C primer are shown in Figure 2.

Significant hybridization of plasmid DNA from clone 104 ee was found to be by a group of linked primers T-1C and primer T-13C, but no detectable hybridization with other undecamers occurred. As shown in Figure 2, numerous of the 39 potential LalF plasmids (pL 2, 4; 13, 17, 20, 30, 31, 34) also hybridize to β by both of these test samples. However, restriction endonuclease analysis revealed that only one of these plasmids, plasmid pL31, also contains an internal BglII fragment of 250 b.p. By the PstI scanning enzyme pL31, the size of this cDNA insert was found to be approximately 1000 b.p.

In the latter Pst1 insert of plasmid pL31, the nucleotide sequence was analyzed by the Maxam-Gilbsrt chemical method (see Mathods Enzymol). 55, 499-550 (1980)] and by the dideoxy chain termination procedure (see Smith, Methods Enzymol. 55, 560-580 (1980) / after subcloning the Sau 3a fragments into the M13 vector. The DNA sequence is shown in Figure 3 under the designation A '. The respective translational reading of said DNA can be predicted from the information available about the amino acid sequence of the interferon protein, the known molecular weight range of LsIF and the relative occurrence of stop-triplets in the three possible reading constructs, and the nucleotide sequence identified LalF, including the presequency or signal peptide. The first ATG translation initiation codon was found at a distance of 60 nucleotides from the 5 'end of the sequence, and is followed by 188 codons later by a TGA termination triplet; then there are 342 untranslated nucleotides at the 3 'end, followed by the poles (A) of the sequence. The putative signal psptid (probably involved in the secretion of mature LalF from leukocytes) is 23 amino acids in length. The mature 165 amino acid LalF has a calculated molecular weight of 19390. The inventors have designated LeXF encoded by plasmid pL31 as LalF A. Sequence data (see A in Figure 4) indicate that the tryptic peptides T1 and T13 of interferon LsIF B correspond to amino acids The actual ONA coding sequences found in these two regions correspond to the sequences represented by the linked T1-C primers and the T 13-C primer (see Figure 8).

H. Direct expression of mature leukocyte interferon A (LsIF A)

I. General

The following method of expressing interferon LsIF A directly in the form of a mature interferon polypeptide is a variant of the method used previously for the preparation of human growth hormone (Goeddel et al., Nature 281, 544-548 (1979)) to the extent that it involves combining synthetic (N-tarminal and complementary ONA).

Oak is shown in Figure 6, the restriction endonuclease cleavage site of Sau3a is preferably located between the mRNA codons 1 and 3 of interferon LalF A. Two synthetic deoxyoligonucleotides have been identified that incorporate the ATG translation initiation codon, restore the codon for amino acid 1 (cysteine) and sticky) EcoRI end. These oligomers

CS 273 181 B2 were ligated to a 34 b.p. fragment. between Sau3a-Avall sites of plasmid pL31. The product obtained by 45 b.p. was ligated to other ONA fragments, and a synthetic-natural gan of 865 bp, which encodes LelF A and which is bound by EcoRI and PstI restriction sites, was constructed. This gene was inserted into plasmid pBR 322 between its EcoRI and PstI restriction sites to give plasmid pLalF A1.

2. Construction of a tryptophan control element (containing the E. coli trp promoter and operator, and a trp rlbosome binding site, but lacking the ATG translation initiation sequence)

The plasmid pGMI carries a tryptophenic (trp) haze of E. coli having an ALE1413 deletion (see Miozzari et al., 0. Bactariology 133, 1457-1465 (1978)) and therefore expresses a fusion protein containing the first 6 amino acids of the trp sequence leader and approximately mid-third. the trp E polypeptide (hereinafter referred to as Le '), and also expresses the trp D polypeptide as a whole, under the control of the tryptophan promoter-operator system. The plasmid (20 [mu] g) was digested with the restriction enzyme PvuII, which cleaved the plasmid on five fresh grains. The gene fragments are then combined with 8 EcoRI linkers (consisting of the own complementary oligonucleotide having the sequence pCATGAATTCATG), which contain the EcoRI restriction pattern needed for later cloning into a plasmid containing the EcoRI site. DNA fragments (20 µg) obtained from pGMI were treated with 10 units of ONA ligase T ₄ in the presence of 200 pmol of 5'-phosphorylated synthetic oligonucleotide pCATGAATTCATG and in a 20 microliter T ₄ DNA ligase buffer (20 mM Tria, pH 7.6, 0, 5 mM ATP, 10 mM anhydrous magnesium chloride, 5 mM dithiothreitol) at 4 ° C overnight. The solution was then heated at 70 ° C for 10 minutes to stop the coupling. The linkers were digested by digestion with EcoRI endonuclease, the resulting fragments, now the 8 EcoRI ends, were separated using PAGE electrophoresis (5% ni gel) and the three largest fragments were isolated after pretreating with ethidium bromide and detection in ultraviolet light by cutting the appropriate portion of the gel layer. containing the desired fragment. Each of said gel fragments is placed together with 300 microliters of 0.1 l TBE buffer (TBE buffer contains 10.8 Tris-base, 5.5 g boric acid and 0.09 g ethylenediaminetetraacetic acid disodium salt (NagEDTA) in 1 liter of water ) into a slaking bag and subjected to an electrophoresis at 100 V for one hour in 0.1 x TBE buffer. The aqueous solution was removed from the dialysis bag, extracted with phenol, extracted with chloroform, treated with 0.2 M sodium chloride solution, and ONA was obtained, after precipitation with ethanol, in an aqueous medium. The obtained EcoRI capture gene 8, containing the trp promoter-oparator, is identified by the following method, which consists in inserting the fragments into a tatracycline-sensitive plasmid; which becomes tetracycline resistant upon said incorporation of the fragment.

Plasmid pBRHI / see Rodrigusz et al., Nuclaic Acids Res. 6, 3267-3287 (1979) / expresses ampicillin resistance and contains a gene for tatracycline resistance, but since it is not linked by the 3 promoter) it does not subsequently express said resistance. The plasmid is therefore sensitive to tetrackylin. By introducing a promoter-operator system into an EcoRI plasmid cleavage site, the plasmid becomes resistant to tetracycline.

