FI82714B - FOERFARANDE FOER FRAMSTAELLNING AV EN TRANSFORMERAD STAM AV E.COLI, SOM FOERMAOR EXPRESSERA ETT HUMANT MOGET LEUKOCYTINTERFERON. - Google Patents

Description

1 82714

A method for producing a transformed E. coli strain capable of expressing mature human leukocyte interferon 5 Partitioned from application 812067

The invention relates to the field of recombinant DNA technology, i.e. to a method for producing a transformed E. coli strain capable of expressing mature human leukocyte interferon, which is human leukocyte interferon (LelF) A, B, C, D, F, H, I or J having an amino acid sequence

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu 15 Met Leu Leu Ala Gin Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn

Gin Phe Gin Lys Ala Glu Thr Ile Pro Vai Leu His Glu Met Ile

Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala

Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin 20 Gin Leu Asn Asp Leu Glu Ala Cys Vai Ile Gin Gly Vai Gly Vai Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu Ala Vai Arg

Lys Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr

Ser Pro Cys Ala Trp Glu Vai Vai Arg Ala Glu Ile Met Arg Ser

Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser Lys Glu, 25

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu

Ile Leu Leu Ala Gin Met Arg Arg Ile Ser Pro Phe Ser Cys Leu

Lys Asp Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Asp

Lys Gin Phe Gin Lys Ala Gin Ala Ile Ser Vai Leu His Glu Met 30 Ile Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr Leu Leu Asp Glu Phe Tyr Ile Glu Leu Asp

Gin Gin Leu Asn Asp Leu Glu Vai Leu Cys Asp Gin Glu Vai Gly

Met Ile Glu Ser Pro Leu Met Tyr Glu Asp Ser Ile Leu Ala Vai

Arg Lys Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys 35 Tyr Ser Ser Cys Ala Trp Glu Vai Vai Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Ile Asn Leu Gin Lys Arg Leu Lys Ser Lys Glu, 2 82714

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Gly Gin Met Gly Arg lie Ser Pro Phe Ser Cys Leu

Lys Asp Arg His Asp Phe Arg lie Pro Gin Glu Glu Phe Asp Gly

Asn Gin Phe Gin Lys Ala Gin Ala Ile Ser Val Leu His Glu Met lie Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala

Ala Trp Glu Gin Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr

Gin Gin Leu Asn Asp Leu Glu Ala Cys Val lie Gin Glu Val Gly

Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser lie Leu Ala Val

Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu lie Glu Arg Lys

Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg

Ser Leu Ser Phe Ser Thr Asn Leu Gin Lys Arg Leu Arg Arg Lys

Asp,

Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu

Met Leu Leu Ala Gin Met Ser Arg lie Ser Pro Ser Ser Cys Leu

Met Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Asp Gly

Asn Gin Phe Gin Lys Ala Pro Ala lie Ser Val Leu His Glu Leu lie Gin Gin lie Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala

Ala Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr

Gin Gin Leu Asn Asp Leu Glu Ala Cys Val Met Gin Glu Glu Arg

Val Gly Glu Thr Pro Leu Met Asn Val Asp Ser lie Leu Ala Val

Lys Lys Tyr Phe Arg Arg lie Thr Leu Tyr Leu Thr Glu Lys Lys

Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg

Ser Leu Ser Leu Ser Thr Asn Leu Gin Glu Arg Leu Arg Arg Lys

Glu,

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Ala Gin Met Gly Arg lie Ser Pro Phe Ser Cys Leu

Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Asp Gly

Asn Gin Phe Gin Lys Ala Gin Ala lie Ser Val Leu His Glu Met lie Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala

Thr Trp Glu Gin Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn

Gin Gin Leu Asn Asp Met Glu Ala Cys Val lie Gin Glu Val Gly

Val Glu Glu Thr Pro Leu Met Asn Val Asp Ser lie Leu Ala Val

Lys Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Thr Glu Lys Lys

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3,82714

Tyr Ser Pro Cys Ala Trp Glu Vai Val Arg Ala Glu lie Met Arg Ser Phe Ser Leu Ser Lys lie Phe Gin Glu Arg Leu Arg Arg Lys Glu,

Cys Asn Leu Ser Gin Thr His Ser Leu Asn Asn Arg Arg Thr Leu Met Leu Met Ala Gin Met Arg Arg lie Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin Ala lie Ser Val Leu His Glu Met Met Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Glu Lys Phe Tyr lie Glu Leu Phe Gin Gin Met Asn Asp Leu Glu Ala Cys Val lie Gin Glu Val Gly Val Glu Glu Thr Pro Leu Met Asn Glu Asp Ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg Ser Phe Ser Phe Ser Thr Asn Leu Gin Lys Arg Leu Arg Arg Lys Asp,

Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Ala Gin Met Gly Arg lie Ser Pro Phe Ser Cys Leu Lys Asp Arg Pro Asp Phe Gly Leu Pro Gin Glu Ghe Phe Asp Gly Asn Gin Phe Gin Lys Thr Gin Ala lie Ser Val Leu His Glu Met lie Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asn Leu Glu Ala Cys Val lie Gin Glu Val Gly Met Glu Glu Thr Pro Leu Met Asn Glu Asp Ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gin Lys lie Leu Arg Arg Lys Asp 4 82714 or equivalent

Cys Asp Leu Pro Gin Thr His Ser Leu Arg Asn Arg Arg Ala Leu

Ile Leu Leu Ala Gin Met Gly Arg Ile Ser Pro Phe Ser Cys Leu

Lys Asp Arg His Glu Phe Arg Phe Pro Glu Glu Glu Phe Asp Gly 5 His Gin Phe Gin Lys Thr Gin Ala Ile Ser Vai Leu His Glu Met

Ile Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala

Ala Trp Glu Gin Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr

Gin Gin Leu Asn Asp Leu Glu Ala Cys Vai Ile Gin Glu Vai Gly

Vai Lys Glu Thr Pro Leu Met Asn Glu Asp Phe Ile Leu Ala Vai 10 Arg Lys Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Met Glu Lys Lys

Tyr Ser Pro Cys Ala Trp Glu Vai Vai Arg Ala Glu Ile Met Arg

Ser Phe Ser Phe Ser Thr Asn Leu Lys Lys Gly Leu Arg Arg Lys

Asp, The method according to the invention is characterized in that the E. coli strain is transformed with an E. coli plasmid containing DNA encoding said mature human leukocyte interferon and which DNA sequence is operably linked to the promoter operator region by well known methods. and growing the transformed E. coli strain in a culture medium under conditions suitable for its survival.

Human leukocyte interferon (LelF) was first invented and prepared in the form of very crude fines by Isaacs and Lindemann [Proc. R. Soc. B 147, ss. 258-267 (1957); U.S. Patent 3,699,222]. Attempts to purify and characterize this substance have been ongoing since then and have led to the production of relatively homogeneous leukocyte interferons derived from leukocytes from 30 normal or leukemia donors (DE Application No. 2,947,134). These interferons belong to a group of proteins known to have the ability to confer antiviral status on target cells.

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In addition, interferon may act to inhibit cell proliferation and alter the immune response. These properties have accelerated the clinical use of leukocyte interferon as a therapeutic agent in the treatment of viral infections and malignancies.

White blood cell interferons have been purified to be substantially homogeneous [Rubinstein et al., Proc. Natl. Acad. Sci. USA 76, p. 640-644 (1979); and Zoon et al., ibid., 76, p. 5601-5605 (1979)], and reported molecular weights of 10 range from about 17,500 to about 21,000. The specific activity of these preparations is remarkably high, 2 x 10® to 1 x 109 units / mg protein, but yields from cell cultures have been depressing. chop. Nevertheless, advances in protein sequencing methods have made it possible to determine partial amino acid sequences [Zoon et al., Science 207, p. 527 (1980); and Levy et al., Proc. Natl. Acad. Sci. USA 77, ss. 5102-5104 (1980)]. The interpretation of glycosylation of different leukocyte interferons is not yet complete, but it is now clear 20 that differences in glycosylation among group members alone are not responsible for the range of molecular weights observed. In contrast, leukocyte interferons differ significantly in amino acid composition and sequence, and in some cases the amino acid homology is less than 80%.

While isolation from donor leukocytes has provided sufficient material for the partial characterization of homogeneous leukocyte interferon and limited clinical trials, it is a completely inadequate source for the amounts of interferon required for large-scale clinical trials and large-scale 6 82714 prophylactic and / or therapeutic use. after. Current clinical trials using human leukocyte-derived interferons in anti-tumor and antiviral trials have been mainly limited to crude (less than 1% pure) formulations, and long delays in producing sufficient quantities even at unrealistic price levels have decisively delayed studies.

However, with the advent of recombinant DNA technology, microbial production of a vast array of useful polypeptides has become possible. Bacteria already modified by this technology have already been made available to enable the production of polypeptide products such as somatostatin, human insulin A and B chains, and human growth hormone [Itakura et al. Science 198, p. 1056-1063 (1977); and Goeddel et al., Nature 281, p. 544-548 (1979)]. Recently, recombinant DNA methods have been used to induce bacterial-20 production of proinsulin and thymosin α-Ι, and several researchers have reported the availability of DNA encoding human leukocyte interferon as well as the resulting proteins with leukocyte interferon activity [ Nagata et al., Nature 284, ss. 316-320 (1980); Mantei et al., Gene 10, p. 1-10 (1980) and Taniguchi et al., Nature 285, p. 547-549 (1980)].

The workhorse of recombinant DNA technology is a plasmid, a non-chromosomal ring of double-stranded DNA found in bacteria and other microbes, often in multiple copies per cell. The information encoded in the plasmid DNA includes the information needed to replicate the plasmid in daughter cells (i.e., "replicon-niH"), and usually one or more selection markers, such as, in the case of bacteria, resistance to antibiotics that allow the host cell to contain identification of the plasmid of interest, clone identification and culture on privileged selective media.

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The advantage of 7,82714 mides is that they can be specifically cleaved by one or more restriction endonucleases, or "restriction enzymes", each of which recognizes a different site on plasmid DNA. The heterologous genes or gene fragments can then be ligated into the plasmid at the ends at the cleavage site or at the reconstructed ends adjacent to the cleavage site; DNA recombination is performed extracellularly, but the resulting "recombinant" plasmid can be introduced into the cell by a process known as transformation, and a recombinant plasmid containing large amounts of heterologous genes is obtained by culturing the transformant. relative to the portions of the plasmid that control the transcription and translation of the encoded DNA message, the resulting expression vector can actually be used to produce the polypeptide sequence encoded by the linked gene, a process termed expression.

Expression is initiated in a region known as a promoter that is recognized and bound by RNA polymerase.

