NO164178B - PLASMID CODING A COMPLETE DEVELOPED HUMAN LEUKOCYTT INTERFERON. - Google Patents

Description

The present invention relates to plasmids which code for a fully developed human leukocyte interferon, of which the amino acid sequence of this interferon includes the partial sequence

Human leukocyte interferon (LelF) was first discovered and developed

presented in the form of me^et impure precipitates by Isaacs and Lindenmann (Proe. R. Soc. B 147, 258-267 [1957]; U.S.P. 3,699,222).

Attempts to purify and characterize the material have been ongoing

since then, and has led to the production of relatively homogeneous leukocyte interferons originating from normal or leukemic donors' leukocytes (DOS No. 2,947,134).

These interferons are a protein family known to

have the property that they bring their target cells in a viral

resistant state. In addition, interferon can prevent cell

division and modulate immune response. These properties have led to the clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and diseases.

Leukocyte interferons have been purified to mainly

homogeneity (Rubinstein et al., Proe. Nati. Acad. Sei. USA

76, 640-644 [1979]; Zoon et al., ibid. 76, 5601-5605 [1979],

and is reported to have a molecular weight range from approx. 17,500 to 21,000. The specific activity of these preparations

8 9

is remarkably high, 2 x 10 to 1 x 10 units/mg

protein, but yields from cell culture methods have been disappointingly low.; Nevertheless, advances in protein sequencing techniques have enabled the determination of partial amino acid sequences (Zoon et al., Science 207, 527 [1980]; Levy et al., Proe. Nati. Acad. Sei. U.S.A. 77» 5102-5104 [1980] ). Elucidation of the glycosylation of different leukocyte interferons is not yet complete, but it is now clear that differences in glycosylation among members of the family

one is not alone responsible for the molecular weight spectrum observed. Instead, leukocyte interferons vary markedly in amino acid composition and sequence, and in some cases amino acid homology is less than 80%.

Although isolation from donor leukocytes has provided sufficient material for partial characterization and limited clinical studies with homogeneous leukocyte interferon, it is a completely unsuitable source for the quantities of interferon needed for large-scale clinical trials and furthermore for broad-scale prophylactic and/or therapeutic application. In fact, previous clinical investigations using human leukocyte-derived interferons in antitumor and antiviral trials have primarily been limited to impure (<1 percent pure) preparations of the material, and long production times for sufficient quantities, even at unrealistic price levels, have critically delayed the investigations on an extended front.

However, with the advent of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. Under control, bacteria are already modified through this technology to produce such polypeptide products as somatostatin, A-

and the B chains of human insulin and human growth hormone (Itakura et al., Science 198, 1056-1063 [1977]; Goeddel et... al., Nature 281, 544-548 [1979]). Recently, recombinant DNA techniques have been used to produce bacterial production of proinsulin and thymosin alpha 1 and several authors

have described results of DNA coding for human leukocyte interferon and resulting proteins with leukocyte interferon activity (Nagata et al., Nature 284, 316-320 [1980];

Mantei et al., Gene 10, 1-10 [1980]; Taniguchi et al., Nature 285, 547-549 [1980].

The tool in recombinant DNA technology is the plasmid, a non-chromosomal loop of double-stranded DNA found in bacteria and other microbes, often in several copies per cell. cell. Included in the information encoded in plasmid DNA is that required to reproduce the plasmid in daughter cells (ie a "replicon") and normally one or more selection characteristics such as, in the case of bacteria, resistance to antibiotics, which enables the clones from the host cell containing the plasmid of interest are recognized and preferably grown in selective medium. The use of the plasmids lies in the fact that they can be cleaved specifically by one or more restriction endonucleases (restriction enzymes), each of which finds a different point on the plasmid DNA. Then, heterologous genes or gene fragments can be inserted into the plasmid through end joining at the cleavage point or at reconstructed ends close to the cleavage point. DNA recombination is carried out outside the cell, but the resulting "recombinant" plasmid can be introduced into it through a process known as transformation, and large quantities of the heterologous gene-containing recombinant plasmid are obtained by culturing the transformant, where the gene is further correctly inserted in relation to parts of the plasmid that direct transcription and transmission of the encoded DNA message, the resulting expression carrier can be used to actually produce the polypeptide sequence that the inserted gene codes for, a process called expression.

Expression is initiated at a region known as the promoter which is recognized and bound by RNA polymerase. In some cases, as with the tryptophan or (trp) promoter, which is preferred in the practice of the present invention, promoter regions are overlapped by "operator" regions and form a combined promoter-operator. Operators are DNA sequences recognized by so-called repressor proteins that serve to regulate the frequency of transcription initiation at a particular promoter. The polymerase moves along the DNA, transcribing the information contained in the sequence from its 5' to 3' end. messenger RNA (m-RNA), which in turn is transferred in a polypeptide with the amino acid sequence that DNA codes for. Each amino acid is encoded by a nucleotide triplet ("codon"). The structural gene is the part that codes for the amino acid sequence of the expressed product. After binding to the promoter, the RNA polymerase first transcribes nucleotides that code for a ribosome binding

point, then a transfer initiation or "start" signal (usually ATG, which in the resulting messenger RNA becomes AUG), then the nucleotide codons within the structural gene itself. So-called stop codons are transcribed at the end of the structural gene, after which the polymerase can form a further frequency of a messenger RNA, which, due to the substance's signal, presence, will remain untransmitted by the ribosomes. Ribosomes attach to the binding point present on messenger RNA, in bacteria normally as mRNA is formed, and produce the coded polypeptide itself by starting with the transfer start signal and ending with the aforementioned stop signal. The desired product is produced if the sequences encoding the ribosome binding point are located correctly in relation to the AUG start codon and if all other codons follow the start codon in phase. The resulting product can be obtained by cleaving the host cell and isolating the product through proper purification from other bacterial protein.

It was noted that the use of recombinant DNA technology (ie, insertion of interferon genes into microbial expression vectors and their expression under the control of microbial gene regulatory elements) would be the most efficient way to provide large quantities of leukocyte interferon,

which, despite the fact that glycosylation characteristics are lacking in material thus prepared from human sources, could be used clinically in the treatment of a wide range of viruses and neoplastic diseases.'-

The method for obtaining a first leukocyte gene according to the present invention contained the following steps:

(1) Partial amino acid sequences from human leukocyte interferon, purified to homogeneity, were used to construct sets of synthetic DNA samples whose codons in the aggregate represented all possible nucleotide combinations capable of encoding the partial amino acid sequences. (2) Bacterial colony banks were prepared containing complementary DNA (cDNA) from induced messenger RNA. Other induced mRNA that had been radiolabeled was hybridized to plasmid cDNA from this bank. Hybridizing mRNA was eluted and examined for transfer to interferon in oocyte assays. Plasmid DNA from colonies which were shown to induce interferon activity in this way is further examined with respect to hybridization to samples prepared as described in (1) above. (3) In parallel with the method of part (2) above, induced mRNA-derived cDNA in plasmids was used to form an independent ratio of transformant colonies. The samples from part (1)

was used to effect the synthesis of radioactively labeled single-stranded cDNA for use as hybridization assays. The synthetic samples hybridized with induced mRNA as a counterpart and were amplified by reverse transcription to form induced, radiolabeled cDNA. Clones from the colony stock that hybridized to radioactively labeled cDNA obtained in this way have been examined further to confirm the presence of a full-length interferon gene.

