SI8212335A8 - Polypeptide with human immune interferons (ifn-gamma)properties. - Google Patents

Description

HUMAN IMMUNOLOGICAL INTERFERON

FIELD OF INVENTION

The present invention relates to the field of reccebinant DNA technology, to means and methods of utilizing such technology in the discovery of DNA sequences and discovered amino acid sequences for human human immune interferon and its production and various products from such production and their applications.

More specifically, the present invention relates to the isolation and identification of DNA sequences encoding for human immune interferon and the construction of carriers for the expression of recombinant DNA containing such DNA sequences that are operatively linked to expression sequences and to engineered expression carriers.

In another aspect, the present invention relates to host culture systems, such as various microorganisms and cell cultures of vertebrates that have been transformed with such expression carriers, and thus are directed to expressing the DNA sequences discussed above. In further aspects, the present invention relates to agents and methods for converting end products of such expression into novel species, such as pharmaceuticals, which are useful for the prophylactic and therapeutic treatment of humans.

In preferred embodiments, the present invention provides specific expression carriers that are properly sequenced to produce human immune interferon and to be excreted from the host cell in the gill form. In addition, the invention relates to various methods that are useful for the production of said DNA sequences, expression carriers. · Host culture systems and end products and their entities as well as their specific and associated embodiments.

The present invention derives in part from the discovery of a DNA sequence and a deduced amino acid sequence encoding for human immune interferon. Furthermore, the present invention provides information on the sequence of the 3'- and 5 ^f flange sequences of the human immune interferon gene, thereby facilitating its in vitro binding into expression carriers. Specifically, a 5'-DNA segment encoding for a putative endogenous signaling polypeptide immediately preceding the amino acid sequence of a putative mature human immune interferon is provided.

These findings, in turn, have enabled the development of means and methods of producing, through recombinant DNA technology, satisfactory amounts of human immune interferon, which again makes it possible to determine its biochemical properties and bioactivity.

The publications and other materials used herein to explain the theoretical reinvention of the invention, and in some cases to provide additional details regarding the implementation of the invention, are incorporated herein by reference, and for convenience, referenced by numbers after the following text and grouped appropriately in the attached bibliography.

The theoretical basis of the invention

A. Human immune interferon

Human interferons can be grouped into three groups based on different antigenic behavior and different biological and biochemical properties.

The first group comprises a family of leukocyte interferons (alpha-interferon, LeIF or IFN-alpha), which are normally produced mainly by constituents of human blood after viral infection. These have been produced microbially and have been found to be biologically active (1, 2, 3). Their biological properties have accelerated their use in clinics as therapeutic agents for the treatment of viral infections and malignancies (4).

In the second group, human fibroblast interferon (betainterferon, FIF, or IFN-beta), which is normally produced by fibroblasts after viral infection, is similarly microbial produced and found to exhibit a wide interval of biological activity (5). . Clinical trials have also highlighted its potential for therapeutic value. Leukocyte and fibroblast interferons exhibit very close similarities in their biological properties despite the fact that the degree of homology at the amino acid level is relatively low. Furthermore, both groups of interferons contain 165 to 166 amino acids and these are acid-stable proteins.

The human immune interferon (gamma-interferon, IIP or IFN-gamma) to which this invention relates is alpha- and beta-interferons, labile on pS 2, and is produced mainly after mitogenic induction of lymphocytes and is also clearly different in in terms of antigenicity. Until recently, human immunological interferon could only be detected in very small quantities, which obviously made it difficult to characterize. Until recently, fairly extensive but still only partial purification of human immune interferon has been reported (6). The compound is said to be produced from lymphocyte cultures stimulated with a combination of phytohemagglutin and phorbol ester and purified by sequential chromographic separation. This process resulted in a product having a molecular weight of 58,000.

Human immune interferon is produced in very small amounts by translation of mRNAs in oocytes, which exhibit interferon activity characteristic of human immune interferon, and this expresses the hope that immune interferon cDNA can be synthesized and cloned (7).

The amount of immune interferon obtained so far is certainly insufficient to conduct unambiguous experiments for its characterization and determination of the biological properties of the purified component. However, in vitro studies performed with crude preparations, as well as in vivo experiments with murine gamma interferon preparations, suggest that the primary function of human interferon may be to serve as an immunoregulatory agent (8, 9). Not only does human interferon have antiviral and anticellular activity common to all human interferons, it also shows a potentiating effect on these activities with alpha- and beta-interferon (10).

Also, the in vitro antiproliferative effect of gamma-interferon on tumor cells has been reported to be approximately 10 to 100-fold greater than other classes of interferons (8, 11, 12). This result, together with its pronounced immunoregulatory role (8, 9), suggests a more pronounced anti-tumor potency for IFN-gamma than for IFN-alpha and IFN-beta. And indeed,

Indeed, in vivo experiments with mouse IFN-gamma mice and preparations show a clear superiority to antiviral-induced interferons in their antitumor effect against osteogenic sarcomas (13).

All of these studies, up to the present invention, had to be done with fairly crude preparations, due to their very low accessibility. However, they certainly suggest very important biological functions for immune interferon.

Not only does Immune Interferon have potent concomitant antiviral activity, it also has strong immunoregulatory and antitumor activity, which clearly highlights a potentially very promising drug for clinic use.

It is understood that the use of recombinant DNA technology will be the most effective way to provide the required large amounts of human immune interferon. Whether or not these materials produced include glycosylation, which is considered a characteristic of native human-derived material, they are likely to exhibit bioactivity that will allow their clinical use in treating a wide range of viral, neoplastic and immunosuppressive conditions or diseases.

B. Recombinant DNA technology

Recombinant DNA technology has reached a certain age of perfection. Molecular biologists can recombine various DNA sequences with some ease, creating new DNA entities that can produce copious amounts of exogenous protein product in transformed microbes. There are agents and methods for in vitro ligation of various DNA fragments with blind or sticky ends, so that expression carriers that are useful in transforming certain organisms are produced, and their effective synthesis of the desired exogenous product is directed. However, on a product-by-product basis, the path remains somewhat painstaking 1 science has not progressed to the point that regular successful production can take place. Indeed, those who achieve successful results without the proper learning of the experimental bases * do so with a considerable risk of inoperability.