In some cases, such as in the tryptophan or "trp" promoter, the "operator" regions overlap the promoter regions to form a combined promoter operator. Operators are DNA sequences that are recognized by so-called repressor proteins, which are responsible for regulating the transcription initiation sequence in a particular promoter. The polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5 'to 3' end into messenger RNA, which in turn is translated into a polypeptide having the amino acid sequence encoded by the DNA. Each amino acid is encoded by a nucleotide triad, or "codon", which is the so-called within the "structural gene" (referred to herein), i.e., the portion that encodes the amino acid sequence of the Expressed Product. After binding to the promoter, the RNA polymerase first transcribes the nucleotides 35 encoding the ribosome binding site, then the translation initiation or "start" signal (usually ATG, which results in AUG in the resulting messenger RNA), then the nucleotide codons themselves. rakennegeenlssä. The so-called stop codon is transcribed at the end of the structural gene, after which the polymerase forms an additional sequence of messenger RNA, which, due to the presence of the stop signal, is not translated from the ribosomes. Ribosomes bind to the messenger RNA at the ordered binding site, usually in bacteria, when mRNA is formed, and form a self-encoded polypeptide that begins at the translation start signal list and ends at the aforementioned stop signal. The desired product is generated if the sequences encoding the ribosome binding site are correctly positioned relative to the AUG start codon and if all other codons follow the start codon in sequence. The resulting product is obtained by digesting the host cell and recovering the product appropriately by purification from other bacterial protein.

It has been found that the use of recombinant DNA technology (i.e., coupling of interferon genes to microbial expression vectors and their expression under the control of regulatory elements of the microbial gene) would be the most efficient way to produce large amounts of leukocyte interferon, despite the lack of a human-derived substance. glycosylation property, could be used clinically in the treatment of viral and tumor diseases over a wide range.

An attempt to obtain a first leukocyte interferon gene in accordance with this invention comprises the following steps: 1) Partially amino acid sequences of homogeneously purified human leukocyte interferon were used to construct 30 sets of DNA probes whose codons in the aggregate represented all possible nucleotide combinations capable of encoding these partial amino acids.

2) Bacterial colony banks containing complementary DNA (cDNA) from induced messenger RNA were prepared. Other induced mRNAs that were

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9,82714 radiolabeled, hybridized to plasmid cDNA from this library. The hybridized mRNA was eluted and translation into interferon was tested in an early egg cell assay. Plasmid DNA from the colony, which was shown to cause interferon activity in this way, has been further tested for hybridization to probes made as described in a) above.

3) In parallel with the attempt shown in 2), the induced mRNA-derived cDNA in plasmids 10 was used to form a library of transformant colonies itself. The probes of 1) were used to initiate the synthesis of radiolabeled single-stranded cDNA for use as hybridization probes. The synthetic probes hybridized as an induced mRNA template and were extended by reverse transcription to form the induced radiolabeled cDNA. Clones from the colony bank that hybridized to the radiolabeled cDNA obtained in this way have been further examined to confirm the presence of the full-length gene encoding interferon. Any of the putative gene fragments of partial length obtained in 1) or 2) can be used as a probe for a full-length gene per se.

4) The full-length gene obtained above was rounded ;;; using synthetic DNA to remove any leader sequence that could inhibit microbial expression of the mature polypeptide, and to allow appropriate placement in the expression vector with respect to the ribosome binding site of the microbial promoter initiation signals. The expressed interferon was purified to a point that allowed its nature to be confirmed and its activity to be determined.

5) The interferon-ni gene fragment prepared as described above was used per se in the assay by hybridizing other highly homologous leukocyte interferon species.

10 82714 Using the methods of recombinant DNA technology outlined above, microbial production of a group of homologous leukocyte terferons (non-glycosylated) was achieved in high yield and purity as mature poly-5 peptides, essentially without the corresponding presequence or any portion thereof. These interferons can be directly expressed, recovered, and purified to levels that make them suitable for use in the treatment of viral and malignant diseases in animals and humans. The members of the group expressed so far have proved to be effective in in vitro experiments, and in the first such chip demonstration also in in vivo experiments, in the latter the first mature leukocyte terferon had to be microbially produced.

The term "mature leukocyte interferon" as used in this application defines a microbially (e.g., bacterially) produced interferon molecule lacking glycosyl groups. The mature leukocyte interferon prepared in accordance with this invention is expressed immediately from the translation initiation signal (ATG) just before the first amino acid codon of the natural product. The "mature" polypeptide thus contains methionine (encoded by ATG) as its first amino acid in its sequence without a substantial change in nature. On the other hand, the microbial host may treat the translation product to omit the initial methionine. Mature leukocyte interferon could be co-expressed with a conjugated protein, other than a conventional leader, whereby the conjugate is specifically cleavable in the intracellular or extracellular environment 30 (see GB Patent 2,0076,676 A). Finally, mature interferon could be prepared in conjunction with a microbial "signal" peptide that transports the conjugate to the cell wall where the signal is processed and the mature polypeptide is secreted. "Expression" of mature leukocyte interferon mer-35 kills an interferon molecule that does not contain glycosyl groups or a presequence that immediately follows human I! 11,82714 leukocyte interferon genome mRNA translation, bacterial or other microbial production.

Certain leukocyte interferon proteins disclosed herein have been determined by the DNA gene analyzed (Figures 3 5 and 8) and by deductive amino acid sequencing (Figures 4 and 9). It is to be understood that for these particular interferons, and for all of the group of leukocyte-terferon proteins belonging to it, there are natural countergene changes and they occur from individual to individual. These changes may be indicated by amino acid difference (s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of any amino acid (s). in the sequence.

From the drawings, Figure 1 shows 2 amino acid sequences common to all interferon species isolated from human leukocyte and purified to homogeneity and labeled with T-1 and T-13. All potential mRNA sequences encoding these peptides are shown as well as the corresponding DNA sequences. Kirjai-20 met A, T, G, C and U denote, respectively, nucleotides containing bases: adenine, thyrine, guanine, cytosine and uracil. The letter N denotes one of nucleotides A, G, C, and U. Polynucleotides are shown to be readable from 5 '(left) to 3' (right-25 le), and when describing double-stranded ("ds") DNA, vice versa. for the base, i.e. the non-coding thread.

Figure 2 is an autoradiogram showing the hybridization of potential LelF plasmids with 32β-labeled synthetic deoxyoligonucleotides.

Figure 3 shows the nucleotide sequences (coding strands) of 8 fragments isolated as candidates for use in the expression of leukocyte interferons and labeled "A" to "H", respectively. The ATG translation start codon and termination triad for each LeIF 35 are underlined. Stop codons, or termination triplets, are followed by 3 'untranslated regions. Of the included i2 8271 4 full-length gene, LelF A lacks one codon found in the others described, as shown in the third A row of Figure 3. The 5 ′ untranslated regions precede the leader sequences. Isolated, fragment E lacks the full presequence of 5 directors, but contains the entire gene, presumably for mature LelF E. Fragment G, as isolated, lacks the full coding sequence.

Figure 4 is a comparison of these 8 LelF protein sequences, 10 of which were predicted from nucleotide sequences. One-letter abbreviations used by the Nomenclature Commission IUPAC-IUB Commission on Biochemical Nomenclature are used: A »alanine; C = cysteine; D = aspartic acid; E glutamic acid; F = phenylalanine; G glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M - methionine; N = asparagine; P = proline; Q * glutamine; R is arginine; S = serine; T = freonine; V * intermediate; W tryptophan; and Y - tyrosine. Numbers refer to amino acid positions; S means signal peptide. Line 20 in the LelF A sequence of position 20 165 is shown at position 44

To align the LelF A sequence directly with the 166 amino acid sequences of other LelFs. Your LelF E sequence was determined by disregarding the extra nucleotide (position 187 in Figure 3) in its coding region. Stars 25 indicate in-phase stop codons. Amino acids common to all LelFs (with the exception of pseudogenic LelF E) are also shown. The underlined residues are amino acids that are also present in human fibroplast interferon.

Figure 5 shows restriction endonuclease maps of these 8 types (A to H) of LelF cloned cDNAs. Hybrid plasmids were constructed by the dC: dG tailing method [Goeddel et al., Nature 287, p. 411-416 (1980)]. Thus, cDNA insertion sequences can be cleaved using PstI. At the end of each cDNA insertion sequence, representative

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i3 827Ί4 homopolymeric dC: dC tails. The positions of the cleavage sites for PvuII, EcoRI, and BglII are indicated.

The shaded areas in the figure represent the coding sequences of mature LelFs; the cross-hatched regions represent the coding sequences of the 5 signal peptides, and the open regions indicate the 3 'and 5' non-coding sequences.

Figure 6 schematically shows the structure of a gene encoding direct microbial synthesis of mature LelF A. Pil contact points and residues are as shown ("Pstl", 10, etc.) The term Hb.p.n means base pair.

Figure 7 (not to scale) schematically shows a digest map of 2 gene fragments used to express mature leukocyte interferon LelF B. The codon sequences indicated are coding strand terminations resulting from extraction with the restriction enzyme Sau3a in the case shown in these 2.

Figures 8 and 9 show the DNA and amino acid (see Figure 4 above, corresponding 1 letter abbreviations) sequences for 5 LelF proteins, including types I and J. In Fig. 20, an asterisk indicates a hyphen omission or gap in the DNA sequence corresponding to the stop codon.

A. Microorganisms used

Two microorganisms were used in the described work: E. coli x 1776, as described in U.S. Patent No. 4,190,495, and E. coli strain K-12 294 (terminal A, thi ", hsr *. Hsm ^), as described in GB Patent 2,066,382 A. Each is deposited in the American Type Culture Collection (ATCC Accession Nos. 31537 and 31446, respectively) All recombinant DNA work was performed according to guidelines suitable for use by the National Institutes of Health *.

The invention is described in the form of its most preferred embodiments using E. coli comprising not only E. coli x 1776 strains and E. coli K-12 strain 35,294 as defined above, but also other known E. coli strains such as E. coli. coli B, or other microbial strains, many of which have been deposited and are available from known depositories of microorganisms, such as the American Type Culture Collection (see also DE Application Publication No. 2,644,432).

5 B. Source and purification of LeIF mRNA

LelF mRNA is obtained from human leukocytes, usually leukocytes from patients with chronic nuclear congenital leukemia, who have been induced to produce interferon by the Sendai or Newcastle disease virus, as described, e.g., in DE-A-2 947 134. A particularly preferred the source and source used in the present work is a KG-1-labeled cell line derived from a patient with acute nuclear congenital leukemia. This cell line, described by HP Koeffler 15 and DW Golde [Science 200, p. 1153 (1978)], grows readily in culture medium containing RPMI (Rosewell Park Memorial Institute) 1640 and 10% FCS (fetal calf serum) inactivated. by heating, 25 mM HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 50 pg / ml gentamicin, and digested into 1 to 3 subcultures twice a week. Cells can be frozen from the above medium and 10% dimethyl sulfoxide. KG-1 is deposited in the American Type Culture Collection (ATCC Accession No. CRL 8031).