To this end, the plasmid pBRHI was digested with restriction endonuclease EcoRI. The enzyme was removed by phenol extraction followed by chloroform extraction and, after ethanol precipitation, ee DNA was recovered in aqueous medium. The DNA molecule obtained was ligated with T ₄ DNA ligase, in separate reaction mixtures, as described above, and with each of the three DNA fragments obtained as described above. The resulting DNA contained in the reaction mixture was used to transform appropriate E. coli K-12, strain 294, using standard techniques (see Harshfleld et al.); Why. Nati. Acad. Sci. USA 71, 3455-3459 (1974)]. The bacteria as are then plated on LB (Luria-Bertani) agar plates containing 20 µg / mol ampicillin and 5 µg / ml tetracycline. Colonies of tetracycline-resistant bacteria are selected, plasmid DNA is isolated, and the presence of the desired fragment is confirmed by restriction enzyme analysis. The plasmid obtained was designated as pBRHtrp.

CS 273 181 82

By digesting the hepatitis B viral genome with the EcoRI and BamHI enzymes, the product obtained is incorporated into the EcoRI and BamHI restriction sites of plasmid pGH6 in a conventional manner (see Goeddel et al.; Natura 281; 544 (1979)] to give plasmid pHS32. The resulting plasmid pHS32 was digested with XbaI / the mixture was extracted with phenol, then extracted with chloroform and ethanol precipitated. The digested plasmid ae is then treated with 1 .mu.l of E. coli DNA polymerase I, Klenow fragment (Boehringer-Mannheim) in an environment of 30 .mu.l of polymerase buffer (i.e., 50 mM potassium phosphate, pH 7.4, 7 mM anhydrous magnesium chloride and 1. mM (1-mercaptoethanol), containing 0.1 mM dTTP and 0.1 mM dCTP, for 30 minutes at 0 ° C and then 2 hours at 37 ° C. In this reaction, two of the 4 nucleotides complementary to the 5 projecting ends of the XbaI cleavage site are added:

5 'CTAGA— 5' CTAGA -, -z

T- 3 TCT -

Two nucleotides, dC and dTj, are incorporated to form an end with two 5 'protruding nucleotides. This linear residue of the plasmid pHS32 (isolated after phenol extraction, chloroform extraction and ethanol precipitation in an aqueous medium) was digested with the EcoRI reetric enzyme. The large plasmid fragment ee is separated from the smaller EcoRI-XbaI fragment by PAGE by electrophoresis and isolated by electroelution. This DNA fragment of plasmid pHS32 (0.2 / µg) was ligated, under conditions similar to those described above, to an EcoRI-Taq1 fragment of the tryptophan operon (0/01 y.ug) obtained from pBRHtrp.

In the above procedure, the binding of the fragment from plasmid pHS32 to the EcoRI Taq1 fragment and the Taq1 protruding tail binds to the XbaI remaining protruding tail, although its bases are not completely paired according to the Watson and Crick principles:

A portion of the thus obtained reaction mixture of ligands is transformed into E. coli 294 cells, then the cells are heat treated and inoculated onto LB agar plates containing ampicillin. Salting yields 24 ampicillin resistant colonies which are cultured further on 3 ml of LB broth (Luria-Bertani) and the plasmid isolated, six of these plasmids having Xbal digestion regenerated with E. coli catalysed mismatching and DNA replication:

-TCTAGA -TCTAGA - »- AGCTCT- -AGATCT It has also been found that these plasmids are cleaved by both the restriction enzyme EcoRI and the enzyme HpaI; and that. provide the expected cleavage fragments. Oedan from these plasmids (designated pTrp14) was used to express heterologous polypeptides as described below.

Plasmid pHGH 107 (Goeddel et al., Nature 281/544 (1979)) contains a human growth hormone gene consisting of 23 amino acid codons produced from synthetic DNA fragments, ¹ and 163 amino acid codons derived from complementary DNA generated by reverse transcription information. RNA for human growth hormone. This gene; although he lacks the codons of the human growth hormone pre-sequence; it comprises an ATG codon for translation initiation. The gene was isolated from 10 µg of plasmid pHGH 107 by a procedure comprising first treating with the restriction enzyme EcoRI and then attaching dTTP and dATP to the obtained fragment by treatment with Klenov fragment 6. coli DNA polymerase I; as described above. The resulting plasmid was isolated by phenol extraction, chloroform extraction and ethanol precipitation, and then digested with BamHI.

CS 273 181 B2

The resulting fragment containing the human growth hormone (HGH :) gene was isolated by PAGE electrophoresis and subsequent electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the structural gene for tetracycline resistance, but lacks the tetracycline promoter-operator system; so; when it incorporates this fragment into a plasmid capable of expression in the following procedure, plasmids containing the insert can be determined by restoring tetracycline resistance. Since the EcoR1 termination of the fragment is complemented by the procedure using Klenow polymerase X, said fragment has one blunt and one sticky end, providing it with the correct orientation when it is later incorporated into a plasmid capable of expression.

Next, an expression capable plasmid pTrp 14 is prepared so that it can receive the HGH gene-containing fragment obtained as described above. For this purpose, plasmid pTrp14 was digested with the XbaI enzyme and the resulting sticky ends of the fragment were complemented with the Klenow polymerase X procedure using dATP triphosphates; dTTP; dGTT ⁵ and dCTP. After extraction of the resulting mixture with phenol, extraction with chloroform and precipitation with ethanol, the obtained DNA is treated with BamHI and the resulting large plasmid fragment is isolated by PAGE electrophoresis and electroelution. Said fragment derived from pTrp 14 has one blunt and one sticky end which provides it with the correct orientation when it is combined with the above-described fragment containing the HGH gene.

The fragment containing the HGH gene and the said fragment pTrp 14AXba-BamHI are combined and ligated together under conditions similar to those described above. The complemented bal and EcoRl ends are weighed together with blunt binding ends to restore both Xbal and EcoRl cleavage mills:

the added XbaI end of the EcoRl end of the HGH gene initiation

-TCTAG AATTCTATG—. —TCTAGAATTCTATG— + -> - AGATC TTAAGATAC —AGATCTTAAGATACX EcoRl

This construction also restores the resistance of the gene to tstracycline. The Oellk plasmid pHGH 107 expresses tetracycline resistance from the upstream promoter of the HGH gene (lac promoter), this resulting construction of a plasmid, designated pHGH 207, allows expression of the tetracycline resistance gene under the control of the tryptophan promoter-operator. The ligation product mixture is transformed into E. coli 294 and resistant colonies are selected after cultivation on LB agar plates with soil containing 5 µg / ml tetracycline.