Plasmid, a non-chromosomal double strand DNA strand found in bacteria and other microbes, often in

- že multiple copies per delia, remains the basic element of recombinant DNA technology, t? the information encoded in plasmid DNA uk 1 j is also learned that is necessary for the reproduction of plasaids in delaying holes (i.e., the source of replication) and usually, in one or more phenotypic loci characteristics such as, in the case of bacteria. antibiotic resistance, which makes it possible for clones of the host cell containing an interesting plasmid to be recognized and preferentially cultured in selected substrates. The usefulness of plasanids lies in the fact that they can be specifically terminated by one or the other restriction endonuclease or restriction enzyme ^a , each of which recognizes a different site on plasmid DNA. Subsequently, fragments of the heterologous gene or gene can be immersed in the plasmid by fusing the ends at the cleavage site or at the reconstructed Jerei near the cleavage site. This is how the so-called replicating carriers of expression are formed. DNA recombination is performed outside the moiety, but the resulting recombinant replicating expression carrier, Or plasmid, can be introduced into the moiety by a process known as transformation and large amounts of the recoenbinant carrier are obtained by culturing the trans formant. You put it, when the gene is properly inserted against the plasmid fragments that control the transcription and translation of the encoded DNA message, the resulting expression carrier can be used to produce the sequence of the polypeptide the gene encodes for, a process known as expression (expression). ).

Expression is initiated in a region known as a promoter that is recognized and bound by SNA polymerase. In the transcription phase of expression, DNA is unwound and expose it as a template for the initiation of messenger RNA synthesis from a DNA sequence. The messenger RNA is again translated into a polypeptide having an amino acid sequence encoded by the mSNA. Each amino acid is encoded by a nucleotide triplet or codon that collectively Sines the structural gene "i.e., that portion that encodes for the amino acid sequence" of the expressed polypeptide product. Translation is initiated at the start 'signal (usually ATG, causing the RNA messenger to become AUG). The so-called stop codons define the end of translation and, therefore, the production of further amino acid units. The product obtained can be obtained by breaking down, if necessary, the demadine fraction in microbial systems, and the product is regenerated by appropriate purification from other proteins.

In practice, the use of recombinant DNA technology may express fully heterologous polypeptides - what is called direct expression - or alternatively, may express a heterologous polypeptide fused to a protein of the amino acid sequence of a homologous polypeptide. In the latter cases, the intended bioactive product is sometimes made bioinactivated within the fused homologous / heterologous polypeptide until it is cleaved in the extracellular environment.

See British Patent Publ. No. 2007676A and Netzel,

American Scientist 68, 664 (1980).

C. Technology of share culture

The science of delia or tissue cultures for the study of genetics and physiology of delia is well known. The means and procedures are affordable to maintain permanent cell lines, made by serial successive transfer of normal cell isolates. For use in research, such steel lines are maintained on a solid support in a liquid support, or growth aid in suspension containing nutrient carriers. Magnification for large preparations seems to present only mechanical problems. For further theoretical background, attention is focused on & icrobilogy, 2nd Edit ion, Harper and Row,

Publishers, Inc., Hagerstown, Maryland (1973), especially pp.

1122 and later and Scientific American 245, 66 and later (1981), which are incorporated herein by reference.

Excerpt from the invention

The present invention is based on the discovery that recanbinant DNA technology can be used to successfully produce human immune interferon, preferably in direct form, and in sufficient quantity to initiate and perform animal testing and clinical testing as prerequisites for market acceptance. The product is suitable for use, in all its forms, for the prophylactic or therapeutic treatment of human whips for viral infections and for malignant and immunosuppressive or immunodeficient conditions. Its forms include various possible oligomemic forms that may include associated glycosylation. The product is produced by genetically engineered micro-organisms or cell culture systems. Thus, it now respects the potential to make and isolate human immune interferon in a more efficient manner than has been possible so far. One significant factor of the present invention is the genetic targeting of a microorganism or cell culture for production to produce human immune interferon in isolable amounts, which is excreted from the host cell in the mature form.

The present invention encompasses the human immune interferon thus produced and the means and methods for its production The present invention is further directed to replicating carriers of DNA expression that provide sequences of the gene encoding for human immune interferon in a form that can be expressed. Furthermore, the present invention is directed to strains of microorganisms or cells of culture of such transformed strains or cultures that can produce human immune interferon. In remote aspects, the present invention is directed to various methods useful for making sequences of genes for said immune interferon, carriers for expressing DNA, strains of microorganisms and cell cultures, and to their specific embodiments. Further, the present invention is directed to making fermentation cultures of said microorganisms and steel cultures. Further, the present invention is directed to making human irraunological interferon, as a direct expression product, extracted from the host cells in the gill form. This approach may utilize the gene encoding the sequence for mature human immune interferon plus 5 'flange DNA encoding for the signal polypeptide. The signaling polypeptide is believed to assist the transport of molecules to the cell wall of the host organism where it breaks down during the secretion process of a mature human interferon product. This realization enables isolation and purification of intended mature

- Immune interferon without the inclusion of methods designed to eliminate the intracellular protein contaminants of the host or cellular debris.

The term used herein mature human immune interferon means the production of human immune interferon in a microbial or cell culture that is not accompanied by a signal peptide or a cross-sectional peptide that directly enables the translation of human immune interferon mRNA. Thus, according to the present invention, a mature human immune interferon having methionine as the first amino acid (present due to the insertion of an ATG starting signal codon in front of a structural gene) is provided, or, when methionine is intra- or extracellularly cleaved, has its normal first amino acid cysteine. Mature human immune interferon can also be produced, according to the methods of this application, together with a conjugated protein distinct from a conventional signaling polypeptide, wherein the conjugate can be specifically cleaved in an intra- or extracellular environment. Vifi British Patent Publication no. 2007676A. Finally,

Finally, mature human immune interferon can be produced by direct expression without the need to disrupt the macaque foreign, artificial polypeptide. It is particularly important that the host is unable or unable to efficiently separate the signal peptide when the expression carrier is intended to express mature human interferon together with its signal peptide. The mature human immune interferon thus produced is regenerated and purified to a level suitable for its use in the treatment of viral,

- 1 ?, malignant and immunosuppressive and immunodeficient states.

Human immune interferon was obtained as follows:

1. Human tissues, for example, human spleen tissue or peripheral blood lymphocytes, are cultured with mitogens to stimulate the production of immune interferon.

2. Cell granules from such cell cultures are extracted in the presence of a ribonuclease inhibitor such that all RNA cytoplasm is isolated.