KG-1 cells were induced to produce leukocyte interferon mRNA by Sendai or Newcastle disease virus following the method described by Rubinstein et al. [Proc. Natl. Acad. Sci. USA 76, p. 640-644 (1979)]. Cells were harvested 5 hours after induction and RNA was prepared by the guanidine thiocyanate-guanidine hydrochloride method [Chirgwin et al., Biochemistry 18, p. 5294-59999 (1979)]. RNA was isolated from uninduced cells in a similar manner. Oligodeoxythymidine (dT) -cellulose chromatography and sucrose gradient-35 ti ultracentrifugation were used to obtain the 12S fraction of poly (A) mRNA, as described by Green et al. [Arch. Biochem. Biophys. 172,

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is 82714 ss. 74-89 (1976)] and Okuyoma et al. [Arch. Biochem. Bio-phys. 188, ss. 98-104 (1978)] have described. The interferon titer of this mRNA was 8,000 to 10,000 units / microgram in the Xenopus laevis early ovum assay 5 [Cavalieri et al., Proc. Natl. Acad. Sci. USA 74, ss. 3287-3291 (1977)].

C. Preparation of a colony bank containing LelF cDNA sequences 5 pg of mRNA was used to prepare double-stranded cDNA by standard methods [Wickens et al., J. Biol. Chem. 253, ss. 2483-2495 (1978), and Goeddel et al., Nature 281, p. 544-548 (1979)]. The cDNA was size fractionated by electrophoresis on 6% polyacrylamide gel, and 230 ng of material ranging in size from 500 to 1,500 base pairs (b.p.) was recovered by electroelution. For a 100 ng aliquot of this cDNA, tails were generated with deoxycytidine (dC) residues as described by Chang et al. [Nature 275, p. 617-624 (1978)], heat-treated plasmid pBR322 with deoxyguanosine (dG) residues at the PstI site [Bolivar et al., Gene 2, pp. 617-624]. 95-113 (1977)], and was used to transform E. coli x 1776. Approximately 130 tetracycline-resistant ampicillin-sensitive transformants per 25 ng of cDNA were obtained.

In another similar experiment, approximately 1,000 tetracycline-resistant ampicillin-sensitive E. coli K-12 strain 294 transformants per ng of cDNA were obtained. In this case, the size-fractionated cDNA material, which ranged in size from 600 to 1,300 base pairs, was recovered by electroelution for cD tail formation.

D. Preparation and use of synthetic oligonucleotides

Knowledge of the amino acid sequences of several tryptic 35 fragments of human leukocyte interferon made it possible to construct synthetic deoxyoliginucleotides complementary to different regions of LeIF mRNA. 2 tryptic peptides, T1 and T13, were selected because their amino acid sequences required only the synthesis of 12 and 4 undecamers, respectively, to correspond to all possible 5 coding sequences (Figure 1). 4 sets of deoxyoligonucleotide probes were synthesized for each sequence containing either 3 (T-1A, B, C, D) or one T-13A, B, C, D) oligonucleotide each. The indicated complementary 11-base deoxyoligonucleotides were chemically synthesized by the phosphotriester method [Crea et al., Proc. Natl. Acad. Sci. USA 75, ss. 5765-5769 (1978)]. 4 individual probes were prepared in the T-13 series. Twelve T-1 probes were prepared in 4 batches of three probes, as shown in Figure 1.

15 T-13 series of 4 single probes and 12 T-1 probes prepared in 4 batches of three primers were each used to initiate the synthesis of radiolabeled single-stranded cDNA for use as hybridization probes. The model mRNA was either 12S RNA from Sendai'11a-induced KG-20 1 cells (8,000 units IF activity / pg) or total poly- (A) mRNA from uninduced leukocytes (<10 units / pg). 32 P-labeled cDNA was prepared from these primers using known reaction conditions [Noyes et al., Proc. Natl. Acad. Sci. USA 76, p. 1770 - 1774,

25 (1979)]. 60 μl reactions were performed at 20 mM

In Tris-HCl (pH 8.3), 20 mM KCl, 8 mM MgCl 2 and 20 mM 6-mercaptoethanol. Reactions contained 1 pg of each primer (i.e., 20 pg of total T1 sets and 4 pg of total T-13 sets), 2 pg of “induced” 30S fraction 30 mRNA (or 10 pg of uninduced poly (A) mRNA), 0.5 mM dATP, dCTP, dTTP, 200 pCi (o32 p) dCTP (Amersham, 2-3,000 Ci / mmol) and 60 units of reverse transcriptase (Bethesda Research Laboratories). The product was separated from the unbound label by gel filtration on a 10 ml 35 SephadeaP * 'G-50 column, treated with 0.3 N NaOH for 30 min at 70 ° C to destroy RNA and neutralized with 178271 4 HCl. Hybridizations were performed as described by Kafatos et al. [Nucleic Acids Res. 7, ss. 1541-1552 (1979)].

E. Identification of clones pL1 to pL30 The method of Birnboim et al. [Nucleic Acids 7, p.

1513-1523 (1973)] a rapid plasmid isolation method for preparing 1 pg of plasmid DNA from each of the 500 individual E. coli K-12 strain 294 transformants (see C). Each DNA sample was denatured and applied to nitro-10 cellulose filters in triplicate following the method of Kafatos et al. (See above).

These 3 sets of nitrocellulose filters containing 500 plasmid samples were hybridized with: a) induced cDNA initiated with the T1 series of primers, b) T-13-initiated induced cDNA, and c) uninduced cDNA. which was prepared using both sets of primers.

Clones were considered positive if they hybridized more strongly to one or both of the induced cDNA probes than to the whole uninduced probe. Of the 500, 30 "positive" clones (pL1-PL30) were selected for further analysis.

11! F. Identification of clones pL31 to pL39; Isolation of plasmid 25 (No. 104) containing the LelF gene fragment E. coli x 1776 transformants were screened by the colony hybridization method of Grunstein and Hogness [Proc. Natl. Acad. Sci. USA 72, ss. 3961-3965 (1975)] 30 using 32 P-labeled induced mRNA as a probe [Lillenhaug et al., Biochemistry 15, p. 1858-1865 (1976)]. Unlabeled mRNA from uninduced cells was mixed with the probe in a ratio of 200: 1 to compete with the uninduced mRNA present in the 32 P-labeled preparation. Hybridization of the labeled mRNA should preferably occur in colonies containing ie 8271 4 induced sequences. 3 classes of transformants were obtained: 1) 2-3% of colonies hybridized to 32β mRNA very strongly; 5 2) 10% hybridized significantly less than class 1), and 3) the remainder gave no detectable hybridization mark.

Positive colonies [classes 1) and 2)] were examined for the presence of interferon-specific sequences in an assay that depends on the hybridization of interferon mRNA specifically to plasmid DNA.

Initially, 60 strongly positive colonies [class 1)] were cultured individually in 100 ml of M9 medium supplemented with tetracycline (20 pg / ml), diaminopimeic acid (100 pg / ml), thymidine (20 pg / ml) and with d-biotin (1 pg / ml). M9 medium contains per liter: 6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl and 1 g NH4Cl. After autoclaving, 1 ml of sterile 1 M MgSO 4 and 10 ml of sterile 0.01 M CaCl 2 were added. 10 cultures were pooled and plasmid DNA was isolated from these 6 pooled batches as described by Clewell et al. [Biochemistry 9, p. 4428-4440 (1970)]. 10 pg of each plasmid DNA batch was digested with HindIII, denatured and covalently bound to DBM (diazobenzyloxymethyl) paper.

1 pg of purified mRNA from induced cells was hybridized to each filter. Unhybridized mRNA was removed by washing. The specifically hybridized mRNA was eluted and translated into Xenopus laevis early oocytes. By this analysis, all 6 batches were negative.

5 batches of 10 colonies each were made from 59 weak colonies [class 2)] and plasmids were prepared from these batches and examined as above.

Of the 6 batches tested, one (K10) hybridized to 35 interferon mRNAs at amounts significantly above background values each time it was tested.

i9 8271 4

To identify a specific interferon cDNA clone, plasmid DNAs were prepared from batch K10 9 colonies and examined individually. Two of the 9 plasmids (Nos. 101 and 104) bind interferon mRNA well above background values. A unique BglII cleavage fragment containing 260 base pairs was isolated from plasmid # 104, labeled with 32p using Taylor et al. [Biochem. Bio-phys. Acta 442, ss. 324-330 (1976)] and was used as a probe to independently screen a transformant of 400 E. coli K-12 strain 294 10 by the in situ colony screening method [Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72, ss. 3961-3965 (1975)]. Nine colonies (pL31 to pL39) were identified that hybridized to different amounts with this probe.

In addition, the labeled 260 bp fragment was used to independently screen 294 transformants of 4,000 E. coli K-12 strain in the same manner. Fifty colonies were identified that hybridized to different amounts with this probe. One contained the LelF G fragment, one contained the LelF H fragment, and one contained the fragment labeled LelF H1 (the apparent countergene of LelF H). The resulting hybrid plasmids are designated "pLelF H", etc.

G. Isolation and Sequencing of the First Full-Length LelF Gene 25 plasmid DNAs were prepared from all 39 potential LelF cDNA clones and rescreened with the same 260 bp DNA probe using the hybridization method of Kafatos et al. ). 3 plasmids (pL4, pL31 and pL34) gave a very strong hybridization signal, 4 (pL13, pL30, pL32 and pL36) moderately hybridized and 3 (pL6, pL8 and pL14) hybridized poorly with this probe.