Plasmid pHGH 207 ss was digested with restriction enzyme EcoR1 and PAGE by alactophoresis and as follows. A 300 bp fragment was isolated by electroelution. containing the trp promoter, operator, and tryptophan barrel ribosome binding glutamine / but lacking the ATG translation initiation sequence. This DNA fragment is cloned into the EcoR1 digestion plasmid of plasmid pLelF A. Expression capable plasmids containing the aforementioned modified trp regulon (the E. coli trp operon from which the attenuation sequence is omitted to increase controllable expression levels) can be cultured to a pre-stage determined levels on a broth containing additional tryptophan in an amount sufficient to suppress the promoter-operator system, then tryptophan is removed; the system is derepressed and the desired product is expressed.

This procedure is described in more detail below and shown in Figure 6. Plasmid pL 31 (250 µg) is digested with PstI and a 1000 b.p. insert fragment is isolated by glacial alactrophoresis on a 6% polyacrylamide gel. About 40 µg of the desired inserted fragment is obtained from the gel by electroelution. which is divided into 3 aliquots for further processing by enzymatic digestion:

A approximately 16 µg sample of this fragment was partially digested with 40 units of BglII endonuclease for 45 minutes at 37 ° C and the reaction mixture 8e purified by PAGE electrophoresis on a 6% 6% gel; obtained about 2 .mu.g of the desired fragment _Y 670 bp

b) the second sample (8 ug _s) Pst I fragment of 1000 bp was digested with AvaII and BglII endonucleases; gel chromatography (PAGE) gave a _y .mu.g of the fragment of 150 bp.

c) 16 µg of the 1000 bp insert fragment was treated with Sau3a and Avall; after isolation elektroforeaou Lo ^ IIM polyacrylamide gel is obtained AEI _Y 0.25 ug (10 pmole)) a fragment of 34 bp Foafortrieeterovou method is further eynthetisují two indicated deoxyoligonucleotides, 5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (Fragment 2). Fragment 2 is phosphorylated as follows; 200 microliters (about 40 pmol) of P) -ATP (Amereham supplier; 5000 Ci / mol activity) is dried and resuspended in 30 microliters of a buffer consisting of 60 mM Trie-HCl (pH 8), 10 mM anhydrous magnesium chloride, and 15 mM J3. mercaptoethanol, and containing 100 pmol of said DNA fragment and 2 units of T4 polynucleotide kinase. The mixture was heated at 37 ° C for 15 minutes; 1 µl of a 10 mM ATP solution is then added, the reaction is allowed to proceed for a further 15 minutes at this temperature and then inactivated by heating the mixture at 70 ° C for 15 minutes. To the reaction mixture ee was added 100 pmol 5 ^of -0H fragment 1 and 10 pmol Sau3a -Avall fragment of 34 bp, and the fragments were ligated together in 50 microliters of medium composed of 20 mM Trie-HCl (pH 7.5); 10 mM anhydrous magnesium chloride, 10 mM dithiothreitol, ^r 0.5 mM ATP, in the presence of 10 units of T4 DNA ligase, at 4 ° C for 5 hours. The mixture was resolved by PAGE electrophoresis on a 6% gel and eluted with a 45 bp product by electroelution. About 30 ng (1 pmol) of the latter product by 45 bp was combined with a 0.5 µg (5 pmol) Avall-BglII fragment of 150 bp and _y 1 ug (2 pmoles) of BglII - Pętla fragment 670 bp

The binding was performed by treatment with 20 units of T4 ONA ligase at 20 ° C for 16 hours. The ligase is inactivated by heating at 65 ° C for 10 minutes and digested with EcoRI and PetI to remove gene polymers. After purification by electrophoresis on 6% PAGE, about 20 ng (0.04 pmol) of the 865 bp product was obtained. Half (10 ng) of this product was ligated into plasmid pBR 322 (0, ^c 3 µg) between its EcoRI and Petl cleavage. ground. Upon transformation of this plasmid into E. coli 294, 70 tetracycline-resistant and ampicillin-sensitive transformants were obtained. Plasmid DNA isolated from 18 of these transformants was digested with EcoRI and PstI. 2 of these 18 plasmids contained a 16 EcoRI-PetI fragment of 865 bp length, one µg of one of these fragments, pLelF A1; ee was digested with EcoRI and ligated to the 300 bp (0.1 µg) EcoRI fragment prepared as described above, containing the trp promoter and the E. coli major ribosome binding rib. Transformants containing the trp promoter were identified using a P-trp assay in conjunction with the Grunetein-Hogness screening method for bacterial colonies. The asymmetrically located XbaI cleavage site in the trp fragment allows the determination of recombinants in which the trp promoter is oriented in the direction of the LelF A gene.

I. In vitro and in vivo efficacy of interferon LelF A

To determine interferon activity, extracts are prepared as follows:

Cultures of X ml volume are grown on L medium containing 5 µg / ml tetracycline to an absorbance value at 550 nm (λ _g0 ) of about 1.0; Then, the culture was diluted into 25 ml of M9 culture medium containing 5 _y / ml tetracycline. When A _ggg reaches 1.0; 10 ml samples are taken and centrifuged, and the resulting cells are suspended in 1 ml of a solution containing 15% aacharoβγ, 50 mM Trie-HCl (pH 8.0) and 50 mM EDTA (ethylenediaminetetraacetic acid). One mg of lysozyme is added to the suspension, and after standing for 5 minutes at 0 ° C, the cells are disrupted by ultrasound. The samples are centrifuged for 10 minutes (15,000 rpm) and the supernatant ea is determined for intarfarone activity by comparing the ee standards with interferon LelF using a cytopathic activity inhibition (CPE) assay. In order to determine the number of interferon (IF) molecules per cell, p

CS 273 181 B2

O takes LalF with a specific activity of 4 10 10 Units / mg.

If shown in Table 1, clone pLelF A trp 25; in which the trp promoter is incorporated

Q in the desired orientation, yields high levels of activity in this assay (in a yield of 2.5 x 10 units per liter). 3a follows from Table 2; interferon produced by E. coli K-12 strain 294 / pLeXF A trp 25 behaves as an authentic human. Interferon LeXF 1 is stable to an acidic pH environment and is neutralized by rabbit antibodies of human leukocyte interferons. The interferon has an apparent molecular weight of about 20,000.