Any sub-length of putative gene fragment obtained in part (1) or (2) can itself be used as a full-length gene probe. (4) The full-length gene obtained above was assembled using synthetic DNA so as to avoid any leader sequence that could prevent microbial expression of the finished polypeptide and enable proper placement in an expression carrier relative to initiation signals and the ribosome binding site for a microbial promoter, expressed interferon was purified to a point that made it possible to establish its character and determine its activity. (5) The interferon gene fragment prepared in the foregoing manner was itself used in research through hybridization of other partially homologous leukocyte interferon species.

By using methods for recombinant DNA technology as described above, microbial production is achieved in high yields and purity of the family of homologous leukocyte interferons (unglycosylated) as finished polypeptides, without the corresponding presequence or other part thereof. These interferons can be directly expressed, isolated and purified to levels that make them suitable for use in the treatment of viruses and malignant diseases in humans and animals. Hitherto expressed interferon types have been shown to be effective in in vitro studies and, in the first such demonstration

tion of its kind, also in in vivo trials, as the latter includes the first finished leukocyte interferon that has been produced microbially.

The term "ready-made leukocyte interferon" used in the context of the present application indicates a microbial

(e.g. bacterially) produced interferon molecule without glycosyl groups. Fully developed leukocyte interferon is expressed immediately from a transfer initiation signal (ATG) just before the first amino acid codon of the natural product. The "finished" polypeptide can thus contain, as the first amino acid in the sequence, methionine (which ATG codes for)

without particularly changing its character. On the other hand, the microbial host can cause the transfer product to remove the first methionine. Ready-made leukocyte interferon could be expressed together with a conjugated protein different from the conventional leader, the conjugate being specifically cleavable in an intra- or extracellular environment (see British Patent No. 2 007 6 76A). Finally, the fully developed interferon could be produced in conjunction with a microbial "signal" peptide that transports the conjugate to the cell wall, where the signal is cleaved off and the finished polypeptide is secreted. "Expression" of finished leukocyte interferon means bacterial or other microbial production of an interferon molecule that does not contain any glycosyl groups

or a presequence immediately associated with mRNA transfer of a human leukocyte interferon genome.

Special leukocyte interferon proteins of these have been defined with the help of determined DNA genes (figures 3 and 8) and deductive amino acid sequencing (figures 4 and 9). For these special interferons, in fact the entire family of leukocyte interferon proteins is included, genetic variations exist and occur naturally from individual to individual. These variations can be detected by (one) amino acid difference(s) in the overall sequence or by omissions, substitutions, insertions, inversions or additions of (one) amino acid(s) in this sequence. For each leukocyte interferon protein below, specific LelF A to LelF J relate to such hereditary variations-

Description of the drawings

Figure 1 indicates 2 amino acid sequences which were common to all interferon species isolated from human leukocytes and purified to a homogeneous state called T-1 and T-13. All potential mRNA sequences that code for these peptides are shown as well as the corresponding DNA sequences. The letters A, T, G, C and U respectively denote the nucleotides containing the bases adenine, thymine, guanine, cytosine and uracil. The letter N denotes any of the nucleotides A, G, C and U. Polynucleotides are indicated read from the 5' (left) to the 3' (right) direction, and where double-stranded ("d.s.") DNA is indicated, etc. -versa for the base or non-coding chain. Figure 2 is an autoradiogram showing hybridization of potential LelF plasmids with P-labeled synthetic deoxyoligonucleotides. Figure 3 shows the nucleotide sequence (coding chains) of 8 gene fragments isolated as candidates for use in the expression of leukocyte interferons, designated "A" to "H" respectively. The ATG transfer initiation codon and termination triplet for each LelF are underlined. The stop codons or termination triplets are followed by 3' untranslated regions. The complete insert for LelF A lacks a codon found in the others shown, as indicated in the third A line in fig. 3. 5' untranslated regions precede the leader sequences. In isolation, fragment E lacks the complete presequence of leader, but contains the complete gene for the presumed finished LelF E. Fragment G, in isolation, lacks the full coding sequence. Figure 4 is a comparison of the 8 LelF protein sequences determined from nucleotide sequences. The one-letter abbreviations recommended by the IUPAC—IUB Commission on Biochemical Nomenclature are used: A, alanine, C, cysteine, D asp-artic acid, E, glutamic acid, F, phenylalanine, G, glycine, H, histidine, I, isoleucine, K, lysine, L, leucine, M, methionine, N, aspargine, P, proline, Q, glutamine, RT arginine., S, serine, T, threonine, V, valine, W, tryptophan and Y, tyrosine. The numbers refer to amino acid positions (S refers to signal peptide). The hyphen in the 165 amino acid LelF A sequence at position 44 is introduced to align the LelF A sequence with the 166 amino acid sequence of the other LelFs. The LelF E sequence was determined by ignoring the extra nucleotide (position 187 in Fig. 3) in the coding region. The arrow indicates in-phase termination codons. Amino acids common to all LelFs (except pseudogene LelF E) are also shown. The underlined residues are amino acids also present in human fibroblast interferon. Figure 5 shows restriction endonuclease maps for the 8 types of LelF cloned cDNAs (A to H). The hybrid plasmids were constructed by the dC:dG construction method (Goeddel, D.V. et al, Nature 287, 411-416 [1980]. Therefore, cDNA inserts can be made using Pstl. The lines at the end of each cDNA insert indicate the flanking homopolymeric dC:dG tails. The positions of PvuII, EcoRI and Bglll restriction sites are indicated. Shaded areas in the figure represent coding sequences for finished LelFs, and the shaded areas indicate signal peptide coding sequences, and the open area shows -

3' and 5' non-coding sequences.

Figure 6 schematically shows the construction of a gene encoding the direct microbial synthesis of finished LelF A. Re-lined ion points and residues are as shown ("Pstl", etc). The term "b.p." denotes "base pairs". Figure 7 (not graded) schematically shows a restriction map of two gene fragments used in the expression of finished leukocyte interferon LelF B. The codon sequences indicated are the coding band termination resulting from degradation with the restriction enzyme Sau3a in the two cases shown. Figures 8 and 9 give the DNA and amino acid (see fig. 4 above with regard to the corresponding one-letter abbreviations) sequences for five LelF proteins thereof, including types I and J. In fig. 9, the asterisk indicates a stop codon in the corresponding DNA sequence and the hyphen an omission or gap in the sequence.