These 39 potential LelF cDNA recombinant plasmids were also screened using 32 P-labeled synthetic 35 undecamers (single T-1 primer lots or single T-13 primers) directly as hybridization probes. The hybridization conditions were chosen to require proper base pairing for detectable hybridization signals [Wallace et al., Nucleic Acids Res. 6, ss. 3543-3557 (1979)]. Thus, plasmid DNA from 39 clones was prepared by a standard method using clarified lysate (Clewell et al., Supra) and purified by Biorad Agarose A-50 column chromatography. Samples of each preparation (3 pg) were linearized by treatment with EcoRI, denatured in alkali, and spotted on two separate nitrocellulose filters, 1.5 pg / spot (Kafatos et al., Supra). Individual synthetic deoxyoligonucleotide primers and pooled primers were phosphorylated (32p) ATP (New England Nuclear, 2,500 Ci / mmol), combined in 30 μl of 50 mM Tris-HCl, 10 mM MgCl 2, and 15 mM β-mercaptoethanol. . 2 units of T4 polynucleotide kinase were added and after 30 minutes at 37 ° C 32 P-labeled primers were purified by chromatography on 10 ml Sephades® G-50 columns. Hybridizations were performed using 106 cpm of primer 20 T-13C or 3 x 10 6 cpm of combined primers T-1C. Hybridizations were performed at 15 ° C for 14 hours in 6 x SSC [1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate; pH 7.2], 10 x Denhardt's solution [0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll] as described by Wallace et al. (see above) has explained. The filters were washed for 5 minutes (3x) at 0 ° C and 6x SSC, dried and placed on X-ray film. The results are shown in Figure 2 for 32β-combined primers T-13C and primers T-1C.

The plasmid DNA of clone 104 was found to give significant hybridization with the combined primers T-1C and primer T-13C, but no detectable hybridization with other undecamers. As shown in Figure 2, many of the 39 potential LelF-35 plasmids (pL2, 4, 13, 17, 20, 31, and 34) also hybridize to both of these probes. Restriction analysis showed

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2i 8271 4, however, that only one of these plasmids, pL31, also contained a 260 bp internal BglII fragment; PstI extraction of pL31 showed a cDNA insertion sequence of approximately 1,000 base pairs.

The entire PstI insertion sequence of pL31 was sequenced as well

By the Maxam-Gilbert chemical method [Methods Enzymol. 65, ss. 499-560 (1980)] and by the dideoxy chain termination method [Smith, Methods Enzymol. 65, ss. 560-580 (1980)] after subcloning of Sau3a fragments into 10 M13 vectors. The DNA sequence ("A") is shown in Figure 3. The appropriate translation coding pattern could be predicted from the available protein sequence information, the known range of LelF molecular weights, and the relative occurrence of termination triplets in the three possible coding diches, which in turn allowed the size of LelF. -prediction of the amino acid sequence containing the precursor signal lipid. The first ATG translation start codon is found 60 nucleotides from the 5 'end of the sequence, followed by 188 codons from the TGA stop colon 20; The 3 'end has 342 untranslated nucleotides followed by a poly (A) sequence. The putative signal peptide (presumably involved in the secretion of mature LeIF from leukocytes) is 23 amino acids in length. The other 165 amino acids that make up mature LeIF have a calculated molecular weight of 19,390. Here, LelF encoded by pL31 is termed "LelF A". From the sequence information ("A") in Figure 4, it can be seen that the tryptic peptides T-1 and T-13 of LelF B correspond to amino acids 145-149 and 57-61 of LelF A, respectively. The actual DNA coding sequences found in these two regions are those represented by the combined primers T-1C and T-13C (see Figure 8).

H. Direct Expression of Mature White Cell Interferon A (LelF A) I. General 35 The method followed for direct expression of LelF A to a mature interferon polypeptide is a modification of 22 8271 4 methods previously used for human growth hormone [Goeddel et al., Nature 281, ss. 544-548 (1979)], insofar as it involved a combination of synthetic (N-termination) and complementary DNA.

As shown in Figure 6, the Sau3a restriction endonuclease site is suitably located between codons 1 and 3 of LelF A. 2 synthetic deoxyoligonucleotides were constructed that combine the ATG translation start codon, reconstruct the codon for amino acid 1 (cysteine) 10, and form a single-stranded EcoRI end. These oligomers were ligated to the Sau3a-AvaII fragment of the base pair of pL31. The resulting 45 bp product was ligated into two additional DNA fragments to construct an 865 bp synthetic-natural hybrid gene encoding LelF A and flanked by EcoRI and PstI cleavage sites. This gene was ligated into pBR322 between the EcoRI and PstI sites to give plasmid pLelF A1.

2. Construction of a tryptophan control element (containing the E. coli trp promoter, operator and trp leader-20 bosomal site but lacking the ATG sequence to initiate translation)

Plasmid pGMI contains an E. coli tryptophan operon containing the deletion ALE1413 [Miozzari et al., Bacte-riology 133, p. 1457-1466 (1978)] and thus expresses a fusion protein comprising the first 6 amino acids of the trp leader and approximately the last 3 amino acids of the trp E polypeptide (hereinafter referred to as "nLE"), as well as the total trp D polypeptide. The plasmid (20 pg) was extracted with the restriction enzyme PvuII, which cleaves the plasmid at five sites. ) or 23 8271 4 including an EcoRI cleavage site for subsequent cloning into a plasmid containing the EcoRI site.

20 pg of DNA fragments from pGM1 were treated with 10 units of T4 DNA ligase in the presence of 20 pmol of 5'-phosphoryl-5 linked synthetic oligonucleotide pCATGAATTCATG and 20 μl of T4 DNA ligase buffer [20 mM Tris (pH 7.6) , 0.5 mM ATP, 10 mM MgCl 2 and 5 mM dithiothreitol) at 4 ° C overnight. The solution was then heated at 70 ° C for 10 minutes to stop the connection. The coupling fragments 10 were digested with EcoRI extraction, and the fragments now with EcoRI ends were separated using 5% polyacrylamide gel electrophoresis (hereinafter "PAGE"), and the 3 largest fragments were isolated from the gel by first staining with ethididium bromide, locating the fragments with ultravio-15. with beam light and by cutting parts of the gene of interest. Each gene fragment was placed with 300 microliters in a 0.1 x TBE dialysis bag and subjected to 100 volt electrophoresis for one hour in 0.1 x TBE buffer (TBE buffer containing 10.8 g Tris base, 5.5 g boric acid, and 20 0, 09 g Na2-EDTA in 1 liter of water). The aqueous solution was collected from a dialysis bag, extracted with phenol, extracted with chloroform and made 0.2 M with sodium chloride, and the DNA was recovered in water after ethanol precipitation. The gene containing the Trp promoter operator with 25 single-stranded EcoRI ends was identified in the following image; followed by insertion of the fragments into a tetracycline-sensitive plasmid which, after insertion into a promoter-operator, becomes tetracycline-resistant.

Plasmids pBRH1 [Rodriques et al., Nucleic Acids

Res. 6, ss. 3267-3287 (1979)] expresses ampicillin resistance and contains a gene for tetracycline resistance, but in the absence of an associated promoter, it does not express this resistance. The plasmid is si-35 sensitive to tetracycline. By introducing a promoter-operator system into the EcoRI site, the plasmid can be made resistant to tetracycline.

pBRH1 was extracted with EcoRI and the enzyme was removed by phenol extraction followed by chloroform extraction and recovered in water after ethanol precipitation. The resulting DNA molecule was combined in separate reaction mixtures with each of the three DNA fragments obtained above and ligated with T4 DNA ligase as described above. The DNA present in the reaction mixture was used to transform 10 valid E. coli K-12 strain 294 by standard methods [Hersh-field et al., Proc. Natl. Acad. Sci. USA 71, ss. 3455-3459 (1974)] and the bacteria were plated on LB (Luria-Bertani) plates containing 20 pg / ml ampicillin and 5 pg / ml tetracycline. Several tetracycline-resistant colonies were selected, plasmid DNA was isolated, and the presence of the desired fragment was confirmed by restriction enzyme analysis. The resulting plasmid is labeled pBRHtrp.

The EcoRI and BamHI extraction product of the hepatitis B virus genome was obtained by conventional methods and cloned into the EcoRI and BamHI sites of plasmid 20 [Goeddel et al., Nature 281, p. 544 (1979)] to form plasmid pHS32. Plasmid pHS32 was digested with XbaI, extracted with phenol and chloroform, and precipitated with ethanol. It was then treated with 1 liter of E. coli DNA polymerase I, Klenow fragment 25 (Boehringer-Mannheim) in 30 μl of polymerase buffer [50 mM

potassium phosphate (pH 7.4), 7 mM MgCl 2 and 1 mM β-mercaptoethanol) containing 0.1 mM dTTP and 0.1 mM cCTP for 30 minutes at 0 ° C and then for 2 hours at 37 ° C. This treatment brings two of the 4 nucleotides complementary to the 5 'end of the XbaI cleavage site complementary to the following: 5' CTAGA-5 'CTAGA-3' T-3 'TCT-25 8271 4 2 nucleotides, dC and dT, were combined to give a head with 2 protruding 5 'nucleotides. This linear residue of plasmid pHS32 (after phenol and chloroform extraction and recovery in water after ethanol precipitation) was digested with EcoRI. The large plasmid fragment was separated from the smaller EcoRI-XbaI fragment by PAGE and isolated after electroelution. This DNA fragment from pHS32 (0.2 pg) was ligated under the same conditions as described above to the EcoRI-TaqI fragment of the tryptophan operon (ca. 0.01 pg) derived from pBRHtrpr.

In a process in which a fragment from PHS32 is ligated to an EcoRI-TaqI fragment as described above, the protruding end of Taq1 is ligated to the remaining protruding end of XbaI, even if it is not completely base-paired according to Watson-Crick: - T CTAGA - TCTAGA- -> -AGC + TCT- AGCTCT- 20

A portion of this annealing reaction mixture was transformed into E. coli K-12 strain 294 cells, heat-treated, and plated on LB plates containing ampicillin.

Twenty-four colonies cultured in 3 ml of 25 LB (Luria-Bertani) medium were selected and the plasmid was isolated. 6 of these were found to have an XbaI site regenerated by E. coli catalyzed DNA repair and the following replication: -TCTAGA - TCTAGA - 30 -> -AGCTCT - AGATCT- These plasmids were also found to be cleaved by both EcoRI and HpaI: and give the expected cleavage fragments-35. One plasmid, designated pTrp14, was used to express heterologous polypeptides as follows.

Plasmid pHGH 107 [Goeddel et al., Nature 281, p. 544 (1979)] contains a gene for human growth hormone consisting of 23 amino acid codons produced from synthetic DNA fragments and 163 amino acid codons derived from complementary DNA. produced by reverse transcription of human growth hormone messenger RNA. This gene, although missing the codons of the "ancestral" sequence of human growth hormone, contains the translation initiation codon ATG. This gene was isolated from 10 μg of pHGH 107 after treatment with EcoRI, followed by treatment with E. coli DNA polymerase I-Klenow fragment and dTTP and dATP as described above. After phenol and chloroform extraction and ethanol precipitation, the plasmid was treated with BamHI.