The efficacy of interferon requires the presence of in vivo makrofégů deadening and natural (NK) cells, "and it seems that ^one way of its effectiveness in vivo involves the stimulation of these cells. Oe therefore possible that ^one interferon produced by E. coli 294 / pLeXF A 25, while having antiviral activity in cell culture assays, may not be effective in infected animals. In addition, the in vivo antiviral activity of bacterially produced, non-glycosylated interferon LelF A could be different from glycosylated LalF derived from human leukocytes. Therefore, the biological efficacy of bacterially synthesized interferon LelF A (2% purity) was compared with that of human leukocyte-coated human skin (LelF; purity 8%) in lethal encephalomyocarditis (EMC) viral infection of stoat (see Table 3).

Table 1. Interferon activity of E. coli extracts

E, coli K-12 strain 294 transformed with plasmid cell density (number of cells per ml) XF efficiency Units / ml culture number of LalF molecules per cell pLelF A trp 25 3.5 χ 10 ⁸ 36 OOO 9 OOO pLeXF A trp 25 1.8 χ 10 ⁹ 250 OOO 12 OOO

Table 2. Comparison of the efficacy of Ε »coli 294 / pLeIF A 25 extracts with the standard LelF ^x

Extract Interferon efficiency (Units / ml) without modification at pH 2 in the presence of rabbit anti-human lysucocyte antibodies E. coli extract 294 / pLeXF A trp 25 500 500 <10 LelF standard 500 500 <10

E. coli 294 / pLeXF A trp 25 extract with an interferon activity of 250,000 units per ml; described in Table 1; was diluted 500-fold with minimum baseline to a specific activity of 500 units / ml.

The leukocyte interferon standard (from the Wadley Institute), forwarded titered to the NIH leukocyte interferon standard, was also diluted to a final concentration of 500 U / ml. Aliquots of 1 ml were adjusted with hydrochloric acid of pH 2 IN * samples were incubated for 52 hours at 4 ° C, neutralized by addition ^of potassium hydroxide solution and fixed IF activity using the standard CPE inhibition assay. □ 25 microliter aliquots of 500 Units / ml (ni15)

CS 273 181 B2 as untreated) were incubated with 25 microliters e rabbit anti-human interferon leukocytárnlho at 37 ^D C for 60 minutes; samples were centrifuged at 12,000-fold for 5 minutes and the eupernatant tested.

Table 3. Antiviral activity of various LsIF preparations against EMC viral infection of stoat

Treatment interferon Survival Dest serum platelet forming units / ml den 2 den 3 de n 4 Control (bacterial proteins 0/3 ¹⁰ ): r 3x10 ^{3 4 *} 0 0 10 10 ⁴ 1200 5> 3.4x10 ⁴ Bacterial 3/3 0 0 0 LelF A 0 0 0 0 0 0 Standard LelF 3/3 0 0 0 0 0 0 0 0 0

Male ostriches with an average weight of 713 g and who had no antibody to the EMC virus prior to infection were used for testing. The animals were infected intramuscularly with a dose of 100 x LDg _{0 of} EMC virus (found in mice). In the control group, stoats died in 134;

158 and 164 hours after infection. Treatment of animals with 10 ⁶ units at 10 ⁶ units was performed by intravenous route at intervals of -4, +2; 23, 29, 48; 72, 168 and 240 hours, based on the time at which the infection was performed. The bacterial leukocyte interferon used was a fraction from the column chromatography of lysate of E. coli 294 / pLeZF A 25 with a specific activity of 7; 4 x 10 units / mg protein. Oako control bacterial proteins were used from the equivalent fraction of column chromatography of E. coli 294 pBR 322 lysate at twice the total protein concentration. Oako standard leukocyte interferon was used by Senda virus-induced interferon from normal human cells, purified chromatographically to a specific activity of 32 x 10 units per mg protein.

Stoats in the control group showed progressive lethargy, 'loss of balance, flaccidity' and paralysis of hind limbs and watering of eyes beginning about 8 hours before death. Interferon-treated stoats did not show any of these abnormalities; they remained active at all times and did not develop viremia. One stoat from the control group that did not develop viremia within 4 days; died at the latest from untreated animals (164 hours after infection); but showed high virus titers in the heart and brain post mortem.

Interstates treated with interferon did not develop antibodies to the EMC virus as determined at 14 and 21 days after infection. These results show that the antiviral efficacy of LelF preparations in infected animals can only be attributed to interferon, since the contaminating proteins in bacterial preparations and in human leukocyte preparations are quite different. These results further suggest that glycosylation is not required for the in vivo antiviral activity of interferon LelF A.

3. Isolation of additional cONAs for other leukocyte interferons of a fully characterized plasmid containing interferon LelF A cONA is excised by DNA enzyme PstI; insulates by electrophoresis and label! Radioactive ³² P 273 ZískáCS 1Θ1 B2 radiolabeled DNA ee is used as a test sample for the selective sorting of other transformants, E. coli 294 obtained by the same procedure as described in Part C; using the in situ screening method of colonies according to Grunstein and Hognees (see above); Colonies are isolated which hybridize in varying amounts with the test sample. By digesting these colonies and the ten hybridizing colonies described in Part G with PstI, plasmid DNA was isolated and characterized by three different methods. Said Pst1 fragments were first characterized on the basis of samples obtained by digestion with restriction endonucleases, enzymes Bgl II, Pvu II and Eco R1. This analysis made it possible to characterize at least eight different types (LelF A, LelF B, LelF C, LelF D, LelF E LelF F, LelF G and LeiF H) shown in Figure 5, which approximately shows the location of the various restriction sites in the The deden of these fragments, LelF D, is probably identical to the DNA fragment described by Nagata and collaborators in Nature 284, 316-320 (1980).

Second, some of these DNAs were tested using the hybridization selection assays described by Gleveland and co-workers in Cell 20, 95-105 (1980), based on the ability to selectively remove LeiF mRNA from KG-1 cell RNA containing poly-A strands. In this assay, LeiF were A, B; C, and F positive. Third, the latter Pst fragments were inserted into an expression plasmid, said plasmid transformed with E. coli 294, and the fragments were expressed. All of the products of the above-mentioned expector were thought to be pra-interferons were positive in the CPE assay for interferon activity, although only the marginal activity was found in the case of the LelF F fragment. In addition to the above characterization, a sequence was determined for all LelF types described.