DESCRIPTION OF PREFERRED EMBODIMENTS

A. Microorganisms used

The work described herein included the use of 2 microorganisms: E. coli x 1776 as described in U.S.P. 4,190,495 and E. coli K-12 strain 294 (end A, thi , hsr , hsm*), as described in British Patent No. 20 55382 A. Each had been deposited with the American Type Culture Collection (ATCC No. 31537 respectively 31446). All recombinant DNA work was performed in accordance with the applicable guidelines from the National Institutes of Health. When using plasmids according to the invention, which plasmids are specified in claim 1's characterizing part, for expression of fully developed human leukocyte interferon, E. coli is preferably used, including not only the strains E. coli x 1776 and E. coli K-12 strain 294 which are indicated above, but also other known E. coli strains, such as E. coli B. ;B. Source and purification of LelF mRNA; LelF mRNA can be prepared from human leukocytes, normally those from patients with chronic spinal leukemia, which leukocytes have been induced to produce interferon with Sendai or Newcastle disease virus as described in e.g. DOS No. 2947134. A particularly preferred source, and the one used in the present work, is a cell line called KG-1 from a patient with acute myeloid leukemia. The cell line described by Koeffler, H.P. and Golde, D.W., Science 200, ;1153 (1978), grows well in a culture medium comprising RPMI (Rosewell Park Memorial Institute) 1640 plus 10% heat-inactivated FCS (fatal calf serum), 25 mM HtiPES buffer (N-2-hydroxyethyl -piperazine-N'-2-ethanesulfonic acid) and 50 µg/ml gentamicin, and subcultivated 1 to 3 possibly twice a week. Cells can be frozen from the aforementioned growth medium plus 10% dimethylsulfoxide. KG-1 is deposited with the American Type Culture Collection (ATCC No. CRL 8031). KG-1 cells were induced to produce leukocyte interferon mRNA with Sendai or Newcastle disease virus following the method described by Rubinstein et al. (Proe. Nati. Acad. Sei. U.S.A. 76, 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, 5294-5299 [1979]). RNA from non-induced cells was isolated in the same way. Oligodeoxythymidine (dT) - cellulose chromatography and sucrose gradient ultracentrifugation were used to obtain the 12S fraction of poly (A) ;mRNA as described by Green et al. (Arch. Biochem. Biophys. ;172, 74-89 [1976] and Okuyuma et al. (Arch. Biochem. Biophys. 188, 98-104 [1978]). This mRNA had an interferon potency of 8000- 10,000 units per microgram in the Xenopus laevis oocyte assay (Cavalieri et al., Proe. Nati. Acad. Sei. U.S.A. 74, 3287-3291 [1977]). ;C. Preparation of colony banks containing LelF cDNA ;sequences ;5 mRNA was used for the production of double-stranded cDNA by standard methods (Wickens et al., J. Biol. Chem. 253. 2483-2495 [1978] and Goeddel et al., Nature 281, 544-548 ; [1979]). cDNA was fractionated by size by electrophoresis on a 6% polyacrylamide gel and 230 ng of material ranging in size from 500 to 1500 bp was isolated by electroelution. A 100 ng portion of this cDNA was appended with deoxycytidine (dC) residues as described by Chang et al., Nature 275. 617-624 (1978), and was treated with 470 ng of plasmid pBR3 22 which had been appended deoxyguanosine (dG) residues at the Pstl point (Bolivar et al., Gene 2, 95-1 13 ; [1977]), and used to transform E. coli x 1776. About 130 tetracycline-resistant, ampicillin-sensitive transformants were obtained per ng cDNA. In a second similar experiment, approx. 1000 tetracycline-resistant, ampicillin-sensitive E. coli K-12 strain 294 transformants per ng cDNA. In this case, size-fractionated cDNA material varying in size from 600 to 1300 b.p. was isolated. by electroelution for dC termination. D. Preparation of synthetic oligonucleotides and their use The knowledge of the amino acid sequence of several tryptic fragments of human leukocyte interferon enabled the construction of synthetic deoxyoligonucleotides complementary to different regions of LelF mRNA. The two tryptic peptides T1 and T13 were chosen because they had amino acid sequences that required the synthesis of only 12 and 4 undecamers, respectively, to fulfill all possible coding sequences (Figure 1). Four sets of deoxyoligonucleotide samples were synthesized for each sequence containing either three (T-1A, B, C, ;D) or one (T-13A, B, C, D) oligonucleotides each. The indicated complementary deoxyoligonucleotides, 11 bases long, were synthesized chemically by the phosphotriester method (Crea et al., Proe. Nati. Acad. Sei. U.S.A. IS, 5765-5769 [1978]). ;Four individual samples were produced in the T-13 series. The twelve T-1 samples were prepared in four stores of three samples as shown in fig. 1. The four individual samples of the T-13 series and the twelve T-1 samples, prepared in four stocks of three primer livers, were used to prime the synthesis of radioactively labeled single-stranded cDNA for use as hybridization samples. Corresponding mRNA was either 12S RNA from Sendai-induced KG-1 cells (8000 units of IF activity per pg) or total poly (A) mRNA from non-induced leukocytes (< 10 units per pg). ;32 ;P-labeled cDNA was prepared from these primers using ;known reaction conditions (Noyes et al., Proe. Nati. ;Acad. Sei. U.S.A. 7_6, 1770-1774 [1979]). 60 µl reaction samples were added in 20 mM Tris-HCl (pH 8.3), 20 mM KCl, 8 mM MgCl 2 , 30 mM 13-mercaptoethanol. The reactions included one µg for each primer (ie 12 µg total for T-1 series, 4 µg total for T-13 series), 2 µg for "induced" 12S fractions mRNA (or 10 µg for non-induced poly (A) mRNA), 0.5mM dATP, dCTP, dTTP, 200 uCi (a<32> P)dCTP (.2-3000 Ci/mmol), among others 60 units of reverse transcriptase. Product was separated from unlabeled material by gel filtration on a 10 ml Sephadex® G-50 column treated with 0.3N NaOH for 30 min. at 70°C to destroy RNA, and neutralized with HCl. Hybridizations were performed as described by Kafatos et al., Nucleic Acids Res. 7, 1541-1552 (1979). ;E. Identification of clones pL1-pL30; The rapid plasmid isolation method of Birnboim et al., Nucleic Acids Res. 7, 1513-1523 (1979), was used to prepare 1 µg of plasmid DNA from each of 500 individual E. coli K-12 strain 294 transformants (see C). Each DNA sample was denatured and applied to nitrocellulose filters in triplicate following the method of Kafatos et al. (see above). ;The three sets of nitrocellulose filters containing the 500 plasmid samples were hybridized with ;a) induced cDNA primed with the T-1 set of primers, ;b) T-13 primed induced cDNA, and ;c) Non-induced cDNA prepared using both set ;primers. Clones were considered positive if they hybridized more strongly to one or both of the induced cDNA samples than to the total uninduced sample. Twenty-two "positive" clones (pL1-pL30) were selected from the 500 for further analysis. F. Identification of clones pL31- pL39 ;Isolation of a plasmid (no. 104) containing a LelF ;gene fragment ;Transformants of E. coli x 1776 were examined by the colony hybridization method of Grunstein and Hogness (Proe. Nati. Acad. Sei. U.S.A. 12_ , 3961-3965 [1975] using <32>P-labeled induced mRNA as a sample (Lillenhaug et al., Biochemistry, 1J5, 1858-1865 [1976]). Unlabeled mRNA from uninduced cells was mixed with samples at a ratio of 200 to 1 to compete with uninduced mRNA that ;32 ;was present in the P-labeled preparation. Hybridization of the labeled mRNA should preferentially occur in colonies containing induced sequences. Three classes of transformants were obtained: ; 