The fragment containing the human growth hormone (HGH) gene was isolated by PAGE followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the tetracycline resistance-20 structural gene, but lacks a tetracycline promoter-operator system, so that when subsequently cloned into an expression plasmid, plasmids containing the insertion sequence can be located by restoring tetracycline resistance. Because the EcoRI end of the 25 fragments is filled in by the Klenow polymerase I method, the fragment has one blunt end and one single-stranded end, ensuring proper orientation when subsequently ligated into the expression plasmid.

Next, expression plasmid pTrp144 was prepared to receive the HGH gene-containing fragment prepared above. Thus, pTrp14 was extracted with XbaI and the resulting single-stranded ends were filled in by Klenow polymerase I method using dATP, dTTP, dGTP and dCTP. The DNA obtained after phenol and chloroform extraction and precipitation with ethanol was treated with BamHI and the resulting large plasma diffraction was isolated by PAGE and electroelution.

Il 27 8271 4 The fragment derived from pTrp14 had one blunt and one single-stranded end, which allowed fusion in the correct direction with the fragment containing the HGH gene described above.

The HGH gene fragment and the pTrp14 Xba-BamHI fragment were combined and ligated together under similar conditions as described above. The filled XbaI and EcoRI ends were joined together by a blunt junction to reconstitute both the XbaI and EcoRI sites: 10 Filled Xbal Filled EcoRI HGH Gene Initiation -TCTAG AATTCTATG- -TCTAGAATTCTAG- -> -AGATC + TTAAGATAC- -AGATCTTAAGAT

15 Xbal EcoRI

This construct also reconstructs the tetracycline resistance gene. Because plasmid pHGH 107 expresses tetracycline resistance from a promoter located 20 upstream of the HGH gene (Lac promoter), this construct, designated pHGH 207, allows expression of the gene for tetracycline resistance in the tryptophan promoter operator engine. Thus, the ligation mixture was transformed into E. coli strain K-12 294 and 25 colonies were selected for LB plates containing 5 pg / ml tetracycline.

Plasmid pHGH 207 was extracted with EcoRI and a 300 bp fragment containing the trp promoter, operator and trp leader ribosome binding site but lacking the ATG sequence to initiate translation by PAGE was recovered, followed by electroelution. This DNA fragment was cloned into the EcoRI site of pLelF A. Expression plasmids containing the above-modified trp-regulon (E. coli-trp operon with the attenuator sequence 35 omitted to adjustably increase expression levels) can be cultured at predetermined levels in 28,871 4 media containing additional tryptophan in amounts of sufficient to attenuate the promoter-operator system, and then remove tryptophan from them to thereby attenuate the system and effect expression of the intended product.

Specifically, and with reference to Figure 6, 250 pg of plasmid pL31 was extracted with PstI and a 1,000 bp insertion sequence was isolated by gel electrophoresis on a 6% polyacrylamide gel. Approximately 40 pg of the insertion cycle was electroextracted from 10 gels and divided into three equal portions for further extraction: a) A 16 pg sample of this fragment was partially extracted with 40 units of BglII for 45 minutes at 37 ° C and the reaction mixture was purified on a 6% polyacrylamide gel. Approximately 2 pg of the desired 670 bp fragment was recovered; b) A second sample (8 pg) of the 1000 bp PstI insertion sequence was digested with Avail and BglII. 1 pg of the indicated 150 bp fragment was recovered after gel electrophoresis; C) 16 pg of the 1000 bp fragment was processed

With Sau3a and Avail. After electrophoresis on a 10% polyacrylamide gel, about 25 pg (10 pmo) was recovered from the 34 bp fragment.

2 indicated deoxyoligonucleotides, 5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (fragment 2) were synthesized by the phosphotriester method. Fragment 2 was phosphorylated as follows: 200 μl (ca. 400 pmo) of (ηρ32ρ) ATP (Amersham; 5,000 Ci / mmol) was thoroughly dried and resuspended in 300 μl of 60 mM Tris-HCl (pH 8). 10 mM MgCl 2 and 15 mM 8-mercaptoethanol containing 100 pmoles of DNA fragment and 2 units of T4 polynucleotide kinase. After 15 minutes at 37 ° C, 1 μl of 10 mM ATP was added and the reaction was allowed to proceed for another 15 minutes. The mixture was then heated at 70 ° C for 35 minutes, combined with 100 pmoles of 5'-OH fragment 1 and 10 pmoles of 34 bp Sau3a-AvaII.

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29 8 2 7 1 4 fragments. Coupling was performed for 5 hours at 4 ° C in 50 silicon 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 10 mM di-thiothreitol, 0.5 mM ATP, and 10 units of T4 DNA. ligase. The mixture was electrophoresed on a 6% polyacrylic-5 amide gel and the 45 bp product was recovered by electroextraction. Approximately 30 ng (1 pmol) of the 45 bp product was combined with 0.5 pg (5 pmol) of the 150 bp Avall-BglII fragment and 1 pg (2 pmol) of the 670 bp BglII-PstI fragment. Ligation occurred at 20 ° C for 16 hours using 21 units of T4 DNA ligase. The ligase was inactivated by heating at 65 ° C for 10 minutes. The mixture was then extracted with EcoRI and PstI to remove gene polymers. The mixture was purified by 6% PAGE. Approximately 20 ng (0.04 pmol) of 865 bp product was isolated. Half of this (10 ng) was ligated into pBR322 (0.3 pg) between the EcoRI and PstI sites. Transformation of E. coli strain K-12 294 yielded 70 tetracycline-resistant, ampicillin-sensitive transformants. Plasmid DNAs isolated from 18 of these transformants were extracted with EcoRI and PstI. 16 of these 18 plasmids contained an EcoRI-PstI fragment of 865 bp in length, 1 pg of one of these (pLelF AI) was extracted with EcoRI and ligated into a 300 bp EcoRI fragment (0.1 pg) containing E. coli -trp promoter and trp-25 leader ribosome binding site prepared as described above. Transformants containing the trp promoter were identified using the 32p-trp probe in conjunction with the Grunstein-Hogness colony screening method. The asymmetrically located XbaI site in the trp fragment allowed the identification of recombinants in which the trp promoter was directed toward the LeiF A gene.

I. In Vitro and In Vivo Activity of LelF A Extracts for the IF assay were prepared as follows: 1 ml cultures were grown in L broth containing 5 pg / ml tetracycline to an A550 of about 1.0 and 827171 was then diluted in 25 ml of M9 medium containing 5 pg / ml tetracycline. 10 ml samples were collected by centrifugation when Asso reached 1.0 and the cell pellets were suspended in 1 ml of 15% sucrose, 50 mM Tris-HCl 5 (pH 8.0) and 50 mM EDTA. : a. 1 mg of lysozyme was added and after 5 minutes the cells were disrupted at 0 ° C by sonication. Samples were centrifuged for 10 minutes at 15,000 rpm and the interferon activity of the supernatants was determined by comparison to LelF standards in a cell disease resistance (CPE) inhibition assay. The specific activity of LeIF at 4 x 108 units / mg was used to determine the number of IF molecules per cell.

As shown in Table 1, clone pLelF A trp 25, to which the trp promoter is inserted at the desired orientation, gives high levels of activity (as high as 2.5 x 10 8 units / liter).

As shown in Table 2, IF produced by E. coli strain K-12 strain 294 / pLeIF A trp 25 behaves like authentic human LelF; it is resistant to treatment at pH 2 and is neutralized by rabbit anti-human leukocyte-lu antibodies. The apparent molecular weight of interferon is about 20,000.

The in vivo efficacy of interferon requires the presence of macrophages and natural killer (NK) cells, and the mode of action in vivo appears to induce stimulation of these cells. Thus, the possibility remained that the interferon produced by E. coli strain K-12 strain 294 / pLeIF A trp 25, while having antiviral activity in a cell culture assay, would not be active in infected animals. In addition, the in vivo antiviral activity of bacterially produced non-glycosylated LelF A could be different from that of glycosylated LelF derived from white blood cells in human coagulated blood fluid after erythrocyte deposition. Therefore, the biological activity of bacterially synthesized LelF A (2% purity) was compared with LelF (8% purity) from 3i 8271 4 from coagulated aqueous humor after erythrocyte sedimentation in lethal monkey mortal myocarditis (EMC). viral infection (Table 3).

5 Table 1

Interferon activity in E. coli extracts E. coli K-12 Cell density IF activity LelF molecule 294; (cells / ml) units / ml chickens 10 transformate culture / cell

pLelF A

with trp 25 3.5 x 108 3 6 0 0 0 9 000

pLelF A

trp 25; lla_1.8 x 109 2 50 0 00_12 000 15

Table 2 Comparison of the activities of E. coli K-12 strain 294 / pLeIF A 25 extracts with standard LelF1 * 20 _

Interferon activity (units / ml) Treat pH 2 Rabbit anti-human mis-leukocyte ;;; _antibodies_ ;; E. coli 294 / pLelF A trp 25- new 500 500 <10

LelF standard_500_500_ <10_: * 'The E. coli K-12 strain 294 / - 30 shown in Table 1 was diluted 500-fold in 250,000 units / ml of pLelF A trp 25 with the minimum amount of medium required to give a specific activity of 500 units / ml. . A leukocyte interferon standard (Wadley Institute) previously titrated against the N1H leukocyte interferon standard was also diluted to a final concentration of 500 units / ml. 1 ml aliquots were adjusted to pH 2 with 1N HCl, 32 8271 4 were incubated at 4 ° C for 52 hours, neutralized by the addition of NaOH, and IF activity was determined by a standard SPE inhibition assay. 25 μl of η aliquots of 500 units / ml samples (untreated) were incubated with 25 μ1 of η-5in anti-human leukocyte interferon for 60 minutes at 37 ° C, centrifuged at 12,000 x G for 5 minutes, and on a precipitate. the liquid was analyzed.

Table 3 10

Antiviral activity of various LelF preparations against EMC virus infection in squirrel monkeys

Surviving Serum 15 Plaque-forming units / ml Treatment_ Day 2 Day 3 Day 4

Comparison 0/3 10 * \ 3 x 104 105 (bacterial 0 ^ 3 0 1,200 '> 3,4x104 proteins) 0) 0 0 20 Bacterial LelF A 3/3 0 0 0 0 0 0 0 0 0

LelF standard 3/3 0 00 0 0 0 '25 _0_0_0_

All monkeys were male (mean weight 713 g) and free of EMC virus antibodies prior to infection. Monkeys were infected intramuscularly with 30,100 x LD50 EMC virus (determined in mice). In the control, the treated monkeys died 134, 158 and 164 hours after infection. Interferon treatments on the 10® unit occurred via the intravenous route at -4, +2, 23, 29, 48, 72, 168, and 240 hours post-infection. Bacterial leukocyte-35 terferon was a column chromatography fraction of E. coli K-12 strain 294 / pLeIF A 25 lysate with specific activity

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33 82 71 4 7.4 x 106 units / mg protein. The reference bacterial proteins were an equivalent column fraction from the lysate of E. coli strain K-12 strain 294 / pBR322 at twice the total protein content. The leukocyte interferon standard was Sendai-5 virus-induced interferon from normal human "coagulated blood fluid, erythrocyte sedimentation," cells chromatographically purified to a specific activity of 32 x 10 6 units / mg protein.