K. Direct expression of second mature leukocyte interferon (LelF B)

The sequence of the first fourteen nucleotides of the isolated fragment containing the mature interferon LelF B gene was found to be the same for both types A and B. From the plasmid pLelF A 25 (a fragment bearing the trp promoter-operator, ribosome binding site and LelP A initiation signal) (· Β) gene, and this fragment was combined with the remainder of the B-sequence to form an expressionable plasmid Maps of protruding restriction sites of the Pst fragment of plasmid pL4 (a plasmid containing the interferon LelF B gene terminated by Pst residues shown in Figure 5); pLelF A 25 is shown in Figures 7a and 7b.

In order to obtain a Sau3a to Pst1 fragment containing approximately 950 b.p · from the sequence shown in Figure 7a; several operations need to be performed since one or more of the intervening Sau 3a restriction sites are present; It is:

1. After digestion, the following fragments are isolated:

a) fragment from Sau3a cleavage site to EcoRI site containing 110 b.p .;

b) a fragment from the EcoRI site to the Iba site containing 132 b.p .;

c) a fragment from the Xba site to the Pst site containing more than 700 b.p.

2. Fragments 1a) and 1b) are ligated together and the Sau 3a and Xba end groups are cut off with XBa and BglII to prevent self-polymerization caused by these terminals (relevant Sau 3a site is within the BglII site; BglII is cleaved to Sau 3a cut off the end). The 242 b.p. fragment was isolated.

3. The products obtained by processes 2) and 1c) are weighed together and, in order to prevent spontaneous polymerization, the end groups are cut off with PstI and BglII. The fragment from Sau3a to the PstI site containing about 950 b.p., shown in Figure 7a, is isolated. This fragment contains that portion of the LelF B gene that is not common to LelF A.

4. A fragment containing approximately 300 b.p. was isolated from plasmid pLelF A25. (from HindIII to Sau3a), containing the trp promoter-operator, ribosome binding site, STG start signal, and cysteine codon for LeiF A.

CS 273 181 B2

5. A fragment from PstI to HindIII containing approximately 3600 bp is isolated from plasmid pBR 322; this fragment contains a replicon and encoded tatracycline but not ampicillin resistance.

6. The fragments obtained in steps 3, 4 and 5 ss bind three times to each other and the plasmid obtained is transformed into E. coli K-12 strain 294.

Minimal screening of transformants / Birnboim et al., Nucleic Acids Rss. 7, 1513-1523 (1979)] and plasmid samples were digested with EcoRI endonuclease. By digesting with ss, three fragments were identified which were identified as 1) a fragment containing EcoRI-EcoRI trp promoters 2) an internal EcoRI to EcoRI fragment of plasmid pL4, and 3) a fragment of plasmid pL4 containing an initiation signal for translation of the protein to EcoRI.

Bacterial extracts from the clones prepared as described above showed a typical value of about 10 x 10 Units of interferon activity per liter at ^Α 550 in the CPE * β test.

1. The Oaden representative clone prepared by this procedure was designated E. coli 294 / pLelF B trp 7.

L. Direct expression of other mature leukocyte interferons (LelF C, 'D, ρ; Η,' I and O)

As with LelF A, other full-length gene fragments containing other types of LelF interferons can be engineered and placed in expression carriers designed for their expression. By means of complete sequence determination by conventional methods, it is determined which restriction site lies sufficiently close to the first amino acid codon of the mature intarfaronic type to allow the procedure described in the above part H to be applied to express mature interferon LelF A, i.e. - amino acids lost by elimination of the anthracite by binding of a synthetic DNA fragment. If this method fails, the following procedure can be used which, in brief, consists of cleavage of the fragment containing the short-term sequence just before the start point of the codon for the first amino acid of the mature polypeptide. It proceeds ee the way it does

1. A Fiber ONA converts to a Fiber ONA in the area surrounding this point:

2. The complementary distance of the primer of the single-stranded ONA is hybridized to the nucleotide region generated according to (1), with the 5 * end of the primer lying on the opposite side of the nucleotide adjacent to the intended cleavage site

3. The portion of the second strand removed in step 1), which lies ^3f away from the primer, is reacted with an ONA polymerase in the presence of adenine, thymine, guanine, and cytoain triphosphates of dsoxynucleotides;

4. Remaining length of single stranded DNA; which protrudes above the intended cleavage site is removed by digestion.

A short strand of eynthetic DNA with an ATG translation initiation signal can then be ligated to the 3 * end of the coding strand as it terminates, for example by binding to the engineered mature interferon gan with the sticky ends, and incorporating the ss gene into an expression plasmid. and control the promoter and its associated ribosome binding site.

In a similar manner to that described in Section K above, gene fragments cocucing LelF C and LelF D coctors are arranged for direct bacterial expression. The expression strategy for these additional leukocyte interferons in each case involves the use of a fragment of approximately 300 b.p. (HindIII to Sau3a); containing the trp promoter-operator, the ribosome binding site, the ATG initiation signal, and the cysteine codon of interferon LelF A from plasmid pLelF A 25. This fragment combines gene fragments from other intarfaron genes encoding their respective amino acid sequences outside the initial cysteiCS 273 181 B2 nu '. which is common to all. Each plasmid thus generated was used to transform E. coli K-12 strain 294. The following ligation was used to construct the appropriate ss genes.

Intsrferon LelF C

The following fragments were isolated from the plasmids pLelF C:

a) a fragment from Sau 3a to Sau 96 of 35 b.p .;

b) a fragment from Sau 96 to PstI of more than 900 b.p.

It was isolated from the plasmids pLelF A-25 as described above in section K4).

c) 300 bp HindIII to Sau 3a fragment;

It was isolated from the plasmids pBR 322 as described above in section K5).

d) a fragment of PstI to HindIII of approximately 3600 b.p.

Construction of the gene

1. We bind fragments a) and c), digest the product with Bgl II and Hind III and isolate the fragment by about 335 b.p.

2. Fragment 1) + b) + d) was ligated in triplicate and the resulting plasmid transformed with E. coli.

A typical clone obtained in this manner was designated E. coli K-12 strain 294 / pLeIF C trp 35.

Interferon LelF D

I isolate the following plasmids from pLelF 0 ss. fragments:

a) 35 bp Sau3a to Ava II fragment;

b) a fragment from Ava II to BglII of 150 b.p .;

c) a fragment from BglII to PstI of approximately 700 b.p.

It was isolated from the plasmids pLelF A25

d) a fragment from HindIII to Sau3a of 300 b.p.

It is isolated from the plasmids

e) a fragment from HindIII to PstI of approximately 3600 b.p.