3. The oligo-dT column isolates the total RNA (mRNA) scatter in polyadenylated form. This mRNA is fractionated by size by using sucrose gradient chromatography and gel electrophoresis.acid-carbamide.

4. The corresponding mRNA (12 to 18 S) is translated into the corresponding single strand complementary DNA (cDNA) from which the double strand cDNA is produced. After poly-dC binding to the tail, this is inserted into a vector, such as a plasmid carrying one or more phenotype markers.

5. Vectors thus made are used to transform bacterial cells by providing a library of colonies. Radiolabeled cDNA made from both induced and non-induced mRNA, ifinduced as described above, is used to specifically probe duplicate colonies libraries. The excess cDNA is then separated and the colonies are exposed to the X-ray film so that the cDNA clones are induced.

6. The corresponding plasmid DNA is isolated and sequenced from the induced cDNA clones.

7. The sequenced DNA is then cut in vitro for insertion into a suitable expression vehicle used to transform the appropriate host cells which are then allowed to grow in culture and express the desired human immune interferon product.

8. The human immune interferon thus produced has, without doubt, 146 amino acids in the fungal form, starting with cisterns, and is of a very basic character. Its monomeric molecular weight was calculated to be 17,140. Perhaps due to the presence of numerous base residues, hydrophobicity, salt bridge type formation and so on, the molecule can be combined into oligomeric forms, e.g., into the form of dimers, trimers or tetramers. The high molecular weights observed early with natural material (6) that cannot be obscured by amino acid sequencing alone may be due to such oligomeric forms as carbohydrate contributions from posttranslational glycosylation.

9. In certain host cell systems, especially when ligation is carried out in an expression carrier so that it is expressed together with its 20 amino acid signal peptide, the mature form of human immune interferon is exported to the cell culture medium, which immeasurably aids isolation and purification.

Description desirable. realization

A. Microorganisms / cell cultures 1. Bacterial strains / promoters

The studies described herein were performed using, inter alia, the micro-organism E. coli K-12 strain 294 (end A, thi ~, hsr ”, ^ hsm ⁺ ), as described in British Patent Publication No. 4/15. 2055382 A. This strain is deposited in American Type Sulture Chollection, ATCC accession no. 31446. However, various other microbial strains are useful, including known E. coli strains such as E. coli B, E. coli X 1776 (ATCC No. 31537) and E. coli W 3110 (F, lambda, protrophic) (ATCC No. 27325), or other microbial strains, many of which have been deposited and (potentially) accessible from known institutions for the deposition of richroorganisms, such as American Type Culture Collection (ATCC) - cf. ATCC list listing. See also German Offenlegungsschrirt 2644432. All other micro-organisms include, for example, Bacilli such as Bacchi Hus subtilis and other enterobacteriacae honey which may be present. referred to as shrimp Salmonella typhimurium and Serratia marcescens, using replicable plasmids to express heterologous gene sequences.

As examples, promoter systems for beta-lactamase and lactose have been suitably used to initiate and support the production of heterologous polypeptides by germs. Details concerning the addition and construction of these proraotor systems have been published in Chang et al. · Nature 275, 617 (1978) and Itakura et al., Science 198 _f , 1056 (1977), which are incorporated herein by reference. 0 More recently, a tryptophan-based system, the so-called trp promoter system, has been developed. Details concerning the creation and construction of this system were published in Goeddel et al. , Nucleic Acids Research &, 4057 (1980) and Kleid et al., - OSSN 133,296, filed March 24, 1980, incorporated herein by reference. Numerous other microbial promoters and details concerning their nucleotide sequences have been discovered and used, enabling the expert to functionally bind them within the plasmid vector have been published - see, e.g., Siebenlist et al., Full 20, 269 (1980). which is incorporated herein by reference.

2. Yeast yeast / yeast promoters

The expression system herein may also utilize the YRp7 plasmid (14, 15, 16), which can be selected and replicated in both E. coli and the yeast, Saccharontyces cerevisiae. For selection in yeast, the plasmid contains the TRP1 gene (14, 15, 16), which is a complement of yeast containing mutations (allows growth in the absence of tryptophan) in this gene, which is above the yen on chromosome IV of yeast (17). The strain used here was RH218 strain (18) deposited in the American Type Culture Collection without restriction (ATCC No. 44076). However, it should be clear that the Saccharomyces cerevisiae maca strain containing the mutation that makes the cell suffer should be an effective environment for the expression of a plasmid containing the expression system. An example of another strain that can be used is pep4-1 (19). This tryptophan. the auxotrophic strain also has a mutation point in the TRP1 gene.

When placed on the 5 'side of the non-yeast 5'-flange gene

- 16 A DNA sequence (promoter) from a yeast gene (for alcohol dehydrogenase 1) can promote expression of a foreign gene in yeast when inserted into a plasmid used to transform the yeast. In addition to the promoter, proper expression of the non-yeast gene in the yeast requires a second yeast sequence set at the 3 * end of the non-yeast gene on the plasmid to allow proper transcription and polyadenylation termination in the yeast. This promoter can be conveniently used in the present invention as well as others - see infra.

In preferred embodiments, the 5'-flange sequence of the yeast (3) phosphoglycerate kinase gene (20) is positioned upstream of the structural gene, which is again accompanied by DNA containing polyadenylation termination signals, for example, the TRPI (14, 15, 16) gene or PGK (20) genes.

Because the 5'-flange sequence (together with 3 'yeast DNA for termination) (infra) can function to promote expression of foreign genes in yeast, it seems likely that the Ξ'-flange sequences of a highly expressed yeast gene can be used to express important gene products. Because in some circumstances yeast expressed up to 65 percent of soluble proteins in the form of glycolytic enzymes (21) and since this high level appears to be due to the production of high levels of single mRNAs (22), it should be possible to use the 5'-flange sequences of mac other glycolytic genes for the purposes of such expression - e.g., enolase, glyceraldehyde - 3-phosphate dehydrogenase, hexokinase. pyruvate decarboxylase, phosphofructokinase, glucose -6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Macaque of the 3'-prtrnbnlh sequences of these genes can also be used for proper termination and mRNA polyaderiylation in such a cf. Supra. Some other highly expressed genes are those for acidic phosphatases (23) and those expressing high levels of production due to mutation in the 5'-flange regions (mutants that increase expression) - usually due to the presence of a ΤΥ1 transposing element (24).