Control monkeys showed persistent dormancy, loss of equal weight, relentless paralysis of the hind limbs, and watering of the eyes that began approximately 8 hours before death. Interferon-treated monkeys did not show any of these abnormalities; they remained active throughout and did not develop viremia. One monkey in the control-15 group that did not develop viremia at 4 days died last (164 hours after infection) but had high virus titers in the heart and brain after death. Interferon-treated monkeys did not develop antibodies to EMC virus as determined 14, 20, and 21 days after infection. These results indicate that the antiviral effects of LelF preparations in infected animals can be attributed to interferon alone, since the contaminating proteins are completely different in bacterial and kaogulated blood-25 preparations. In addition, these results indicate that glycosylation is not required for the in vivo antiviral activity of Lei F A.

: J. Isolation of cDNAs for additional leukocyte interferons from a plasmid containing 30 DNa of fully characterized LelF A cDNA was cut with PstI, isolated electrophoretically and labeled with 32p. The resulting radiolabeled DNA was used as a probe to screen additional transformants of E. coli strain K-12 strain 294, which were obtained in the same manner as in Part C, by the in situ colony screening procedure of Grunstein and Hogness (see above). 8271 4 by. Colonies that hybridized to the probe in varying amounts were isolated. Plasmid DNA from these colonies and the 10 hybridized colonies referred to in Section 6 above were isolated by digestion with PstI and 5 were characterized by 3 by a different method.

First, these Pst fragments were characterized according to their restriction endonuclease extraction patterns with the enzymes BglII, PvuII and EcoRI. This analysis allowed for at least 8 different types of classifications (LelF A, B, C, D, 10 E, F, G, and H) shown in Figure 5, which approximately shows the location of the different cleavage sections relative to the hitherto known presequence and LelF A relative to the coding sequence of. One of these, LelF D, is believed to be the same as that described by Nagata et al. [Nature 15 284, ss. 316-320 (1980)].

Second, certain DNAs were tested according to Gleveland et al. [Cell 20, p. 95-105 (1980)] by describing the ability to selectively remove LelF mRNA from KG-1 cell-20 RNA containing poly-A by a hybrid ring selection assay. LelF A, B, C and F were positive in this assay.

Third, the latter Pst fragments were ligated into an expression plasmid, E. coli K-12 strain 294 was transformed with this plasmid, and the fragments were expressed. The expression products believed to be pre-interferons were all positive by CPE analysis for interferon activity, although the activity was quite borderline for the LelF F fragment. In addition to the above, all of the described LelF types are sequenced. 30 K. Direct expression of another mature white blood cell interferon (LelF B)

The sequence of the isolated fragment containing the gene for mature LelF B shows that the first nucleotides of type A and type B 14 are identical. A fragment with the trp promoter operator, a ribosome binding site, and the start of the LelF A (* B) gene was then separated from pLelF A25 and ligated with the remainder of the B sequence in the expression plasmid. Branched cleavage maps for the Pst fragment of pL4 (plasmid containing the LelF B-Pst-terminal gene shown in Figure 5) and pLelF A25 are shown in Figures 7a and 7b, respectively.

To obtain an approximately 950 base pair Sau3a-PstI fragment from the sequence shown in Figure 7a, several steps were required due to the presence of one or more intermediate Sau3a cleavage sites, i.e.: 1) The following fragments were isolated: a) 110 base pairs from Sau3a to EcoRI ; b) 132 base pairs from EcoRI to Xba; and c)> 700 base pairs from Xba to Pst.

2) Fragments 1a) and Ib) were joined and excised

With Xba and BglII to prevent self-polymerization via the Sau3a and Xba major terminals (the appropriate Sau3a site was within the BglII site; BglII cleaves leaving the Sau3a-yk internal end). A 242 bp fragment was isolated. 3) The product of 2) and lc) was joined and cut

With PstI and BglII, again to prevent self-polymerization. A fragment of about 950 base pairs, from Sau3a to PstI (Figure 7a), was isolated. This fragment contained that part of the LelF B gene which is not common to LelF A.

4) A fragment of about 300 base pairs (from HindIII to Sau3a) containing the trp promoter operator, ribosome binding site, ATG initiation signal and cysteine codon of LelF A was isolated from pLelF A 25.

5) A fragment of about 3,600 base pairs from PstI

HindIII was isolated from pBR322. This contained a replicon and encoded tetracycline but not ampicillin resistance.

6) The fragments obtained in steps 3), 4) and 5) were tri-ligated and the resulting plasmid was transformed into E. coli strain K-12 294.

36 8271 4

Transformants were mini-screened [Birnboim et al. (Nucleid Acids Res. 7, pp. 1513-1523 (1979)) and plasmid samples were extracted with EcoRI to give 3 fragments characterized by: 5 1) EcoRI-EcoRI-trp- promoter; 2) the internal EcoRI fragment of pL4; and 3) a protein translation initiation signal to the EcoRI fragment of pL4.

In the CPE assay, bacterial extracts obtained from 10 clones made as described above typically give about 10 x 10 6 units of interferon activity / liter in A550 * 1. A representative clone prepared in this manner is E. coli strain K-12 294 / pLeIF B trp 7.

L. Direct expression of other mature leukocyte interferons (LelF C, 15 D, E, F, H, I and J)

In particular, other full-length gene fragments containing other types of LeiF can be prepared and inserted into an expression vector for expression, as in the case of LelF A. Complete sequencing by conventional methods indicates whether the cleavage site is located close enough to the first amino acid codon of the mature interferon-type to allow adequate recourse to the method used in Part H above to express mature LelF A, i.e., to remove the presequence by N-segment replacement cleavage. for amino acids lost in deletion of the presequence by coupling a synthetic DNA fragment. Without accomplishing this, the following method may be used, briefly comprising cleaving a fragment containing the pre-sequence just 30 points before the point where the codon for the first amino acid of the mature polypeptide begins, by: 1) converting double-stranded DNA to single-stranded DNA in the region surrounding that point; 2) hybridizing a primer complementary to the single-stranded DNA length of the single-stranded DNA formed in step 1) so that the 5 'end of the primer is 37,827 4 opposite the nucleotide associated with the intended cleavage site; 3) reconstructing the portion of the second strand removed in step 1) located 3 'to the primer by reacting with DNA polymerase in the presence of adenine-containing deoxynucleotide triphosphates; and 4) extracting the remaining single-stranded length of DNA that protrudes beyond the intended cleavage point.

A short piece of synthetic DNA terminating at the 3 'end of the coding strand and having the translation start signal ATG can then be inserted, e.g., into a specially engineered gene for mature interferons, and the gene can be ligated into an expression plasmid and inserted into a promoter. and under the control of an associated ribosome binding site.

In the same manner as used in Part K above, gene fragments encoding LelF C and LelF D were appropriately engineered for direct bacterial expression. The expression strategy for these additional leukocyte interferons in each case involved recourse to a fragment of about 300 base pairs (from HindIII to Sau3a) containing the trp promoter operator, ribosome binding site, ATG initiation signal, and 25 cysteine codon of LelF A: from. Gene fragments from other interferon genes encoding their respective amino acid sequences over an initial cysteine common to all were combined. Each plasmid obtained was used to transform E. coli strain K-12 294. The links to generate the corresponding genes were as follows:

LelF C

The following fragments are isolated from pLelF C: a) 35 base pairs from Sau3a to Sau96; b)> 900 base pairs from Sau96 to PstI; 35 c) isolating a fragment of about 300 base pairs (HindIII-

Sau3a) pLelF from A 25 as in part K 4) above; and 38 8 2 71 4 d) isolating a fragment of about 3,600 base pairs from part K 5) above;

Structure 1) a) and c) are added. Digest with BglII and 5 HindIII and isolate the product of about 335 base pairs.

2) Triple-link 1) + b) + d) and transform with E. coli with the obtained plasmid.

A representative clone made in this way is E. coli strain K-12 294 / pLeIF C trp 35.

10 LeiF D

The following are isolated from pLelF D: a) 35 base pairs from Sau3a to Avail; b) 150 base pairs from Avail to BglII; and c) about 700 base pairs from BglII to Pstlre.

15 pLelF A 25 is isolated from: d) 300 base pairs from HindIII to Sau3a; The following are isolated from pBR322: e) about 3,600 base pairs from HindIII to PstI.

Construction: 1) a) + b) are coupled, cut with BglII and the 185 bp product 1) is purified.

2) 1) + d) is coupled, cut with HindIII and BglII and the product of about 500 base pairs is purified 2).

3) 2) + c) + e) are connected and transformed into E.

25 coli of the resulting plasmid.

A representative clone made in this way is E. coli strain K-12 294 / pLeIF D trp 11.

Lei F F

In particular, a fragment containing Lei F F can be processed for direct expression by reassembly, as appropriate, by complete homology of amino acids 1-13 of LelF B and LelF F. The Trp promoter-containing fragment a) with appropriately shaped ends is obtained from the pHGH 207 described above via PstI-35 and XbaI extraction, followed by isolation of a fragment of about 1,050 bp 39,827 4. The second fragment b) is obtained larger from the two fragments obtained from the extraction of plasmid pHKY 10 with PstI and Bgl. The fragment a) contains approximately half of the gene encoding ampisil-5 line resistance; fragment b) contains the remainder of the gene and the entire gene for tetracycline resistance, except for the associated promoter. Fragments a) and b) are combined with T4 ligase and the product is treated with XbaI and BglII to remove dimerization to form fragment 10 c), which contains the trp promoter operator and genes for tetracycline and ampicillin resistance.

Approximately 580 bp fragment d) is obtained by extraction of pLelF F with Avail and BglII. This contains codons for amino acids 114-166 of LelF F.

Fragment e) (49 base pairs) is obtained by extracting pLelF B with XbaI and AvaII. Fragment e) encodes amino acids 1-13 of LelF F.

Fragments c), d) and e) are triple ligated in the presence of T4 ligase. The cohesive ends 20 of the corresponding fragments are such that the recombinant plasmid forms a ring correctly, placing the tetracycline resistance gene under the control of the trp promoter operator together with the gene for mature LelF F, so that bacteria transformed from the desired plasmid can be selected. A representative clone prepared in this manner is E. coli K-12 strain 294 / pLeIF F trp 1.