32 (1) 2-3% of the colonies hybridized very strongly to P-mRNA, ;(2) 10% hybridized significantly less than class 1, and ;(3) The rest gave no detectable hydridization signal. ;The positive colonies (class ( 1) and (2)) were examined for the presence of interferon-specific sequences through a investigation that depends on hybridization of interferon mRNA specifically to plasmid DNA. First, 60 strong positive colonies (class 1) were grown individually in 100 ml of M9 medium supplemented with tetracycline (20 ug/ml), diaminopimelic acid (100 ug/ml), thymidine (20 ug/ml), and d-biotin ( 1 µg/ml). The M9 medium contained per liter Na2HPC>4 (6g) , KH2P04 (3g) , ;NaCl (0.5g) and NH4C1 (1g). After autoclaving, 1 ml of sterile IM MgSO4 and 10 ml of sterile 0.01 M CaCl2 were added. Ten cultures were pooled and plasmid DNA isolated from the six pools ("pools") as described by Clewell et al., Biochemistry 9, 4428-440 [1970]. Ten µg of each plasmid DNA stock was digested with HindIII, denatured and covalently bound to DBM (diazobenzyloxymethyl) paper. A pg of purified mRNA from induced cells was hybridized to each filter. Unhybridized mRNA was removed by washing. The specifically hybridized mRNA was extracted and transferred into Xenopus laevis oocytes. In this sample, all 6 stores were negative. 5 stocks of 10 colonies each and one stock of 9 colonies each were prepared from 59 weakly positive colonies, (class 2) and plasmids were prepared from the stocks and examined as above. Among the 6 pools examined, one (K10) hybridized to interferon mRNA' at levels significantly higher than background levels every time it was tested. To identify the specific interferon cDNA clone, plasmid DNAs were prepared from the 9 colonies of stock K10 and examined individually. 2 of the 9 plasmids (no. 101 and no. 104) bound interferon mRNA well above the background level. From plasmid No. 104, a unique Bglll restriction fragment containing 260 ;3 2 ;b.p. isolated, labeled with P using the method described by: Taylor et al., Biochim. Biophys. Acta 44 2, 324-330 (1976), and used as a sample to independently test 400 E. coli 294 transformants by an in situ colony assay method (Grunstein and Hogness, Proe. Nati. Sei. U.S.A. 72, 3961 -3965 [1975]). 9 colonies (pL31-pL39) which hybridized to varying degrees with this sample were identified. ;Additionally, 260 b.p. was marked. fragment used to independently test 4,000 E. coli 294 transformants in the same manner. ;50 colonies were identified as hybridizing to varying degrees with this sample. One contained the LelF G fragment, one contained the LelF H fragment and one contained a fragment called ;LelF Hl, an obvious allele of LelF H. The resulting hybrid plasmids are called "pLelF H", etc. ;G. Isolation and sequencing of a first full-length ;LelF gene ;Plasmid DNA was prepared from all 39 potential LelF cDNA clones and tested again with the same 260 b.p. DNA sample using the hybridization method of Katatos et al. (see above). Three plasmids (pL4, pL31, pL34) gave very strong hybridization signals, 4 (pL13, pL30, pL32, pL36) hybridized moderately, and 3 (pL6, pL8, pL14) hybridized weakly with the sample. ;The 39 potential LelF cDNA recombinant plasmids were also ;32 ;tested using P labeled synthetic undecamers (individual T-1 primer stocks or individual T-13 primers) directly as hybridization probes. The hybridization conditions were chosen so that perfect base pairing would be required for detectable hybridization signals (Wallace et al., Nucleic Acids Res. 6, 3543-3557 [1979]). Thus, plasmid DNA from 39 clones was prepared through a standardized lysate method (Clewell et al., see above) and purified by Biorad Agarose A-50 column chromatography. Samples (3 µg) of each preparation were linearized by treatment with Eco RI, denatured in alkali and deposited on 2 separate nitrocellulose filters, 1.5 µg per deposition (Kafatos et al., see above). Individual synthetic deoxyoligonucleotide primers and ;32 ;primer pools were phosphorylated with (γ P)ATP as follows: ;32 50 pmol oligonucleotide and 100 pmol (γ P)ATP (New England Nuclear, 2500 Ci/mmol) were combined in 30 µl 50mM Tris-HCl, 10 mM MgCl 2 , 15 mM &-mercaptoethanol. 2 units of T4 polynucleotide kinase were added, and after 30 min. at 37°C, 32 <32>P-m,erked primers were purified by chromatography on 10 ml Sephadex R G-50 columns. Hybridizations were made with 10 3 cpm of primer T-13C or 3 x 10<6> cpm of primer stock T-1C. The hybridizations were performed at 15°C for 14 h in 6 x SSC [1 x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.2], 10 x Denhardfs [0.2% bovine serum albumin, 0.2% poly-polyvinylpyrrolidone, 0 .2% Ficoll] solution, as described by Wallace et al (see above). Filters were washed for 5 min. ;(3 times) at 0°C in 6 x SSC, dried, and exposed to x-ray film. The results are shown in fig. 2 for " 32 P primer pool T-13C and primer T-1C. Plasmid DNA from clone 104 was found to give significant hybridization with primer pool T-1C and primer T-13C, but no detectable hybridization with the other undecamers. As shown in Figure 2" several of the 39 potential LelF plasmids (pL2, 4, 13, 17, 20, 30, 31, 34) also hybridized with both of these samples. However, restriction analysis showed that only one of these plasmids, pL31, also contained a 260 bp internal BglII fragment. Pstl treatment of pL31 showed that the size of the cDNA insert was approximately 1000 bp. The entire Pstl insert of pL31 was sequenced by both Maxam-Gilbert's chemical method (Methods Enzymol. 65, 4 99 -560 ; [1980] and the dideoxy chain termination method (Smith, Methods Enzymol. 6_5, 560-580 [1980]) after subcloning Sau3a fragments into an M13 vector. The DNA sequence is shown ("A") in Fig. 3. The correct translation frame could is predicted under control from the protein sequence information, via the known range of LelF molecular weight is and the relative occurrence of stop triplets. in the 3 possible reading frames, and it again made it possible to predict that the LelF amino acid sequence included a pre- or signal peptide. 1 the first ATG transfer initiation codon is found 60 nucleotides from the 5' end of the sequence and follows, 188 codons later, of a TGA termination triplet; where there are 342 non-translated nucleotides at the 3' end, followed by a poly (A) sequence. The presumed signal peptide (presumably involved in the secretion of finished LelF from leukocytes) is 23 amino acids long. The 165 amino acids that make up the finished LelF have a calculated molecular weight of 19 3 90. LelF, which is encoded by pL31, is called "LelF A". One can see from the sequence data ("A") in fig. 4 that the tryptic peptides T1 and T13 in LelF B correspond to amino acids 145 - 149 and respectively 57 to 61 of LelF A. The actual DNA coding sequence found in these 2 regions are those indicated by the primer supply Tl-C and primer T13-C (see fig. 8). H. Direct expression of fully developed leukocyte interferon A (LelF A) 1. In general, the procedure followed to express LelF A directly as a finished interferon polypeptide is a variant of that previously used for human growth hormones (Goeddel et al., Nature 281, 544-548 [1979]), provided that the in - involves the combination of synthetic (N-terminal) and complementary DNAs. ;As shown in fig. 6, a Sau3a restriction endonuclease site readily locates between codons 1 and 3 of LelF A. 2 synthetic deoxyoligonucletides were constructed that contain an ATG transfer initiation codon, rendering the codon for amino acid 1 (Cysteine), creating an EcoRI binding terminus. These oligomers were bound to a 34 b.p. Sau3a - Avall fragment of pL31. The resulting 45 b.p. product was ligated to 2 additional DNA fragments to construct an 865 b.p. synthetic-natural hybrid gene encoding LelF A and delimited by EcoRI and Pstl restriction sites. This gene was inserted into pBR322 between the EcoRI and Pstl points and gave the plasmid pLelF Al. 2. Construction of the tryptophan control element (containing the E. coli trp promoter, operator and trp leader ribosome binding site but lacking an ATG sequence for initiation of transfer); Plasmid pGM1 carries the E. coli tryptophan operon containing the deletion &LE1413 (Miozzari et al., J. Bacteriology 133, 1457-1466 [1978] and consequently expresses a fusion protein containing the first 6 amino acids of the trp leader and approximately the last third of the trp E polypeptide (hereinafter referred to in context as LE') , as well as the trp D polypeptide in its entirety, all under the control of the trp promoter-operator system. The plasmid, . 