All the genes mentioned above are thought to be transcribed by RNA polymerase II (24). It is possible that promoters for RNA polymerase I and III, which transcribe genes for ribosomal RNAs, 5S RNAs and tRNAs (25, 24), may also be useful in such expression constructs.

Finally, many yeast promoters also contain transcriptional control so that they can be switched off or on by varying growth conditions. Some examples of such yeast pro-rotors are genes that produce the following proteins: Alcohol dehydrogenase II, isocitochrora-c, acid phosphatqase, degradative enzymes that track nitrogen metabolism, glyceraldehyde-3phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization (22). Such a control region will be very useful in controlling the expression of a protein product - especially when their production is toxic to yeast. It should also be possible to position the control region of a 5 '~ flange sequence with a 5' flange sequence containing a promoter from a highly expressed gene. This will be given by the hybrid promoter and this should be possible since the control region and the promoter appear to be physically distinct DNA sequences.

... bir b * ' ¹ *

λ ...-- ..-- - ..- ---- ^ - -; ·., ·· - '. . ,. z .. ^μ <¥ · ί. ·· ίι, · 5- · .'k- * ·· · - ”<<'-v- ·· -' ·.

- '* ^ / 3 ^ (31 (Steel culture systems / steel-culture vector ~ j | ^' * 7 · - ^ 77'λν «'' ^ E ^ ophthalmology of vertebrate cells in culture (tissue culture)., _ ..... have become a routine procedure lately (see Tissue. / ·· '. ··. ·.' Γ 1 · ....- · · '/ ·

A »* f 'J iCulture / Academie Press, Kruse and Patterson eds,

1973,)>. The COS-7 monkey kidney fibroblast line was used here as a 'host' for the production of immune interferon (25a),. However, the experiments described here can be performed c ^^ elJ ^^ p ^ lini Ji ^ ko j a, .can. yes - replicates ^ expresses a compatible vector, e.g., WI38, ΒΗΚ, 3T3, CHO,

VERO and HeLa steel lines. Further, what is required of the expression vector is the origin of the replication and the promoter located in front of the gene to be expressed, together with any ribosomal binding sites required, RNA cleavage sites, polyadenylation sites, and transcriptional terminator sequences. Although these essential elements of the SV40 have been utilized herein, it should be clear that the invention, although described as a preferred embodiment, is not limited to such sequences. For example, the origin of replication of other viral ones (e.g.,

Polyoma, Adeno, VSV, BPV and so on) vectors, as well as steel sources of DNA replication that can function in the non-integrated state.

· ·

, B. Vector Systems -7.1: / 7

77 /// K,., K Direct expression of mature immune ihterfe: w :: 'Λ.ί.> ... · ^ ί,: · a *! ⁴ »- v. ·. , - ·,. '' χ-i ·

-UU C?% £ ··· '«*» ·'

Used'procedure; to obtain direct expression of IFN-gamma in E. coli in the form. the mature interferon-mediated polypentide (minus the signal sequence) was a variant of that which is .λ.'s, /; ^: K · · »/ '/'':

the wound was used for human growth hormone (26) and human </ · - -19 - 'g · / leukocyte interferon (1), as it did not include a combination of synthetic (N-terminal) and cDNA.

f · &

As inferred from the nucleotide sequence p69, as described below, and based on comparison with the known cleavage site between the signal peptide and the mature polypeptide for several IFN-alpha (2), IFN-gamma has a hydrophobic signal peptide of 20 amino acids followed by 146 amino acids of mature IFN-gamma follow (Figure 5). As shown in Figure 7, the BstNI restriction endonuclease site is conveniently located at amino acid 4 of the mature IFN-gamma. Two synthetic deoxyoligonucleotides have been constructed that incorporate the ATG codon to initiate translation, the codons for amino acids 1,2 and 3 (cysteine-tyrosine-cysteine) and create in the EcoRI cohesive end. These deoxyoligonucleotides were ligated for fragments of 100 base pairs of BstNI-Pst1 p69 to construct a synthetic-natural hybrid gene with 1115 base pairs encoding for IFN-gamma and bound by EcoRI and Pst1 restriction sites. This gene was inserted into plasmid pLeIF A A trp 103 between the EcoRI and Pst1 sites to produce a plasmid for expression of pIFN-gamma trp 48. In this plasmid, the IFN-gamma gene is expressed under the control of the E. coli trp promoter. (pLeIF A trp 103 is a derivative of pLeIF A 25 in which the distal EcoRI site relative to the LeIF A gene is separated. Using the procedure for separating this EcoRI site, j <is described (27).

2. Expression in yeast

For the expression of such a heterologous gene; such as cDNA for immunological interferon in yeast, it was necessary to construct a vector of plasmid containing four compo-

nente. The first component is the part that allows the transformation of both E. coli and yeast and so must contain a gene that can be selected from any organism. (In this case, this is the gene for ampicillin resistance from E. coli and the gene ·

Yeast TRPI). This component also requires a source of replicates from both organisms to be maintained as plasmid DNA in both organisms. {In this case, this is an E. coli source from pBR322 and ars1 source from chromosor III yeast).

The second component of the plasmid is the 5′-flange sequence from the highly expressed yeast gene so as to promote transcription of the downstream set structural gene. In this case, the 5'-flange sequence used is that used from the yeast 3-phosphoglycerate kinase (PGK) gene. The fragment was constructed in such a way that it separates ATG from the PGK structural sequence as well as S bp (base pairs) upstream of this ATG. This sequence has been replaced with a sequence containing both the Xbal and EcoRI restriction sites for the proper fusion of this 5′-flange sequence for the structural gene.

The third component of the system is a structural gene constructed in such a way that it contains both ATG translational start and translational stop signals. The isolation and construction of such a gene is described below.

The fourth component is the yeast DNA sequence containing the 3′-flange yeast gene sequence, which contains the correct * signals for transcription termination and polyadenylation.

·: ~ 21 When all these components are present, immune immunosuppressant interferon is produced.

3. Expression in mammalian cell culture

The strategy for the synthesis of immune interferon in mammalian cell culture is based on the development of a vector capable of both autonomous replication and expression of a foreign gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture is achieved by providing. sources of DNA replication (derived from the SV40 virus), and providing helper function (T antigen) by introducing a vector into a cell line endogenously expressing this antigen (28, 29). The last promoter of the SV40 virus is preceded by a structural interferon gene, thus providing gene transcription.