LelF H

The complete LelF H gene can be engineered into mature leukocyte interferon for Expression-30 as follows: 1) Plasmid pLelF H is extracted with HaeII and Rsal and an 816 bp fragment extending from amino acid 10 of the signal peptide to the 3 'non-coding region is isolated.

2) The fragment is denatured and subjected to repair-35 synthesis with the Klenow fragment of DNA polymerase I [Klenow et al., Proc. Natl. Acad. Sci. USA 65, pp. 168 40 82714 (1970)] using the synthetic deoxyribo-oligonucleotide primer 5'-dATG TGT AAT CTC TCT.

3) The resulting product is digested with Sau3A and a 452 bp fragment representing amino acids 1 to 5,150 is isolated.

4) Sau3a and PstI extraction of pLelF H and the resulting 500. isolation of a base pair fragment yields a gene encoding amino acids from 150 to the end of the coding sequence.

5) The fragments isolated in steps 3) and 4) are joined to form a fragment: 1166 met cys asp stop

ATG TGT .. -1- ... GAT TAG ... PstI

15 Sau3a encoding 166 amino acids of LelF H.

6) pLelF A trp 25 is extracted with XbaI, blunt-ended with DNA polymerase I and the product is extracted with 20 PstI. The resulting large fragment can be isolated and ligated with the product of step 5) to form an expression plasmid capable of expressing mature LelF H after transformation of E. coli K-12 strain 294 or other host bacteria.

25 LelF I

The human genome phage Charon 4A recombinant library constructed by Lawn et al. [Cell 15, p. 1157 (1978)], were screened for leukocyte interferon genes by the methods described by Lawn et al. (supra) and 30 Maniatis et al. [Cell 15, p. 687 (1978)]. The radioactive LeiF probe, derived from the cDNA clone LelF A, was used to screen about 500,000 plaques. This screening yielded 6 LelF genomic clones. After re-screening and plaque purification, one of these clones, X.HLeIF2 35, was selected for further analysis.

41 8271 4 Using the method described above, soil experiments can be used to promote the isolation of other LelF clones from the human genome. These, in turn, can be used to produce additional leukocyte interferon proteins in accordance with this invention.

1) The 2,000 base pair EcoRI fragment of the ^ JLeIF2 clone was subcloned into pBR325 at the EcoRI site. The resulting plasmid, LelF I, was digested with EcoRI and a 2,000 base pair fragment was isolated. Deoxyoligonucleotide dATTCTGCAG

10 (an EcoRI-PstI converter) was ligated to this 2,000 base pair EcoRI fragment and the resulting product was digested with PstI to give a 2,000 base pair fragment containing PstI ends. This was digested with Sau96 and a 1,100 bp fragment with one PstI and one Sau96 end was isolated.

2) Plasmid pLelF C trp 35 was extracted with PstI and XbaI. The large fragment was isolated.

3) A small XbaI-PstI fragment from pLelF C trp 35 was extracted with XbaI and Sau96. A 40 bp 20 XbaI-Sau96 fragment was isolated.

4) The fragments isolated in steps 1), 2) and 3) were ligated to form the expression plasmid pLelF I trp 1.

LelF J

1) Plasmid pLelF J contains a 3.8 kilobase 25 HindIII fragment of human genomic DNA containing

LeiF J gene sequence. A 760 bp DdeI-RsaI fragment was isolated from this plasmid.

2) Plasmid pLelF trp 7 was digested with HindIII and

Ddei and a 340 bp HindIII-Ddel fragment was isolated.

3) Plasmid pBR322 was digested with PstI, blunt-ended by incubation with DNA polymerase 1 (with the Klenow fragment) and then extracted with * HindIII. A large fragment (about 3,600 base pairs) was isolated.

42 8271 4 4) The fragments isolated in lies 1), 2) and 3) were ligated to form the expression plasmid pLelF J trp 1.

M. Cleaning

The concentration of leukocyte interferon in bacterial extracts 5 can be increased by sequential: 1) polyethyleneimine precipitation, in which most of the cellular protein, including interferon, remains in the supernatant; 2) ammonium sulfate fractionation, in which the interferon exits the solution in 55% saturated ammonium sulfate; 3) suspending the ammonium sulfate pellet in 0.06 M potassium phosphate and 10 mM Tris-HCl (pH 7.2) and dialyzing against 25 mM Tris-HCl (pH 7.9) (interferon activity remains in solution); 4) by chromatography of the supernatant obtained above, the pH being adjusted to 8.5, on a DEAE-cellulose column (eluting with a direct gradient of NaCl from 0 in 0.2 mM Tris-HCl; pH 8.5) ; 5) adsorption on Cibachrome Blue Agarose'11a or hydroxylapatite and elution with 1.5 M KCl or 0.2 M phosphate solution, respectively (optional); 6) molecular size sorting on a Sepha-dea G-75 column; and 7) cation exchange chromatography on CM cellulose in 25 mM ammonium acetate at pH 5.0 developed with an ammonium acetate gradient (to 0.2 M ammonium acetate).

The above process gives a substance with a purity of more than 95%.

The substance can also be purified by size exclusion chromatography, reverse phase (RP-8) high performance liquid chromatography, or affinity chromatography with immobilized anti-interferon antibodies.

Alternatively, the material from step 4) above can be loaded onto a monoclonal antibody column prepared as described by Milstein [Scientific American 243, p. 66 (1989)] and eluted with 0.2 M acetic acid, 0.1%. with Triton® and 0.15 NaCl.

In an alternative preferred embodiment, the leukocyte interferon prepared by the method described above can be purified by the following steps: 1) Frozen cell pellets containing the expressed leukocyte interferon are disrupted by hand or with a suitable size reducing device. The partially thawed cells are suspended in 4 volumes of buffer A containing 0.1 M Tris adjusted to pH 7.5-8.0, 10% (w / v) sucrose, 0.2 M NaCl, 5 mM EDTA, 0.1 mM PMSF and 10-100 mM MgCl 2. The suspension is kept at about 4 ° C. The suspension is passed through a homogenizer at about 6,000 psi, followed by a second pass at less than 1,000 psi. The effluent from the homogenizer from each run is cooled in an ice bath.

2) Polyethyleneimine is slowly added to the homogenized product to a concentration of about 0.35% and allowed to stand for about 30 minutes. The solid is removed by centrifugation or filtration. The temperature of this step is monitored or carried out quickly enough so that the temperature of the liquid (filtrate) on top of the precipitate remains below 10 ° C. The liquid (filtrate) on top of the precipitate is concentrated by ult-25 fat filtration to about 1/10 of the original volume. Particulate matter or mist on retention is removed as a suitable filter, such as a microporous membrane.

3) The clarified solution is applied directly to a monoclonal antibody column with a flow of about 5-8 cm / h (e.g. 25-40 ml / h in a 2.6 cm diameter column). After loading, the column is washed with about 10 column volumes of 25 mM Tris-HCl (pH 7.5-8.5) containing NaCl (0.5 M) and a surfactant such as Tritori ^ 35 X- 100 (0.2%) or equivalent. After washing, the column is rinsed with about 10 column volumes of a solution containing 0.15 M NaCl and a surfactant such as Triton® X-100 (0.1%: ista) or equivalent. The column is eluted with 0.2 M acetic acid containing a surfactant such as Tritori-X-100 5 (0.1%) or equivalent. The protein peak from the monoclonal antibody column (as determined by UV absorbance or other suitable assay) is pooled and the pH is adjusted to about 4.5 with NaOH or 0.1 M Tris base.

4) The combined interferon peak is loaded onto a cation exchanger such as Whatman CM52 cellulose or equivalent equilibrated with a suitable buffer such as ammonium acetate (pH 4.5) (50 mM). After loading, the column is washed with equilibration buffer-15 Ia until the UV absorbance of the effluent has reached a level at which little additional protein elutes from the column. The column is then eluted with 25 mM ammonium acetate / 0.12 M sodium chloride or a combination that optimizes interferon recovery and gives a lyophilized cake with satisfactory appearance and satisfactory solubility properties.

The monoclonal antibodies used in the preferred embodiment described above can be prepared by the methods described by Staehel et al. [Proc. Natl. Acad. Sci. USA 78, ss. 1848-1852 (1981)]. Monoclonal antibodies are purified and covalently bound to Affigel-10 as described below.

Preparation and Purification of Monoclonal Antibodies from Peritoneal Fluid 5 female Balb / c mice were seeded with 5-10 x 10® hybridoma cells from the medium logarithmic growth stage. Approximately 5 x 10® viable cells obtained from the fluid produced by the mice were implanted intraperitoneally into each of 10 or more mice. Peritoneal fluid was collected repeatedly (2-4 times) from already 35 mice. Transfers and collections up to 3

It 45 8271 4 can be performed from one group of mice to another. Peritoneal fluid from mice was pooled from each inoculation.

Cells and organic debris were removed from the aqueous humor by centrifugation at low speed (500 -5,000 x G) for 15 minutes. Centrifugation was performed at 90 ml rpm at 90,000 rpm. The liquid on top of the precipitate was frozen and stored at -20 ° C. After thawing, excess fibrin and particulate matter were removed by centrifugation at 35,000 rpm for 90 minutes. Aliquots of aqueous humor from each inoculum were tested for specific antibody activity by a solid phase antibody binding assay (Staehelin et al., Supra) and pooled if found to be satisfactory.

The protein content of the combined solutions was estimated by estimating that 1 mg of protein gives an absorbance of 1.2 at 280 nm in a cuvette with a travel of 1.0 cm. Peritoneal fluids with high levels of antibodies contain 30 to 35 mg protein / ml. This corresponds to 4-7 mg specific antibody / ml. The liquid was diluted with 20 PBS [0.01 M sodium phosphate (pH 7.3) and 0.15 M NaCl] to a protein concentration of 10-12 mg / ml.

To each 100, 90 ml of saturated ammonium sulfate solution at room temperature was added slowly and vigorously with stirring at 0 ° C. The suspension was kept on ice for 40-60 minutes, then centrifuged for 15 minutes at 10,000 rpm at 4 ° C. The liquid on top of the precipitate was decanted and drained well. The protein pellets were dissolved in 0.02 M Tris-HCl (pH 7.9) / 0.04 M NaCl (buffer I). The protein solution was dialyzed for 16 to 18 hours at room temperature against 100 volumes of buffer I at least once with buffer change. The dialyzed solution was centrifuged at 15,000 rpm for 10 minutes to remove insoluble material. About 30-35% of the initial total protein in the aqueous humor 35 was recovered, as estimated by absorption at 280 nm.