20 } iq, was treated with the restriction enzyme PvuII which cleaves the plasmid at 5 sites. Gene fragments were then combined with EcoRI linkers (consisting of a self-complementary oligonucleotide with the sequence: pCATGAATTCATG) which provided an EcoRI cleavage point for a later cloning into a plasmid containing an EcoRI site. The 20 µg DNA fragments obtained from pGM1 were treated with 10 units of T4 DNA ligase in the presence of 200 pmol 5<1->phosphorylated synthetic oligonucleotide pCATGAATTCATG and in 20 µl T4 DNA ligase buffer (20 mM Tris, pH 7.6, 0.5 mM ATP , 10 mM MgCl2, 5 mM dithiothreitol) at 4°C overnight. The solution was then heated for 10 min. at 70°C to stop the ligation. The chains were cleaved by EcoRI treatment and the fragments, now with EcoRI ends, were separated using 5% polyacrylamide gel electrophoresis (hereafter PAGE), and the 3 largest fragments, isolated from the gel by first treatment with ethidium bromide, localized the fragment with ultraviolet light, and the parts of interest were cut from the gel. Each gel fragment together with 300 microliters of 0.1 x TBE, was placed in a dialysis bag and subjected to electrophoresis at 100 V for one hour in 0.1 x TBE buffer (TBE buffer contains: 10.8 g Tris base, 5.5 g boric acid, 0.09 g Na2EDTA in 1 liter H20). The aqueous solution was collected from the dialysis bag, phenol extracted, chloroform extracted and made 0.2 M sodium chloride, and DNA was isolated in water after ethanol precipitation. The trp promoter-operator-containing gene with EcoRI-binding ends was identified in the next described method, which involves the introduction of fragments into a tetracycline-sensitive plasmid which, after promoter-operator insertion, becomes tetracycline resistant. Plasmid pBRHI (Rodriguez et al., Nucleic Acids Res. 6, 3267-3287 [1979]) expresses ampicillin resistance and contains the gene for tetracycline resistance, but, as it is not associated with any promoter, does not express that resistance. The plasmid is therefore tetracycline sensitive. By introducing a promoter-operator system at the EcoRI point, the plasmid can be made tetracycline resistant. pBRHI was treated with EcoRI and the enzyme removed by phenol extraction followed by chloroform extraction and isolated in water after ethanol precipitation. The resulting DNA molecule was separated into reaction mixture is combined with each of the 3 DNA fragments obtained above and ligated with T4 DNA ligase as previously described. The DNA present in the reaction mixture was used to transform competent E. coli K-12 strain 294 through standard techniques (Hersh-field et al., Proe. Nati. Acad. Sei. U.S.A. 71, 3455-3459 ; [1974]) and the bacteria plated on LB (Luria-Bertani) plates containing 20 µg/ml ampicillin and 5 ug/ml tetracycline. Several tetracycline-resistant colonies were selected, plasmid DNA isolated and the presence of the desired fragment confirmed by restriction enzyme analysis. The resulting plasmid is called pBRHtrp. ;An EcoRI and BamHI treatment product of the viral genome of hepatitis B was obtained by conventional means and cloned into the EcoRI and BamHI sites of plasmid pGH6 (Goeddel et al., Nature 281, 544 [1979]) yielding plasmid pHS32. Plasmid pHS 32 was digested with XbaI, phenol extracted, chloroform extracted and ethanol precipitated. It was then treated with 1 µl E. coli DNA polymerase I Klenow fragment in 30 µl polymerase buffer (50 mM potassium phosphate pH 7.4, 7 mM MgCl2, 1 mM (3-mercaptoethanol) containing 0.1 mM dTTP and 0.1 mM dCTP for 30 min .at 0°C and then 2 hours at 37°C. This treatment causes 2 of the 4 nucleotides complementary to the 5' overhang of the XbaI cleavage site to fill in: ;;2 nucleotides, dC and dT, was inserted, giving an end with 2;5' overhanging 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 µg), was ligated under conditions similar to those described above to the EcoRI-Taq1 fragment of the tryptof an operon (~0.01 µg), derived from pBRHtrp. ;In the procedure for ligation of the fragment from pHS32 to ;EcoRI - Taql fragm either as described above, the Taq1 protruding end is ligated to the Xbal remaining end, although not the complete Watson-Crick base pair: ;A portion of this ligation reaction mixture was transferred to E. coli 294 cells, heat-treated and plated on LB plates containing ampicillin. 24 colonies were selected, grown in 3 ml LB (Luria-Bertani) media, and plasmid isolated. 6 of these were found to have the XbaI position regenerated via E. coli catalyzed DNA repair and replication: ;These plasmids were also found to cleave ;both with EcoRI ;and HpaI and give the expected restriction fragments. A plasmid named pTrpl4 was used for expression of heterologous polypeptides as discussed below. ;The plasmid pHGH 107 (Goeddel et al., Nature 281, 544 [1979] contains a gene for human growth hormone built up from 2 3 amino acid codons produced from synthetic DNA fragments and 163 amino acid codons obtained from complementary DNA produced through reverse transcription of human growth hormone messenger RNA . This gene, although lacking the codons for the "pre" sequence of human growth hormone, contains an ATG translation initiation codon. The gene was isolated from 10 µg pHGH 107 after treatment with EcoRI followed by 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 'human growth hormone (HGH)' gene-containing fragment was isolated by PAGE followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the tetracy. clin-resistant structural gene," but lacks the tet-cyclin promoter operator system, so that upon subsequent cloning in a expression plasmid, the plasmids containing the insert can be located by restoring the tetracycline resistance..: Because the EcoRI end of the fragment is inserted through the Klenow polymerase I method, the fragment has a blunt and an attached end, which ensures correct orientation at later insertion into an expression plasmid. The expression plasmid pTrpl4 was then prepared to receive the HGH gene containing fragment prepared above. Thus, Trpl4 XbaI was processed and the resulting sticky ends filled in by the Klenow polymerase I method using dATP, dTTP, dGTP and dCTP. After phenol and chloroform extraction and ethanol precipitation, the resulting DNA was treated with BamHI and the resulting large plasmid fragment isolated by PAGE and electroelution. The pTrpl4-derived fragment has a blunt and an attached end which