The vector used to achieve IFN-gamma expression consisted of pBR322 sequences that provided a selective marker for selection in E. coli (resistance to ampicillin) as well as in the E. coli source for DNA replication. These sequences were derived from plasmid pML-1 (28) and encompassed a region comprising EcoRI and BamHI restriction sites. The SV40 source was also derived from a PvuII-HindIII fragment with 342 base pairs spanning this region (30, 31) (both ends translate to EcoRI ends). These sequences, in addition to encoding a viral source for DNA replication, encode the promoter for both early and late transcription transcription units. The orientation of the * SVlon source of the source was such that the promoter for the later transcriptional unit was positioned adjacent to the gene encoding for interferon. "

- 22 Brief Description of Drawings

Figure 1 describes centrifugation with a sucrose gradient of peripheral blood (PBL) induced Poly (A) + RNA. Two peaks were observed for interferon activity (as shown in the shaded areas) with sizes 12S and t

S. The position and ribosomal RNA markers (centrifuged independently) were labeled above the absorbance profile.

Figure 2 shows the electrophoresis of PBL-induced Poly (A) +

SNA assisted acid-urea-agarose. Only one peak of activity was observed, which interacted with 18S SNA.

The positions of ribosomal RNA markers that were electrophoresed in the adjacent line.and visualized by staining with ethidium bromide were marked above the activity profile.

Figure 3 shows hybridization schemes of 96 colonies with induced and non-induced P-labeled cDNA probes.

single trans formants were cultured in a microtiter plate, replicates were mounted on two nitrocellulose membranes, and then the filters were hybridized with P-cDNA probes made either from induced mSNA (above) or from mSNA isolated from uninduced PBL cultures (uninduced, below). The filters were washed to separate non-hybridized RNAs and then exposed on an X-ray film. This filter set represents 86 such sets (8300 independent colonies). One example of an induced clone of brand-ran as _& H12.

Figure 4 is a restrictive endonuclease map of the insert cDNA

clone 69. cDNA insert is bound to the help of Pst! sites (points at both ends) and oligo dC-DG tails (simple lines). Number I says

- 23 fractions produced by restriction endonuclease cleavage were assessed by electrophoresis using a 6 percent assay.

of acrylamide gels. The positions of the sites were confirmed by nucleic acid sequencing (shown in Figure 5). The coding region of the largest open reading frame is framed and the shaded region represents the putative sequence of the 20-residue signal peptide, while the dotted region represents the sequence of the mature IIP. (145 amino acids). The 5 'end of the mRNA is to the left while the 3' end is to the right.

Figure 5 illustrates the nucleotide sequence of the plasmid p69 insertion sDNA, however, it is also illustrative of the most common allelic form of IFN-gamma. A complete amino acid sequence of the longest reading frame is also shown. The putative signal sequence is shown by means of residues labeled Sl to S20.

Figure 6 is a comparison of the mRNA structure of IFN-gamma with the structure of leukocyte (IFN-alpha) and fibroblast (IFN-beta) interferon. mRNA of clone 69 (immunolabeled) contains significantly higher amounts of untranslated sequences.

Figure 7 is a schematic diagram of the construction of the IFN-gamma expression plasmid pIFN-gamma trp 48. The starting material is a Pst cDNA insert with 1250 base pairs from plasmid p69.

Figure 8 shows a diagram of a plasmid used for IFN-gamma expression in monkey portions.

Figure 9 shows Southern hybridization of eight different ones

EcoRI of digested human genomic DNA hybridized with

-X 32 ' ^with -xP-marked. Ddel fragment with 600 base pairs from the tyhe cDNA insert p69. EcoRI fragments are clearly hybridized to the probe in each DNA sample.

- 23ce »

Image. 10 describes Southern hybridization of human genomic DNA digested with six different restriction endonucleases''' · '\ ^: '>; and 32 '· -. '-4'. · · · 'Hybridized with P-marker probe from p69.

Figure 11 schematically illustrates the restriction map of the Hindlll insert vector pB1 at 3.1 kbp from which the PGK promoter was isolated. The insertion of the EcoRI site and the Xbal site in the 5′-flange DNA of the PGK gene is indicated.

Figure 12 illustrates the 5′-flange sequence plus the initial coding sequence for the PGK gene before inserting the Xbal and EcoRI sites.

Figure 13 schematically illustrates the techniques used to insert the Xbal site in position - 8 in the PGK promoter and to isolate the fragment with the 39 bp 5′-flange sequence of the PGK containing this Xbal end and Sau3A end.

Fig. 14 schematically illustrates the construction of a 300 bp fragment containing the 39bp upper fragment _f supplementary PGK 5'-flange sequence (265bp) from Pvul for Sau3A (see Fig.

11), and an EcoRI site adjacent to Xbal.

18 schematically illustrates the construction of a 1500 bp PGK promoter fragment (HindIII / EcoRI) containing, in addition to the fragment constructed in FIG. 14, Hindlll for a 1300bp Pvul fragment from the PGK 5 'flange sequence (see Fig. 11).

Figure 16 illustrates the composition of an expression vector for human immunostained interferon in yeast, which contains a modified PGK promoter, sDNA IFN-gamma, and the terminator region of the yeast PGK gene as described herein in more detail.

Detail description

A. Source of IFN-gamma mRNA

Peripheral blood lymphocytes (PBL) are derived from human donors by leucophoresis. PBLs were further purified by Ficoll-Kypaque gradient centrifugation and then cultured at 5x10 ⁶ cells / ml in RPMI 1640, 1% L-glutamine, mM HEPES, and 1% penicillin-streptomycin solution (Glbco, Grand Island, NY). These cells were induced to produce IFN-gamma by the mitogen staphylococcal enterotoxin B (1 / ug / ml) and cultured for 24 to 48 hrs / 37 ° C in 5% CO ₂ · Desacetiitimosin-alpha-1 (0.1 ^ ug / ml) was added to PBL cultures to increase the relative yield of IFN-gamma activity.