Construction of the gene

1. Weigh the fragments a) + b), ‘the product was excised with BglII and after purification the product was 185 b.p. (1).

2. Bind fragments 1) + d), cut out with HindIII and BglII and purify the product by approximately 500 b.p. (2).

3. Binding of 2) + c) + e) fragments is performed 3 a and the obtained plasmid is transformed with E. coli.

A typical clone prepared in this way was designated E. coli K-12 strain 294 / pLeIF D trp 11.

Interferon LelF F

The LelF F-containing fragment can be constructed for direct expression by preferably using the amino acid sequence of 1 to 13 LelF 8 interferons, which is exactly the same as that of LelF F interferons, from the pHGH 207 plasmids described above. restriction enzymes Pst I and Xba I digestion and subsequent isolation prepare a fragment of about 1050 bp (a) containing the trp promoter and having the desired configuration at its ends. The second fragment (b) is isolated as a larger fragment from a mixture obtained by digesting the plasmids pHKY 10 with PstI and BglII endonucleases. Fragment a) comprises approximately half of the gene encoding ampicillin resistance; fragment b) contains the remainder of this gene and the entire tetracycline resistance gene except the linked promoter. Fragments a) a

CS 273 181 B2

b) is ligated with T4 ligase and the product is digested with enzymes XbaI and BglII to remove dimarisac products. This results in fragment c) containing the trp promoter-operator and genes for tetracycline and ampicillin resistance.

By digesting the plasmids pLelF F with the enzymes Avall and BglII, a fragment of d) having approximately

580 b.p. and containing codons for amino acids 14 to 166 of interferon LelF F.

Digestion of plasmids pLsIF B by XbaI and Avall enzymes yielded a 49 bp fragment which encodes amino acids 1-13 of interferon LelF F.

Performs ss triple ligations of fragments c), d) and e) in the presence of T4 ligase. The cohesive ends of the respective fragments are such; that the ss fragments correctly encircle to form a composite plasmid that carries the tetracycline resistance gene under the control of the trp promoter-operator, together with the gsns for the mature interferon LelF F. The bacteria transformed with this plasmid can therefore be selected based on said resistance in their agar culture. plates containing tatracycline. A typical clone prepared in this way was designated E. coli K-12 strain 294 / pLaIF F trp 1.

Interferon LsIF H

The full length LelF H gene for the expression of mature leukoeytar interferon LelF H can be constructed as follows:

1) Plasmid pLsIF H was subjected to digestion with the enzymes RsaI and Hasil and isolating a 816 bp fragment extending from the signal peptide amino acid 10 to 3 ⁷ non-coding region.

2. The fragment is dsnaturated and subjected to synthesis synthesis using Klenow DNA Polymarase I fragment (see Klenow et al., Proc.). Nati. Acad. Sci. USA 65, 168 (1970)], using the 8-synthetic dsoxyrbobo-oligonucleotide 5 ⁷ -dATG TGT AAT CTG TCT as primer.

3. The resulting product was digested with Sau3a and the 452 bp fragment was isolated. amino acids interferon 1 to 150.

4. Plasmid pLelF H ss digests Sau3a and PstI and isolates the resulting fragment of 500 b.p .; obtaining a gene encoding amino acids from ISO to the end of the coding sequence.

5. The fragments isolated in steps 3) and 4) are ligated together to form a fragment

166 met cya asp stopl

ATG TGT ... _J- .... GAT TGA _..._ Pst I

Sau 3a encoded. 166 amino acids of interferon LelF H.

6. Plasmid pLsIF A trp 2S was digested with the restriction enzyme XbaI, terminated with the digestion ends and DNA polymorph I and digested with PstI endonuclease. The resulting large fragment is isolated and ligated with the product obtained in step 5). An expression-capable plasmid is obtained which, upon transformation into E. coli K-12 strain 294 nsbo into another host bacterium, can express mature interferon LelF H.

Interferon LelF I

The recombinant gen Charon 4A human genome, constructed by Lawn and coworkers (see Cell 15, 1157 (1978)), was screened for leukocyte interferon genes using procedures described by Lawns and coworkers (see above) and Maniatissm and coworkers (see Cell 15, 687 (1978)]. A rioactive LsIF assay, obtained from the interferon LelF A cONA clone, was used to screen approximately 500,000 plaques from which six LsIF genomic clones were selected. After repeated screening and plaque cleaning, it was selected

CS 273 181 B2 one of the listed clans, HLalF 2, for further analysis.

Using the method described above, other test samples can and preferably be used to isolate the other laukocyte intarfarone proteins of the invention.

Construction of the LelF I interferon gene:

1. EcoRI fragment of clone AhLsXF 2 at 2000 b.p. ee clones into plasmid pBR 325 at its restriction site. The resulting plasmid containing LalF I was digested with EcoRI and the ss fragment of 2000 b.p. was isolated. This ts EcoRI fragment of 2000 b.p. The dAATTCTGCAG (EcoRI-PstI converter) was ligated, the resulting product was digested with PstI and the ss fragment of 2000 b.p. containing PstI ends. This fragment was digested with Sau96 and a 1100 bp fragment having one PstI and one Sau96 konac was isolated.

2. Plasmid pLelF C trp 35 was digested with PstI and XbaI and the larger fragment isolated.

3. A small XbaI - PstI fragment from plasmid pLalF C trp 35 was digested with XbaI and Sau96 and the ss XbaI - Sau96 fragment was isolated by 40 b.p.

4. Ligate the fragments isolated in steps 1), 2) and 3) to ligate to generate an expression plasmid pLelF I trp 1.

Interferon LelF 3

To construct a plasmid capable of expressing LelF 3:

Plasmid pLelF 3 comprises a HindIII fragment of 3.8-kilobase human ganoma DNA that comprises the LelF 3 gene sequence. The DdsI-Rsal fragment □ 760 b.p.

2. The plasmid pLalF B trp 7 was digested with HindIII and DdsI and the HindIII -IdeI fragment of 340 b.p. was isolated.

3. Plasmid pBR 322 was digested with PstI, fragments with sticky ends incubated with 8 DNA polymerase I (Klenow fragment) and digested with HindIII. A large fragment of approximately 3600 b.p. is isolated.