«82714

A solution containing 30 to 40 mg protein / ml was then applied to a column of DEAE cellulose equilibrated with buffer I. At least 100 ml of column mattress volume 5 was used for each gram of protein added. The antibody was eluted from the column with a linear gradient of NaCl containing 0.02 M Tris-HCl (pH

7.9) and 0.04-0.5 M NaCl. The combined peak fractions, eluting with 0.06 to 0.1 M NaCl, were concentrated by mixing with an equal volume of saturated ammonium sulfate at room temperature and centrifuging. The protein pellets were dissolved in 0.2 M NaHCO 3 (pH n.

8.0) / 0.3 M NaCl (buffer II) followed by dialysis at room temperature against the same buffer which was changed three times. The dialyzed solutions were centrifuged at 20,000 x G for 15 minutes to remove any insoluble material.

The protein concentration was set between 20 and 25 mg / ml with buffer II.

Preparation of immunoadsorbents

Affigel-10 (BioRad Laboratories, Richmond, Ka-20 lyfornia) was washed on a sintered glass filter three times with ice-cold distilled water. The gel slurry (ca. 50% in cold water) was transferred to plastic tubes and sedimented briefly by centrifugation. The liquid on top of the precipitate was sucked off. The packed gel was mixed with an equal volume of purified antibody solution and vortexed at 4 ° C for 5 hours. After the reaction, the gel was centrifuged, then washed twice with buffer III (0.1 M NaHCO 3 / 0.15 M NaCl) to remove unbound antibody. The protein assay of the combined washes indicated that more than 90% of the antibodies were gel-bound.

To block unreacted sites, the gel was mixed with an equal volume of 0.1 M ethanolamine HCl (pH 8) and vortexed at room temperature for 60 minutes. The gel slurry was washed free of reactants with PBS and stored in PBS in the presence of 0.02% (w / v) sodium azide at 4 ° C.

N. Intraperitoneal administration

LeIF can be administered parenterally to subjects in need of anti-tumor or anti-viral treatment and to those with immunosuppressive conditions. The dose and dosage amounts may be similar to those commonly used in clinical trials of substances of human origin, e.g. about 1 to 10 x 106 units per day, and in the case of substances with a purity greater than 1%, suitably e.g. x Up to 107 units daily.

As an example of a suitable dosage form for a predominantly homogeneous bacterial LeiF in a form administered parenterally, 3 mg of LeIF having a specific activity of e.g. 2 x 10 8 units / mg in 25 ml of 5 N human serum albumin is dissolved in a bacteriological filtrate. and the filtered solution is aseptically dispensed into 100 ampoules, each containing 20 x 10 6 units of pure interferon, suitable for parenteral administration. The ampoules are preferably stored refrigerated (-20 ° C) before use.

The compounds of this invention may be treated according to known methods to prepare pharmaceutically useful compounds, the polypeptide being combined in admixture with a pharmaceutically acceptable carrier. Suitable carriers and their formation are described by E. W. Martin in Remington's Pharmaceutical Sciences. Such combinations contain an effective amount of an interferon protein prepared in accordance with this invention, together with a suitable amount of a carrier, to produce pharmaceutically acceptable combinations suitable for effective administration to a subject to be treated. The preferred route of administration is parenteral.

Claims (15)

A process for producing a transformed strain of E. coli that expresses a human mature leukocyte interferon which is human leukocyte interferon (LelF) A, B, C, D, F, H, I or J with the amino acid sequence Cys Asp Leu Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu 10 With Leu Leu Ala Gin With Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr Ile Pro Vai Leu His Glu With Ile Gin Gin Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gin 15 Gin Leu Asn Asp Leu Glu Ala Cys Vai Ile Gin Gly Vai Gly Vai Thr Glu Thr Pro Leu With List Glu Asp Ser Ile Leu Ala Vai Arg List Tyr Phe Gin Arg Ile Thr Leu Tyr Leu List Glu List List Tyr Ser Pro Cys Ala Trp Glu Vai Vai Arg Ala Glu Ile With Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu Ser Leu Arg Ser List Glu, 20 Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile Leu Leu Ala Gin With Arg Arg Ile Ser Pro Phe Ser Cys Leu Asp Asp Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Asp Lys Gin Phe Gin Lys Ala Gin Ala Ile Ser Vai Leu His Glu Met 25 Ile Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Leu Asp Glu Thr Leu Leu Asp Glu Phe Tyr Ile Glu Leu Asp Gin Lein Asn Asp Leu Glu Vai Leu Cys Asp Gin Glu Vai Gly Vai Ile Glu Ser Pro Leu With Tyr Glu Asp Ser Ile Leu Ala Vai Arg List Tyr Phe Gin Arg Ile Thr Leu Tyr Leu Thr Glu List List 30 Tyr Ser Ser Cys Ala Trp Glu Vai Arg Ala Glu Ile With Arg Ser Phe Ser Leu Ser Ile Asn Leu Gin Lys Arg Leu Lys Ser Lys Glu, 54 8271 4 Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Gly Gin With Gly Arg lie Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Arg lie Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin Ala Ile Ser Val Leu His Glu With lie Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu Glu List Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys Val lie Gin Glu Val Gly Val Glu Glu Thr Pro Leu With Asn Glu Asp Ser lie Leu Ala Val Arg Lys Tyr Phe Gin Arg lie Thr Leu Tyr Leu lie Glu Arg Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie With Arg Ser Leu Ser Phe Ser Thr Asn Leu Gin Lys Arg Leu Arg Arg List Asp, Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu With Leu Leu Ala Gin With Ser Arg lie Ser Pro Ser Ser Cys Leu With Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Pro Ala lie Ser Val Leu His Glu Leu lie Gin Gin lie Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gin Lein Asn Asp Leu Glu Ala Cys Val With Gin Glu Glu Arg Val Gly Glu Thr Pro Leu With Asn Val Asp Ser lie Leu Ala Val List List Tyr Phe Arg Arg lie Thr Leu Tyr Leu Thr Glu List List Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie with Arg Ser Leu Ser Leu Ser Thr Asn Leu Gin Glu Arg Leu Arg Arg List Glu, Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Ala Gin With Gly Arg lie Ser Pro Phe Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin Ala lie Ser Val Leu His Glu Met lie Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gin Ser Leu Glu List Phe Ser Thr Glu Leu Asn Gin Gin Leu Asn Asp With Glu Ala Cys Val lie Gin Glu Val Gly Val Glu Glu Thr Pro Leu With Asn Val Asp Ser lie Lieu Ala Val List Lys Tyr Phe Gi n Arg lie Thr Leu Tyr Leu Thr Glu List List II 55 8271 4 Tyr Ser Pro Cys Ala Trp Glu Vai Val Arg Ala Glu lie With Arg Ser Phe Ser Leu Ser Lys lie Phe Gin Glu Arg Leu Arg Arg Lys Glu, Cys Asn Leu Ser Gin Thr His Ser Leu Asn Asn Arg Arg Thr Leu With Leu With Ala Gin With Arg Arg lie Ser Pro Phe Ser Cys Leu List Asp Arg His Asp Phe Glu Phe Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin Lys Ala Gin Ala lie Ser Val Leu His Glu Met With Gin Gin Thr Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Glu Lys Phe Tyr lie Glu Leu Phe Gin Gin With Asn Asp Leu Glu Ala Cys Val lie Gin Glu Val Gly Val Glu Glu Thr Pro Leu With Asn Glu Asp Ser lie Leu Ala Val Arg List Tyr Phe Gin Arg lie Thr Leu Tyr Leu With Glu List List Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu lie With Arg Ser Phe Ser Phe Ser Thr Asn Leu Gin Lys Arg Leu Arg Arg Lys Asp, Cys Asp Leu Pro Gin Thr His Ser Leu Gly Asn Arg Arg Ala Leu lie Leu Leu Ala Gin With Gly Arg lie Ser Pro Phe Ser Cys Leu Lys Asp Arg Pro Asp Phe Gly Leu Pro Gin Glu Glu Phe Asp Gly Asn Gin Phe Gin List Thr Gin Ala lie Ser Val Leu His Glu With He Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala Ala Trp Glu Gin Ser Leu Leu Glu List Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asn Leu Glu Ala Cys Val He Gin Glu Val Gly With Glu Glu Thr Pro Leu With Asn Glu Asp Ser He Leu Ala Val Arg List Tyr Phe Gin Arg He Thr Leu Tyr Leu Thr Glu List List Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Ala Glu He With Arg Ser Leu Ser Phe Ser Thr Asn Leu Gin Lys He Leu Arg Arg Lys Asp and Cys Asp Leu Pro Gin Thr His Ser Leu Arg Asn Arg Arg Ala Leu He Leu Leu Ala Gin With Gly Arg He Ser Pro Phe Ser Cys Leu List Asp Arg His Glu Phe Arg Phe Pro Glu Glu Glu Phe Asp Gly His Gin Phe Gin Lys Thr Gin Ala He Ser Val Leu His Glu With He Gin Gin Thr Phe Asn Leu Phe Ser Thr Glu Asp Ser Ser Ala 56 82714 Ala Trp Glu Gin Ser Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gin Gin Leu Asn Asp Leu Glu Ala Cys Vai Ile Gin Glu Vai Gly Vai Lys Glu Thr Pro Le u With Asn Glu Asp Phe Ile Leu Ala Vai Arg List Tyr Phe Gin Arg Ile Thr Leu Tyr Leu With Glu List List Tyr Ser Pro Cys Ala Trp Glu Vai Vai Arg Ala Glu Ile With Arg Ser Phe Ser Phe Ser Thr Asn Leu Lys Lys Gly Leu Arg Arg Lys Asp, characterized by transforming an E. coli strain with an E. coli plasmid containing a DNA encoding said human mature leukocyte interferon and which DNA sequence is operably linked to a promoter-operator region by well-known methods and culturing the transformed E. coli strain in a culture medium during pre-culture. heights, suitable for keeping it alive. 2. A method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon A (LelF A). Method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon B (LelF B). Method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon C (LelF C). 5. A method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon D (LelF D). Method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon F (LelF F). A method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon H (LelF H). Method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon I (LelF I). Il 57 8271 4 9. A method according to claim 1, characterized in that the E. coli plasmid encodes human mature leukocyte interferon J (LelF J). Method according to claim 1, characterized in that the E. coli plasmid is pLelF A trp 25, pLelF B trp 7 or pLelF F trp 1. Method according to claim 1, characterized in that the E. coli plasmid is pLelF C trp 35 or pLelF D trp 11. A method according to claim 1, characterized in that the E. coli plasmid is pLelF G or pLelF H. 13. A method according to claim 1, characterized in that the E. coli plasmid is pLelF I trp 15. or pLelF J trp 1. Method according to any of claims 1-13, characterized in that the E. coli strain to be transformed is E. coli K-12 strain 294.