B. Isolation of RNA messenger

Total RNA from PBL cultures was extracted essentially as described in Berger, SL et al. (33). Cells were granulated by centrifugation and then resuspended in 10 mM NaCl, mM Tris-HCl (pH 7.5), 1.5 mM MgCl ₂ and 10 mM ribonucleoside vanadyl complex. Cells were dissolved by the addition of NP-40 (1 percent final concentration), and the nuclei were granulated by centrifugation. The superatantant contained total RNA which was further purified by multiple extractions with phenol and chloroform. The aqueous phase was brought to 0.2 M in NaCl and the total RNA was then precipitated by the addition of two volumes of ethanol. RNA from uninduced (unstimulated) cultures was isolated by the same procedures. Oligo-dT cellulose chromatography was used to purify the DNA from total RNA preparations (34). Typical yields of T-2 liter cultured PBLs were 5-10 milligrams total RNA and 50-20 micrograms

Poly (A) + RNA.

C. Size mRNA fractionation

Two methods were used to fractionate mRNA preparations.

These methods were used independently (rather than uniquely) and each led to significant enrichment of IFN-garaa mRNA.

Sucrose gradient centrifugation in the presence of denaturation formamide was used for mRNA fractionation. Gradients of 5 percent to 25 percent sucrose in 70 percent formamide (32) were centrifuged at 154,000 x g for 19 hours at 20 ° C. Successive fractions were then separated (0.5 ml) from the top of the gradient, ethanol-stained, and an aliquot was injected into Xenopus laevis oocyte 2a mRNA translation (35). After 24 h at room temperature, the incubation medium was then evaluated for antiviral activity in a standard assay to inhibit cytopathic effect using Vesicular Stomatitis Virus (Indiana strain) and Encephalomyocarditis Virus on WISH (human amnion) cells as described by Stewart (36), except that the samples were incubated with cells for 24 hours (instead of 4) before the virus attack. Peaks of activity in the RNA fractionated with sucrose gradient were observed consistently (Figure 1). The edema peak was sedimented with a calculated size of 125 and contained 100-400 units / ml of antiviral activity (in addition to the IFN-alpha standard) per microgram of injected RNA. The second activity peak was sedimented as 16S in size and contained about half the activity of the slow-moving sediment. Each of these activity peaks appears to have been due to IFN-gamma, since no activity was observed when the same fractions were evaluated on the να 26-line front line (MDBK) that was not protected by human IFN-gamma assistance. Both IFN-alpha and IFN-beta activity would be easily detected with the MDBK assay (5).

The mRNA fractionation (200 µg) was also performed by electrophoresis using acidic carbamide-agarose gels. The piece of agarose gel (37, 38) consisted of 1.75 percent agarose, 0.025 M sodium citrate, pH 3.8, and 6 M urea. Electrophoresis was performed for 7 hours at 25 milliamps 1 4 ° C.

The gel was then fractionated with the blade of the blade. The individual cutouts were melted at 70 ° C and extracted twice with phenol and once with chloroform. The fractions were then precipitated with ethanol and subsequently evaluated for IFN-gamma mRNA by injection into Xenopus laevls oocytes and antiviral assay.

Only one peak of activity was observed in the samples fractionated on the gel (Figure 2). This peak commigrated with 18S RNA and had an activity of 600 units / ml per microgram of injected RNA. This activity also appears to be specific for IFN-gamma, since it did not protect MDBK cells.

The difference in size between activity peaks observed on sucrose gradients (12S and 16S) and acidic urea gels (18S) can be explained by the observation that these independent fractionation procedures were not performed under conditions of total denaturation.

D. Construction of a colony library containing IFN-gamma sequences / Ug mRNA fractionated on gel was used to make double strand cDNAs using standard procedures (26,

39). cDNA was fractionated in size at 6 percent

- 27 polyacrylamide gel. Two size fractions were electroeluted, 800-1500 bp (138 ng) and jireko 1500 bp (204 ng). Sections of 35 ng of cDNA of each size were expanded with deoxyC residues using terminal deoxynucleotidyl transferase (40) and tempered with 300 ng of plasmid pBR322 (41), which was similarly tailored to deoxyG residues at the Pst1 site (40). Each tempered mixture was then transformed into E. coli K12 strain 294. Approximately 8000 transformants with 800-1500 bp cDNA and 400 transformants with over 1500 bp cDNA were obtained.

E. Testing the colony library for induced cDNAs

The colonies were individually inoculated into holes on microtiter plates containing LB (58) + 5 µg / ml tetracycline and stocked at −20 ° C after addition of DMSO up to 7 percent. Two copies of the colony library were cultured on nitrocellulose filters and DNA from each colony was fixed to the filter using the Grunstein-Hogness procedure (42).

P-tagged cDNA probes were made using 18S size mRNA fractionated on gel from induced and non-induced PBL cultures. The oligo ^dT -j2-i8 P ^rimer 1 reaction conditions described previously were used (1). Filters containing 8000 transformants from a 6001500 bp cDNA clip and 400 transformants from a cDNA clip size via

1500 bp were hybridized with 20 z 10θ cpm induced

3 ² p-cDNA. Duplicate filter set is hybridized with *

32 x 10 cpm uninduced P-cDNA. Hybridization lasted for 16 hours using the conditions described in Fritsch et al. (43). The filters were extensively washed (43) and then exposed on a Kodak XR-5 X-ray film with DuPont Lightning intenSv- · - «· · '. . \ .-. _T A, g .. '· *? · + _Λ ·.> * ·· - *. <- · !. · -.-7 ^ *. ·. , · .. '' · ™ 4 * 0- * ⁱⁿ ', ·' zipper sittings for 16-48 hours. the hybridization scheme of each colony was compared with two probes. Approximately 40 percent of the colonies were clearly hybridized with both probes, while approximately 50 percent of the colonies failed to hybridize with one of the probes (shown in Figure 3). 124 colonies were significantly hybridized with the probe but were undetectable or quite poorly hybridized with the non-induced probe. These colonies were individually inoculated into holes on microtiter plates, cultured and transferred to nitrocellulose filters, and hybridized with the same two probes as described above. Plasmid DNA isolated from each of these colonies by rapid procedure (44) was also bound to nitrocellulose filters and hybridized to the induced and uninduced probe.

DNA from 22 colonies hybridized with the probe only and these colonies were termed induced colonies.

F. Characterization of induced colonies

Plasmid DNA was made from 5 Induced Colonies (46) and used to characterize cDNA inserts. Restriction endonuclease mapping of five induced plasmids (p67, p68, p69, p71, and p72) suggested that four had similar restriction nuclease maps. These four (p67, p69, p71 and p72) all had four Ddel sites,

HinfI sites, and one Rsal site in the cDNA insert. The fifth plasmid (p68) contained an ordinary Ddel fragment and appeared to be a short cDNA clone relative to the other four. The homology suggested by the restriction nuclease map was confirmed by 32 by hybridization. A P-tagged DNA probe (47) was made from a 600 bp p67 plasmid Ddel fragment and used for hybridization (42) of other induced colonies.