4. Ligate the fragments isolated in steps 1), 2) and 3) to ligate to construct an expression plasmid pLelF 3 trp 1.

M. Čištěni

The content of leukocyte interferon in bacterial extracts can be increased by sequential purification:

1. Polyethylsimine precipitation; in which most cellular proteins, including interferon, remain in the supernatant.

2. Ammonium sulphate fractionation in which the interferon falls out of a 55% saturated ammonium sulphate solution.

3. Suspending the ammonium sulfate palates in a buffer composed of 0.06 M potassium phosphate and 10 mM Tris-HCl, pH 7.2, and dialysis against 25 mM Tris-HCl, ¹ pH7.9 (intarferon activity remains in solution).

4. Chromatography of the supernatant obtained above, at pH adjusted to 8.5, on a DEAE cellulose column (linear gradient elution with 0 to 0.2 M sodium chloride solutions in 25 mM Tris-HCl, pH 8; 5).

5. Adsorption on agarose (Cibachrome Blua Agarose) or on hydroxyapatite and elution

1.5 M potassium chloride solution or 0.2 M phosphate solution.

D

6. Molecular screening on Sephadax G-75 column.

CS 273 181 B2

7. Cation exchange chromatography on CM-cellulose in a 25 mM ammonium acetate solution at pH 5.0 eluting with an ammonium acetate gradient (up to 0.2 M ammonium acetate solution).

The above-mentioned purification procedures result in a material having a purity of greater than 95%.

F

The crude material can also be purified by molecular size chromatography, reverse phase high-pressure liquid chromatography (RP-8) or affinity chromatography on immobilized anti-interferon antibodies.

Alternatively, the material obtained in step 4 above can be applied to a monoclonal antibody-containing column prepared as described by Milstein in Scientific American 243/66 (1980) and the interferon eluted with 0.2 M acetic acid,

0.1% Triton solution and 0.15 M sodium chloride solution.

In an alternative preferred embodiment, the leukocyte interferon produced as described above can be purified by the following purification operations:

1. Frozen cell pellets containing expressed leukocyte interferon are broken either manually or in a suitable grinding apparatus. The partially thawed cells are suspended in 4 volumes of Buffer A / containing 0.1 M Tris-base solution adjusted to pH 7/5 to 8.0 10% (w / v) sucrose solution, 0.2 M sodium chloride solution, 5 mM EDTA solution, 0.1 mM PMSF solution, and 10 to 100 mM anhydrous magnesium chloride solution. The suspension is maintained at about 4 ° C.

The slurry is then passed through a homogenizer at about 41.4 MPa and then again at a pressure of less than 6/9 MPa. The effluent from the homogenizer from both passages is collected while cooling with an ice bath.

2. Polyethyleneimine is slowly added to the homogenate to a concentration of about 0.35% and the mixture is allowed to stand for about 30 minutes. The solids were collected by centrifugation or filtration. This step is carried out under constant temperature control or sufficiently fast so that the temperature of the supernatant (or filtrate) is still below 10 ° C. The supernatant (filtrate) ss concentrates the ultrafiltration to approximately 1/10 of the original volume. Small particles or haze in the retained thickened solution can be removed by filtration through a suitable filter, such as a microporous membrane.

3. Apply the clarification solution directly to the monoclonal antibody column at a flow rate of 5 to 8 cm / hr. (For example, 25 to 40 ml / hr on a 2.6 cm diameter column). After application of the solution, the column is washed approximately ten times the column volume of a 25 mM Tris-HCl solution of pH 7.5 to 8/5 / containing 0/5 M sodium chloride solution and a surfactant solution such as 0.2% Triton X-100 solution. or equivalent substances. After washing, the column is rinsed ten times the column volume of a solution containing 0.15 M sodium chloride and a surfactant such as Triton X-100 (0.1% solution) or equivalent. The column was then eluted with a 0.2 M acetic acid solution containing a surfactant such as Triton X-100 (0.1% solution) or equivalent. The protein fraction from the monoclonal antibody column (determined by UV absorbance or other suitable analytical method) was collected and the pH adjusted to about 4.5 with LN sodium hydroxide solution or 1/0 M Tris-base solution.

4. Combine interferon fractions onto a cation exchanger column, as a CM 52 cellulose column (Whatman brand) or equivalent previously equilibrated with a suitable buffer, such as a 50 mM ammonium acetate solution at pH 4.5. After interferon loading, the column is washed with the buffer used to equilibrate until the UV absorbance of the effluent has reached such a level that some of the desired protein is eluted from the column. The column is then eluted with a solution of 25 mM sodium acetate containing 0.12 M sodium chloride or another combination that optimizes the recovery of interferon and provides, after lyophilization, a product having satisfactory properties such as appearance and solubility.

The monoclonal antibodies used in the preferred purification of the invention described above can be prepared by the procedures described by Staehelins et al. In Proc.

CS 273 181 B2

Nati. Acad. Sci. USA 78, 1848-1852 (1981). Monoclonal antibodies prepared from the ascites fluid are purified and covalently bound to Affigel-10 as described below:

Each of the five Balb / c 9Θ female mice inoculates 5-10 x 10 ^S hybridoma cells from the medium growth phase of the strain. The 5 x 10 ⁵ viable cells obtained from the mouse production fluid were inoculated intraperitoneally with another group of ten or wolf mice.

Ascites fluid was collected (2x to 4x) from each mouse of the latter group. Up to three cell transfers can be performed followed by removal of ascites fluid from one group of mice to another. The ascites fluid collected from the mice at each cell transfer is collected.

Cells and their debris are removed from the ascites fluid by centrifugation at low speed (500-10000 times g) for 15 minutes. Centrifugation is then continued at 18,000 rpm for 90 minutes. The supernatant is frozen and stored at -20 ° C.

After thawing, additional fibrin and fine precipitated material are removed by centrifugation at 35,000 rpm for 90 minutes. All lots of ascites fluid from each transform are tested for specific antibody activity by determining the solid phase antibody binding (see Staehelin et al., Supra, cited above) and matching ee fractions.

The protein concentration in the combined solutions is determined. on an approximation basis, 1 mg of protein at 280 nm exhibits an absorbance of 1.2 in a 1.0 cm cuvette. Ascitic fluids with high antibody levels contain 30 to 35 mg protein per ml. This amount is equivalent to 4-7 mg specific antibody / ml. The ascites fluid at this concentration is diluted with PBS solution (0.01 M sodium phosphate solution at pH 7.3 and 0.15 M sodium chloride) to a concentration of 10-12 mg protein per ml.