enables recombination in the correct orientation with the HGH gene-containing fragment previously described. The ;HGH gene fragment and the pTrpl4 &Xba-BamHI fragment were combined and joined under similar conditions to those described above. The inserted XbaI and EcoRI ends were ligated together by butt-end ligation, recreating both the XbaI and EcoRI sites. ;This construct also recreates the tetracycline-resistant gene. As the plasmid pHGH 107 expresses tetracycline resistance from a promoter located upstream of the HGH gene (the lac promoter), which construction is called pHGH 207, this enables expression of the gene for tetracycline resistance under the control of the tryptophan promoter operator. Thus, the ligation mixture was transferred into E. coli 294 and colonies selected on LB plates containing 5 µg/ml tetracycline. ;Plasmid pHGH 207 was EcoRI treated and a 300 b.p. fragment containing the trp promoter, operator and trp leader-riboson binding site, but lacking an ATG sequence for initiation of translation, was isolated from PAGE followed by electroelution. This DNA fragment was cloned into the EcoRI site of pLelF A. Expression plasmids containing the above modified trp regulon (the E. coli trp operon from which the attenuator sequence has been removed to controllably increase expression levels) can be grown to predetermined levels in a nutrient medium. containing tryptophan in sufficient amounts to repress the promoter-operator system, then tryptophan is stripped to turn the system back on and cause expression of the intended product. More precisely, and with reference to fig. 6, 250 µg of plasmid pL31 was treated with Pstl and 1000 b.p. the insert, isolated by gel electrophoresis on a 6% polyacrylamide gel. About. ;40 µg insert was electroeluted from the gel and divided into 3 aliquots for further processing: a) A 16 µg sample of this fragment was partially treated with 40 units of Bglll for 45 min. at 37°C and the reaction mixture purified on a 6% polyacrylamide gel. About 2 µg of the desired 670 bp fragment was isolated. b) Another sample (8 pg) of the 1000 bp Pstl insert was cleaved with Avall and Bglll. One pg of the indicated 150 bp fragment was isolated after gel electrophoresis. c) .16 pg of the 1000 bp fragment was treated with Sau3a and AvalI. After electrophoresis on a 10% polyacrylamide gel, approximately 0.25 pg (10 pmol) of the 34 bp fragment was isolated. The two indicated deoxyoligonucleotides, 5'-dAATTCATGTGT ( fragment 1) and 5'-dGATCACACATG (fragment 2) were synthesized by the phosphotriester method. Fragment 2 was ;32 phosphorylated as follows. 200 µl (<<v4 0 pmol) of (γ P) ATP (5000 Ci/mmol) was dried oa resuspended in 30 µl of 60 mM Tris-HCl (pH8), lOmM MgCl2, 15 mM (3-mercaptoethanol, containing 100 pmol DNA fragment and 2 units of T4 poly-nucleotide kinase. After 15 min. at 37°C, 1 µl of lOmM ATP ; added and the reaction allowed to continue for a further 15 min. The mixture was then heated at 70°C for 15 min., combined with 100 pmol of 5'-OH fragment 1 and 10 pmol of 34 b.p. S au3a - The Avall fragment. Ligation was carried out for 5 hours at 4°c in 50 µl 20mM Tris-HCl (pH7.5] lOmM MgCl2, 10mM dithiothreitol, 0.5mM ATP and 10 units of T4 DNA ligase. The mixture was electrophoresed on a 6% polyacrylamide gel and 4 5 bp product isolated by electroelution. About 30 ng (1 pmol) of the 45 bp product was combined with 0.5 µg (5 pmol) of the 150 bp Avall - Bglll fragment and 1 pg (2 pmol) of the 670 bp Bglll - Pstl fragment. The ligation was performed at 20°C for 16 hours using 20 units of T4 DNA ligase. The ligase was inactivated by heating to 65°C for 10 min. The mixture was then treated with EcoRI and Pstl to remove the polymers from the gene. The mixture was purified by 6% PAGE. ;Approximately 20 ng (0.04 pmol) of 865 bp product was isolated.Half (10 ng) of this was ligated into pBR322 (0.3 µg) between the EcoRI and Pstl sites.Transformation of E. coli 294 ;ga 70 tetracycline-resistant, ampicillin-sensitive transformants. Plasmid DNA isolated from 18 of these transformants was treated with EcoRI and Pstl. 16 a v the 18 plasmids had an EcoRI - Pstl fragment 865 b.p. in length. One pg of one of these, pLelF Al, was treated with EcoRI and ligated to 300 bp. The EcoRI fragment (0.1 pg) containing the E. coli trp promoter and the trp leader ribosome binding site, prepared as described above. Transformants containing the trp promoter were further determined using a <32>P-trp assay in conjunction with the Grunstein-Hogness colony-picking method. An asymmetrically positioned XbaI point in the trp fragment enabled the determination of recombinants in which the trp promoter was oriented in the direction of the LelF A gene. ;IN. In vitro and in vivo activity of LelF A ; Extracts were prepared for IF determination as follows: 1 ml cultures were grown in L medium containing 5 pg/ml tetracycline to an A^q value of approx. 1.0, then diluted to 25 ml M9 medium containing 5 pg/ml tetracycline. 10 ml samples were withdrawn by centrifugation when A^q reached 1.0, and cell pellets were resuspended in 1 ml 15% sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA. 1 mg of lysozyme was added, and after 5 min. at 0°C cells were lysed by ultrasound. The samples were centrifuged for 10 min. (15,000 pmdr./min.) and the interferon activity in the supernatants was determined by comparison with LelF standards by the cytopathic effect (CPE) inhibition measurement. To determine the number of IF molecules per cell, a LelF specific activity of 4 x loo units/mg was used. As shown in Table 1, clone pLelF A trp 25, in which the trp promoter was inserted in the desired orientation, gives high levels of activity (as high as 2.5 x 10 g units/liter). As shown in Table .2, IF produced by E. coli K-12 strain 294/pLeIF A trp 25 acts similarly to authentic human LelF, and it is stable when treated at pH 2 and is neutralized by rabbit anti-human leukocyte antibodies. The interferons have an observed molecular weight of approx. 20,000. The in vivo activity of interferon requires the presence of macrophages and natural killer (NK) cells, and the in vivo action appears to involve stimulation of these cells. Thus, it remained possible that the interferon produced by E. coli 294/pLeIF A 25, while having anti-viral activity in the cell culture assay, would not be active in infected animals. Furthermore, the in vivo antiviral activity of the bacterially produced non-glycosylated LelF A could be different from the glycosylated LelF from human "buffy coat" leukocytes. Therefore, the biological activity of bacterially synthesized LelF A (2% pure) was compared with buffy coat LelF (8% pure) in the lethal encephalomyocarditis (EMC) virus infection of squirrel monkeys (Table 3). ;<*> 250,000 units/ml extract of E. coli 294/pLeIF A trp 25 described in Table 1 was diluted 500 times with minimal essential medium giving a specific activity of 500 units/ml. A leukocyte interferon standard (Wadley Institute), previously titrated against the NIH leukocyte interferon standard, was also diluted to a final concentration of 500 U/ml. One ml aliquots were adjusted to pH 2 with IN HCl, incubated at 4°C for 52 h, neutralized by addition of NaOH and IF activity determined by standard CPE inhibition measurement. 25 µl aliquots of the 500 units/ml samples (untreated) were incubated with 25 µl rabbit anti-human leukocyte interferon for 60 min. at 37°C, centrifuged at 12,000 x g for 5 min. and the supernatant measured.