- 29 All five restriction nuclease mappings. colonies underwent cross-hybridization with this probe, as did 17 other colonies out of 124 selected in induced / non-induced testing. The length of the cDNA insert in each of these cross-hybridized plasmids was determined by PstI digestion and gel electrophoresis. The clone with the longest cDNA insert appears to have been clone 69 with an insert length of 1200-1400 bp. This DNA was used for all four. a further experiment, and its restriction endonuclease map is shown in Fig. 4.

It was demonstrated that the cDNA was inserted into the p69 IFN-gamma cDNA based on its expression product, produced in three independent expression systems, resulting in antiviral activity, as described in more detail below.

G. Sequential analysis of the p69 insert cDNA.

The complete nucleotide sequence of the plasmid p69 insert cDNA was determined by the method of termination of the deoxynucleotide sequence (48) after subcloning the fragments into the M13 vector mp7 (49) and using the Maxam-Gilbert chemical process (52). The longest open reading frame encodes for the 166 amino acid protein shown in Figure 5. The first encoded residue is the first meth codon encountered at the 5 'end of the cDNA.

The first 20 residues at the amino terminus probably serve as a signal sequence to secrete the remaining 146 amino acids. This putative signal sequence has conspecific properties with other okar actors and with anim signal sequences such as size and hydrophobicity. Furthermore, the four amino acids found as a putative cleavage sequence (ser-leu-gly-cys) are identical with the four residues that

- 30 are located at the breakpoint of several leukocyte interferons (LeIF B, C, D, F, and H, (2)). The encoded mature 4 amino acid sequence 146 amino acids have the same molecular weight of 17,140.

There are two potential glycosylation positions (50) in the encoded protein sequence, at amino acids 28 to 30 (asn-gly-thr) and amino acids 100 to 102 (asn-tyrser). The existence of these positions is consistent with the observed glycosylation of human IFN-gamma (6, 51). Furthermore, only two cysteine residues (positions 1 and 3) are spatially too close to form a disulfide bridge, which is consistent with the observed stability of IFN-gamma in the presence of such reducing agents as beta-mercaptoethanol (51). The derived mature amino acid sequence is generally quite basic, with a total of 30 residues of lysine, arginine and histidine and only 19 total residues of aspartic acid and glutamic acid.

The mRNA structure of IFN-gamma as inferred from the DNA sequence of plasmid p69 is clearly different from IFN-alpha (1, 2) or IFN-beta (5) mRNA. As shown in Figure 6, the IFN-gamma coding region is shorter while the 5 'untranslated and 3' untranslated regions are much longer than either IFN-alpha or IFN-beta.

H. Direct expression of mature human interferobe in E. col

For reference Figure 7, 50 ^ g of plasmid p69 was digested with Pstl and an insert of 1250 base pairs was isolated by gel electrophoresis · on a 6 percent polyacrylamide gel. About 10 ng of this insert was electroeluted from the gel.

The '.tt' ^ ug of this Pstl fragment was partially digested from unit BstNI (Bethesda Research Labs) for 15 minutes at 37 C and the reaction mixture was purified on a 6% polyacrylamide gel. Approximately 0.5 µg of the desired BstNI - Pstl fragment of 1100 base pairs was isolated.

Two indicated deoxyligonucleotides, 5 '-dAATTCATGTGTTATTGTC and 5'-dTGACAATAACACATG (Figure 7), were synthesized by the phosphoester ester procedure (53) and phosphorylated as follows. 100 pmol of each deoxynucleotide was combined in 30 µl of 60 mM Tris-HCl (pH 8), 10 mM MgCl ₂ , 15 mM beta32 mercaptoethanol and 240 µCi (gamma-P) ATP (Amersham, 5000 Ci / mmol). 12 units of T4 polynucleotide kinase were added and the reaction was allowed to proceed at 37 ° C for 30 minutes.

J

1 µl of 10 mM ATP was added and the reaction was allowed to proceed for another 20 minutes. After extraction with 0-OH / CHCl _{2, the} oligomers were combined with a 0.25 µg BstNI-Pst1 fragment of 1100 base pairs and precipitated with ethanol. These fragments were subjected to ligation at 20 ° C for 2 h in 30 ^ ul of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl, 10 mM dithiothreitol.

0.5 mM ATP and 10 units of T4 DNA ligase. The mixture was digested for 1 hour with 30 units of Pstl and 30 units of EcoRI (to eliminate ligation of cohesive terminals leading to polymerization) and electrophoresis was performed on a 6 percent polyacrylamide gel. Electro-eluting isolated a product of 1,115 base pairs (110,000 cpm).

Plasmid pbeiP A trp 103 (Fig. 7) is a derivative of plasmid * pLeiF A 25 (1) in which it is married 1 EcoRI site from the LelF gene separately (27). 3 yug pLeiF A trp 103 was digested with 20 units of EcoRI and 20 units of Pstl for 90 minutes at 37 ° C and subjected to electrophoresis on 6 percent polyacryl-3? amide gel. A large (about 3900 base pairs) vector fragment was isolated by electroelution. The EcoRI-Pstl IFN-gamma DNA fragment of <115 base pairs was ligated in 0.15 µg of this vector made. Transforming E. coli K-12 strain 294 (ATCC No. 31446) yielded 120 tetracycline resistant colonies. Plasmid DNA was made from 60 of these transformants and digested with EcoRI and Pstl.

Three of these plasmids contained the desired EcoRI-Pst1 fragment of 1,115 base pairs. DNA sequence analysis showed that these plasmids had the desired nucleotide sequence at the junctions between the trp promoter, synthetic DNA, and cDNA.

One of these pIFN-gamma trp 48 plasmids was selected for further study, This plasmid was used to transform E. coli K-12 strain W3110 (ATCC No. 27325).