To each 100 ml of the diluted solution at 90 ° C, 90 ml of saturated ammonium sulfate solution was slowly added at room temperature with vigorous stirring. The suspension is ice-cooled for 40 to 60 minutes and then centrifuged for 15 minutes at 4 ° C at 10,000 rpm. The supernatant is decanted off and the centrifuged substance is dried well. Dissolve the protein pellets in a solution containing 0.1 M Tris-HCl (pH 7.9) and 0.04 M sodium chloride (buffer I). The protein solution is dialysed for 16-18 hours at room temperature against 100 volumes of said buffer I with at least one buffer change. The dialysed solution is centrifuged at 15,000 rpm for 10 minutes to remove insoluble material. By determining the absorption at 280 nm, 30-35% of the original amount of total protein present in the ascites fluid is recovered.

The solution containing 30 to 40 mg protein per ml is then applied to a DEAE-cellulose column equilibrated beforehand with buffer X. A column layer of at least 100 ml is used for each gram of protein loaded. The antibody is eluted from the column by gradient elution with sodium chloride solution containing 0/02 M Tris-HCl, pH 7.9, with a linear gradient from 0.04 M to 0/5 M sodium chloride. The pooled protein fractions eluted with a 0.06-0.1 M sodium chloride solution were concentrated by precipitating the protein with an equal volume of an ammonium sulfate solution saturated at room temperature and centrifuging the product. The obtained protein pellets were dissolved in a solution containing 0.2 M sodium bicarbonate (pH about 8.0) and 0.3 M sodium chloride (buffer XI) and the solution was dialyzed at room temperature against the same buffer, which was changed three times. The dialysed solution is centrifuged at 20,000 times g for 15 minutes to remove a small amount of insoluble material, and the protein concentration is adjusted to 20-25 mg / ml with buffer II. Said antibody solution is used to prepare an immunoadsorbant as follows.

Affigel-10 (available from BioRad Laboratories / Richmond, Calif.) Is washed on an eintral glass filter three times with ice-cooled isopropanol and then three times with ice-cold distilled water. The gel slurry (about 50% ni in ice water) was transferred to the plastic tubes and the adsorbent was sedimented by brief centrifugation; the supernatant is aspirated. To

An equal volume (relative to gel) of the purified antibody solution was added to the gel-filled tube, and the tube was rotated about 4 hours at 4 ° C for about 5 hours to mix the contents. After completion of the reaction, the gel is centrifuged and washed twice with buffer IXI (0.1 M sodium bicarbonate solution and 0.15 M sodium chloride solution) to remove uncoupled antibody. Protein determination in the combined wash solutions showed that more than 90% of the antibody bound to the gel.

To block unreacted sites, the so treated gel is mixed with an equal volume of 0.1 M ethanolamine hydrochloride solution (pH 8) and the tube is again rotated at room temperature for 60 minutes to mix the contents. The gel slurry was washed until the ractants were removed with PBS and stored in PBS in the presence of 0.02% (ra / V) sodium azide at 4 ° C.

N. Parenteral administration of interferons

Interferon LelF can be administered parenterally to persons in need of anti-tumor or antiviral therapy and those who develop immunosuppressive conditions. Vaccination and dose numbers are parallel to those commonly used in clinical trials of human-derived interferons θ, for example about (JL to 10) x 10 units per day, and in the case of materials with a purity greater than 1%, expediently up to this of purity, for example 5 x 10 units per day.

Jeden as one example of a suitable dosage form for a substantially homogeneous bacterial interferon LelF, parenteral formulations can be mentioned: 3 mg of LelF having a specific activity, for example 2 x 10 units / mg, are dissolved in 25 ml of a 5N human serum albumin solution; the ss solution is filtered through a bacteriological filter and the filtered ss solution is aseptically divided into θ

100 ampoules of ampoules each containing 6 x 10 units of pure interferon suitable for parenteral administration. The vials are preferably stored refrigerated (-20 ° C) prior to use.

The compounds of the present invention can be formulated by known methods into pharmaceutically acceptable compositions wherein the active polypeptide is used in admixture with a pharmaceutically acceptable carrier. Suitable formulas and their formulation are described, for example, in Remington's Pharmaceutical Sciences E.V. Martin, to which we refer. The pharmaceutical compositions of the invention comprise an effective amount of the interferon protein together with an appropriate amount of a vehicle to allow the preparation to be effectively administered to the host. The container of the preferred modes of administration is parenteral.

Claims (6)

A method for producing E.coli cells capable of producing mature human leukocyte intsrferon LelF A, B, C, D, F, Η, X or β with an amino acid sequence from positions 1 to 166 as shown in Figures 4 and 9, characterized by with that a) incubating E. coli cells treated with calcium chloride DNA with an expression vector selected from the group consisting of plasmid pLalF A trp 25 DNA, plasmid pLeXF B trp 7 DNA, plasmid pLsIF C trp 35 DNA, plasmid pLeXF D trp 11 DNA, plasmid pLalF F trp 1 DNA, plasmid pLeXF H DNA, plasmid pLalF-trp 1 DNA and plasmid pLelF-trp 1 DNA in ligation buffer for 30 minutes and b) transforming E. coli cells on a solid layer of tetra cyclin-containing agar is selected. 2. The method of item 1 for the production of E. coli cells capable of producing mature human leu cocytic interferon LelF A having an amino acid sequence from position 1 to 166 as shown in Figure 4, characterized in that E. coli cells are incubated with plasmids pLelF. And suffer 25 DNA. CS 273 181 B2 3. The method of item 1 for producing ¹ E. coli cells capable of producing mature human leukocyte interferon LelF B, C, D or F having an amino acid sequence from position 1 to 166 as shown in FIG. 4, characterized by incubating E. coli DNA cells with an expression vector selected from the group consisting of plasmid pLelF B trp 7 DNA, plasmid pLelF C trp 35 DNA, plasmid pLelF D trp 11 DNA, and plasmid pLelF trp 1 DNA. 4. The method of item 1 for producing E. coli cells capable of producing mature human leukocyte interferon LelF H and an amino acid sequence from position 1 to 166 as shown in Figure 4, characterized in that E. coli cells are incubated with plasmid pLelF H. DNA. 5. The method of item 1 for producing E. coli cells capable of producing mature human leukocyte interferon LelF I or 3 with the amino acid sequence as shown in Figure 9, characterized in that E. coli cells are incubated with plasmid pLelF I trp 1 DNA. or plasmid pLelF 3 trp 1 DNA. 6. The method of item 1, wherein the E. coli strain is E. coli K-12 strain 294 (ATCC 31446).