All monkeys were male (mean weight 713 g) and had no EMC virus antibodies prior to infection. The monkeys were infected intramuscularly with 100 x LD,-,, EMC virus (determined in

oU

mouse). Control-treated monkeys died 134, 158 and 164 hours after infection. Interferon treatments with 10^ units intravenously were performed at -4, +2, 23, 29, 48, 72, 168 and 240 hours, relative to the infection. The bacterial leukocyte interferon was a column chromatography fraction from a lysate of E. coli 294/pLeIF A 25 with a specific activity of 7.4 x 10^ units/mg protein. The control bacterial proteins were an equivalent column fraction from a lysate of E. coli 294/pBR322 at 2 times the total protein concentration. The leukocyte interferon standard was Sendai virus induced interferon from normal human "buffy coat" cells, purified chromatographically to a specific activity of 32 x 10 units/mg protein.

The control monkeys showed increasing lethargy, loss of balance, flaccid paralysis of the hind limbs and watering of the eyes which began approx. 8 hours before, dead. Interferon-treated monkeys showed none of these abnormalities and remained active throughout and did not develop viremia. The one monkey in the control group that did not develop viremia after 4 days died at the latest (164 hours after infection), but showed high concentrations of virus

in heart and brain after death. The interferon-treated monkeys did not develop antibodies against EMC virus when measured 14 and 21 days after infection. These results show that the antiviral effects of the LelF preparations in the infected animals can be attributed to interferon alone because the accompanying proteins are completely different in the bacterial and in the "buffy coaf" preparations. In addition, these results show that glycosylation is not necessary for in vivo the antiviral activity of LelF A.

J. Isolation of cDNAs for additional leukocyte interferons

DNA from the fully characterized LelF A cDNA-containing plasmid was digested with Pstl, isolated electrophoretically and labeled with 3 2P. The resulting radiolabeled DNA was used as a sample to select additional E. coli

2 94 transformants, obtained identically to those in part C, by the in situ colony selection method of Grunstein and Hogness

(see ahead). Colonies were isolated that hybridized to varying degrees with the sample. Plasmid DNA from these colonies and the 10 hybridizing colonies discussed in Part G above was isolated by Pstl cleavage and characterized through 3 different methods. First, these Pst fragments were characterized by their restriction endonuclease treatment patterns with the enzymes Bgl II, Pvu II and Eco RI. This analysis enabled the classification of at least 8 different types (LelF A, LelF B, LelF C, LelF D, LelF E, LelF F, LelF

G and LelF H), shown in Figure 5, which approximately indicate the location of various restriction positions in relation to those known for sequence and coding sequence of LelF A. One of these, LelF D, is of the same type as that reported by Nagata et al., Nature, 28_4, 316-320 (1980), but has a Val residue at position 114, while that indicated by Nagata has an Ala residue at this position.

Second, certain of the DNAs were tested by the hybridization-selection assay described by Cleveland et al., Cell 20, 95-105 (1980) for the ability to selectively remove LelF mRNA from poly-A-containing KG-1 cell RNA. LelF A, B, C and F were positive in this determination. Third, the latter Pst fragments were inserted into an expression plasmid, E. coli 294 was transferred with the plasmid, and the fragments were expressed. The expression products believed to be preinterferons were all positive in the CPE assay for interferon activity, although the activity was marginal in the case of the LelF F fragment. In addition to the foregoing, all the described LelF types are sequence determined.

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

The sequence of the isolated fragment consisting of the gene for finished LelF B shows the first 14 nucleotides of types A and B as identical. Accordingly, a fragment from pLelF A25 carrying the trp promoter operator, ribosome binding site and the start of the LelF A (=B) gene was isolated and combined with the remaining part of the B sequence in an expression plasmid.

The conspicuous restriction map for the Pst fragment of pL4

(a plasmid consisting of LelF B Pst terminated gene indicated in Fig. 5) and pLelF A25 are respectively shown in Fig. 7a and 7b.

To achieve the approximate 950; b.p. Sau3a to Pstl fragment from the sequence shown in fig. 7a, additional steps were necessary because one or more intervening Sau3a restriction sites were present, i.e.:

1. The following fragments were isolated:

a) 110b b.p. from Sau3a to EcoRI; b) 132 b.p. from EcoRI to Xba;

c) >700 b.p. from Xba to Pst.

2. Fragments (la) and (lb) were ligated and cleaved with Xba and

Bglll to prevent self-polymerization through Sau3a and Xba end caps (the relevant Sau3a point was within a Bglll point; Bglll parts to leave a Sau3a attaching end). A 242 b.p. fragment was isolated.

3. The product from (2) and (lc) was ligated and cleaved with Pstl

and Bglll, again to prevent self-polymerization. An approximate 950 b.p. fragment, Sau 3a to Pstl from fig. 7a, was isolated. This fragment consisted of the part of the LelF B gene that was not shared with LelF A.

4. An approximate 300 b.p. fragment (Hindlll to Sau3a)

consisting of trp promoter-operator, ribosome binding site, ATG start signal and cysteine codon of LelF A was isolated from pLelF A25.

5. An approximate 3600 b.p. fragment Pstl to Hindlll bla

isolated from pBR322. This included the replicon and encoded tetracycline but not ampicillin resistance.

6. The fragments obtained in steps 3, 4 and 5 were triple ligated and the resulting plasmid transferred into E. coli K-12 strain 2 94.'

Transformants were selected - (Birnboim et al., Nucleic Acids Res. 1_, 1513-1523 [1979] and plasmid samples cleaved

with EcoRI. The digestion gave 3 fragments characteristic of: 1) EcoRI-EcoRI trp promoter fragment; 2) The internal EcoRI to the EcoRI fragment from pL4, and 3) the protein translation start signal to the EcoRI fragment from pL4.

In the CPE determination, bacterial extracts from clones prepared in the previous way typically show approx. 10 x 10 units of interferon activity per liters at A55Q =1. A representative clone prepared in this way is E. coli 294/pLeIF B trp 7.

L. Direct expression of additional ferridimtykylated leukocyte interferons (LelF C, D, F, H, I and J)

Additional full-length gene fragments comprising other LelF types can be assembled and engineered into expression vehicles for expression as in the case of LelF A. Complete sequencing by conventional means will clarify whether a restriction site is sufficiently close to the first amino acid codon of the finished interferon type to enable easy use of the methodology used, in part H' above for the expression of finished LelF A, i.e. elimination of the presequence by restriction digestion and, replacement of codons for N-terminal amino acids that are lost, in presequence eliminationf by ligation of a synthetic DNA fragment. If: this does not work, the following method can be used. This includes cards. cleavage of the sequential fragment just before the "point" where the code is, the first amino acid of the finished polypeptide is obtained by:

1. the double-stranded DNA is transferred to single-stranded DNA

in an area surrounding this point,

2. to the single-stranded region formed in step (a), a complementary primer length of single-stranded DNA is hybridized, with the 5' end of the primer lying opposite the nucleotide which lies up to the intended cleavage point,

3. the part of the second chain which is eliminated in step 1, and which lies in the 3' direction from the primer by reaction with DNA polymerase is restored in the presence of adenine, thymine, guanine and cytosine-containing deoxynucleotide triphosphates, and

4. the remaining single-stranded length of DNA that protrudes beyond the intended cleavage point is degraded.

A short length of synthetic DNA ending at the 3•-end of the coding strand with the translation initiation signal ATG can then be ligated to, i.e. blunt end to, the resulting assembled gene for the finished interferons and the gene inserted into an expression plasmid and brought under the control of a promoter and its associated ribosome binding site.

In a similar manner as used in Part K above, gene fragments encoding LelF C and LelF D were generated for direct bacterial expression. The expression strategy for these additional leukocyte interferons involved in each case the use of the approximately 300 b.p. fragment HindIII to Sau3a) consisting of trp promoter operator, ribosome binding point ATG start signal and cysteine codon of LelF A from PLelF A25. For this, gene fragments from the additional interferon genes that code for their respective amino acid sequences past the first cysteine that was common to all were combined. Each resulting plasmid was used to transform E. coli K-12 strain 294. Ligaments to form the respective genes were as follows:

LelF C

Isolate the following fragments from pLelF C:

(a) 35 b.p. from Sau 3a to Sau 96

(b) >900 b.p. Sheep 96 to Pst I

(c) Isolate an approximately 300 b.p. fragment (Hind III -

Sheep 3a) from pLelF A-25 as in part K (4) above.

(d) Isolate it approximately 3600 b.p. fragment from part

K (5) before.

Structure:

(1) Lies (a) and (c). Cleave with Bglll, Hindlll and isolate the approximately 335 b.p. product. (2) Repeat (1) + (b) + (d) and transform E. coli with the resulting plasmid.

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

LelF D

Isolate from pLelF D:

a) 35 b.p. from Sau3a to Avall

b) 150 b.p. from Avall to Bglll

c) approximately 700 b.p. from Bglll to Pstl

Isolate from pLelF A25:

d) 300 b.p. from Hindlll to Sau3a

Isolate from pBR322:

e) approximately 3600 b.p. from Hindlll to Pstl Construction: (1) Liger (a) + (b), split with Bglll and clean a 185 b.p. product (1). (2) Liger (1) + (d), cleave with Hindlll, Bglll, and purify the approximate 500 b.p. product (2). (3) Liage (2) + (c) + (e) and transform E. coli with the resulting plasmid..

A representative clone prepared in this way is E. coli

K-12 strain 294/pLeIF D trp 11.

LelF F

The LelF F-containing fragment can be constructed for direct expression through reconstruction that is easily carried out completely homologous for amino acids 1-13 of LelF B and LelF F. A trp promoter-containing fragment (a) with approximately identical ends is obtained from pHGH207 described above via Pst I and Xba I cleavage, followed by isolation of the ca. 1050 b.p. fragment. A second fragment (b) is obtained as the largest of the fragments resulting from Pst I and Bglll cleavage of the plasmid pHKY 10. Fragment (a) contains approx. half of the gene encoding ampicillin resistance; fragment (b) contains the remainder of the gene and the entire tetracycline resistance gene except for the associated promoter. Fragments (a) and (b) are combined via T4 ligase and the product is treated with XbaI and BglII to eliminate dimerization, forming a fragment (c) comprising the trp promoter operator and genes for tetracycline and ampicillin resistance.

A fragment (d) of about 580 b.p. obtained by Avall and Bglll cleavage of pLelF F. This includes codons for amino acids 14-166 of LelF F.

A fragment (e) (49 b.p.) is obtained by XbaI and Avall cleavage of pLelF B. Fragment (e) encodes amino acids 1-13

by LelF F.

Fragments (c), (d) and (e) are triple ligated in the presence of

T4 ligase. The attaching ends of the respective fragments are such that the assembled plasmid circularizes correctly, bringing the tetracycline resistance gene under the control of the trp promoter operator together with the gene for finished LelF F, so that bacteria transformed with the desired plasmid can be selected on tetracycline-containing plates. A representative clone prepared in this manner is E. coli K-12 strain 294/ pLelF F trp 1.

LelF H

The complete LelF H gene can be engineered for expression as

a finished leukocyte interferon as follows:

1. Plasmid pLelF H is subjected to Haell and Rsal digestion by isolation of 816 b.p. the fragment extending from signal peptide amino acid 10 to the 3' non-coding region. 2. The fragment is denatured and subjected to repair synthesis with Keinow fragment of DNA polymerase I (Klenow et al., Proe. Nat. Acad. Sei. U.S.A. 65, 168 [1970], using the synthetic deoxyribooligonucleotide primer

5'-dATG TGT AAT CTG TCT.

3. The resulting product is cleaved with Sau3a, and a 452

b.p. fragment consisting of amino acids 1 to 150 is isolated.

4. Sau3a and Pstl digestion of pLelF H and isolation of the resulting 500 b.p. fragment gives a gene that codes for amino acids 150 to the end of the coding sequence. 5. Fragments isolated in steps (3) and (4) are ligated to form a fragment:

which encodes the 166 amino acids of LelF H.

6. pLelF A trp 2 5 is digested with XbaI, end blunted with DNA polymerase I and the product is cleaved with Pst I. The large resulting fragment can be isolated and ligated with

the product from step (5) and forms an expression plasmid which, after transformation of E. coli K-12 strain 294 or other host bacteria, is able to express fully developed LelF H.

LelF I

The Phage XCharon 4A recombinant material of the human genome engineered by Lawn et al., Cell 15, 1157 (1978), was screened for leukocyte interferon genes by the methods described by Lawn et al. (supra) and Maniatis et al., Cell 15, 687 (1978). A radioactive LelF sample derived from the cDNA clone LelF A was used to examine approx. 500,000 records. 6 LelF genome clones were obtained in this selection. After reut selection and plate purification, one of these clones, A.HLeIF2, was selected for further analysis.

Using the method described above, other samples can advantageously be used to isolate additional LelF clones from the human genome. These can in turn be used to produce further leukocyte interferon proteins according to this invention. 1. 2000 b.p. The EcoRI fragment from clone \HLeIF2 was sub-cloned into pBR325 at the EcoRI position. The resulting plasmid LelF I was digested with EcoRI7 and 2000 b.p. the fragment isolated. The deoxyoligonucleotide dAATTCTGCAG (an EcoRI- Pstl converter) was ligated to 2000 b.p. the EcoRI fragment, and the resulting product digested with PstI to give a 2000 b.p. fragment that contained Pstl ends. This was cleaved with Sau96, and a 1100 b.p. fragment was isolated that had a Pstl and a Sau96 end. 2. The plasmid pLelF C trp 35 was cleaved with PstI and XbaI. The large fragment was isolated. 3. The small XbaI - Pstl fragment from pLelF C trp 35 was cleaved with XbaI and Sau96. A 4 0 b.p. XbaI - Sau96 fragment was isolated. 4. Fragments isolated in steps (1), (2) and (3) were ligated to form expression plasmid pLelF I trp 1.

LelF J

1. The plasmid pLelF J contains a 3.8 kilobase HindIII fragment from human genomic DNA containing the LelF J gene sequence. A 760 b.p. DdeI - RsaI fragment was isolated from this plasmid. 2. The plasmid pLelF B trp 7 was cleaved with HindIII and DdeI and a 340 b.p. Hindlll - Ddel fragment isolated. 3. Plasmid pBR322 was cleaved with PstI, end blunted by incubation with DNA polymerase 1 (Klenow fragment), then digested with HindIII. The large (~3600 b.p.) fragment was isolated. 4. Fragments isolated in steps (1), (2) and (3) were ligated to form the expression plasmid pLelF J trp 1.

Claims (8)

1. Plasmid encoding a fully developed human leukocyte interferon, of which the amino acid sequence of this interferon includes the partial sequence Cys-Ala-Trp-Glu-Val-Val-Arg-Ala-Glu-Ile-Met-Arg-Ser, characterized in that the plasmid contains a first DNA sequence that codes for a fully developed. leukocyte interferon which is operably linked to a second DNA sequence capable of providing microbial expression of the interferon. 2. Plasmid according to claim 1, characterized in that the interferon is selected from the group comprising human leukocyte interferon (LelF), A, B, C, D, F, H, I and J. 3. Plasmid according to claim 1, characterized in that the first DNA sequence codes for human leukocyte interferon A (LelF A). 4. Plasmid according to claim 3, characterized in that it is plasmid pLelF A trp 25. 5. Plasmid according to claim 1, characterized in that the first DNA sequence codes for human leukocyte interferon B (LelF B). 6. Plasmid according to claim 5, characterized in that it is plasmid pLelF B trp 7. 7. Plasmid according to claim 1, characterized in that the first DNA sequence codes for human leukocyte interferon D (LelF D). 8. Plasmid according to claim 7, characterized in that it is plasmid pLelF D trp 11.