I. Structure of genes with the IFN-gamma coding sequence

The structure of the gene encoding for IFN-gamma has been analyzed

Southern hybridization .. D; by this method (54), 5 grams of micro-human lymphoma of apparent high molecular weight DNA (made as in 55) is digested to complete with various restriction endonucleases, electrophoresed on 1.0 percent agarose gels (56) and placed on a nitrocellulose 32 filter ( 54). A P-labeled DNA probe (47) was made from a Ddel fragment of a 600 bp cDNA insert p69 and hybridized (43) with an ntrocellulose-DNA probe. 10 counts per minute of the probe were hybridized for 16 hours and then washed as described (43). Eight genomic DNA samples from different human donors were digested with EcoRI restriction endonuclease 32 and hybridized with a p69 P-labeled probe. As shown in Figure 9, two were observed

-V—

- 33 clear hybridization signals with sizes of 8.8 kilobase pairs (kbp) and 2.0 kbp as compared by comparison with mobility with Hindlll digested lambdaDNA. This could be the result of two IFN-gamma genes or one gene that is shared by the EcoRI site. Because p69 cDNA does not contain the EcoRI site, an intervening sequence (intron) with an internal EcoRI site would be necessary to explain one gene.

To differentiate between these two possibilities, a second Southern hybridization was performed with the same probe versus five other 3endonuclease digests of one human DNA (Figure 10). Two hybridized DNA fragments with two other endonuclease digests, PvuII (6.7 kbp and 4.0 kbp) and HincII (2.5 kbp and 2.2 kbp), were observed. However, three endonuclease digestion schemes provide only one fragment of hybridized KB DNA: Hindlll (9.0 kbp), Bglll (11.5 kbp) and BamHI (9.5 kbp). Two IFN-gamma genes would have to be bound at an unusually close distance (less than 9.0 kbp) to reside within the same HindIII hybridizing fragment. This result suggests that only one homologue of the IFN-gamma gene (unlike many related IFN-alpha genes) is present in human generated DNA and that this gene is interrupted by one or more introns containing EcoRI, PvuII and HincII sites. This prediction 32 is supported by the hybridization of a P-tagged (47) fragment made exactly from the 3 * untranslated cDNA region from p69 (the 130 bp Ddel fragment from 860 bp to 990 bp in Figure 5) against the EcoRI digest of human genomic DNA.

¹ *

Only a 2.0 kbp fragment of hlbridbed with this probe suggested that this fragment contains 3 * untranslated sequences, whereas an 8.8 kbp EcoRI fragment contains 5 * sequences.

* ίϊ-'Λ · '* · ”-.- τί ·'. · '? - «·· 3 'ΛΧ“'<< · '-. - "<< - · 'ν - ·. ·>. = - * · .- ^; ·. ^; -. ~ - · - '''- -. T »'.

The structure of the IFN-gamma gene (one gene with at least one intron) is clearly different from IFN-alpha (multiple genes (2) without introns (56)) or IFN-beta (one gene without introns (57)).

J. Making bacterial extracts

The precocious E. coli W3110 / pIFN-gamma trp 48 culture in Luria broth + 5 micrograms per ml of tetracycline was used to inoculate the M9 (58) medium containing 0.2 percent glucose, 0.5 percent casamino acids, and 5 micrograms per ml tetracycline at 1: 100 dilution. Indolacrylic acid was added to a final concentration of 20 micrograms per ml when Α ₅₅ θ was between 0.1 and 0.2. Samples of 10 ml were defended by centrifugation at Α ^ ₅ θ = 1.0 and are currently resuspended in 1 ml of phosphate buffered saline containing 1 mg per ml of bovine serum albumin (PBS-BSA). the cells were opened by sonication and the debris was cleared by centrifugation. The supernatants were stacked at 4 ° C until analyzed. The activity of interferon in the supernatant was determined to be 250 units / ml by comparison with the IFN-alpha standard after the cytopathic effect inhibition test (CPE).

K. Transforming yeast / strains and substrates

Yeast strains were transformed as described previously (59). The E. coli strain JA300 (thr leuB6 thi thyA - - R trpC1117 hsdm hsdR str) (20) was selected to select plasmids containing the functional TRPI gene. The yeast strain RH218 having the genotype (tolerated by 2 SUC2 mal CHOPI) (18) was used as the yeast host for transformation. RH218 was deposited without restriction in the American Type Culture Collection, ATCC No.

g> · '- 35 -' L <L «-U X. v _. - .. T * -. b

44076. M9 (minimum substrate) with 0.25 percent casino acids (CAA) and LB (rich substrate), as described by Miller (58), were used with the addition of 20 µg / ial ampicillin (Sigma) and subsequently autoclaved and cooled. .

The yeast was re-cultivated on the following grounds:

YEPD containing 1 percent yeast extract, 2 percent peptone, and 2 percent glucose +3 percent Difco agar.

ΥΝΒ + CAAs contained 6.7 grams of yeast nitrogen base (without amino acids) (ΥΝΒ) (Difco), 10 mg adenine, 10 mg uracil.

grams of CAA, 20 grams of glucose and +. 30 grams of agar per liter.

L. Construction of yeast expression vector

1. 10 South YRp7 (14, 15, 16) was digested with EcoRI.

The resulting sticky ends of DNA were made by bluntly using DNA Polymerase I (Klen fragment). The vector and insert were chromatographed on a 1 percent agarose (SeaKem) gel, excised from the gel, electroelected, and extracted 2x with equal volumes of chloroform and phenol before ethanol precipitation. The resulting blunt-ended DNA molecules were then subjected to ligation in a final volume of 50 µl for 12 hours at 12 ° C. This ligation mix was then used to transform E. coli strain JA300 to resistance to ampicillin and tryptophan prototrophy. Plasmids containing the TRPI gene in both orientations were isolated. pFRW1 had a TRP gene in the same j orientation as YRp7 while pFRW2 contained a TRP gene in the opposite orientation.

*) ig pFRW2 was linearized with HindIII and subjected to electrophoresis on a 1% agarose gel. The linear molecules were eluted from the gel and then 200 ng was subjected to ligation with 500 ng 3.1 kb Hindlll plasmid insert pB1 (13)

&. · -;

• ci »

· Which is a restriction fragment containing yeast 3-phosphoglycerate kinase gene. Ligation mike was used to transforce E. coli strain 294 for ampicillin resistance and tetracycline sensitivity. A plasmid made from one such recombinant had an intact TRPI gene at 3.1 kbp

A HindIII fragment from the pB1 DNA insert at the HindIII site of the tetracycline resistance gene. This plasmid is pPRM31. 5 ^ ag pPRM31 was completely digested with EcoRI, extracted twice with phenol and chloroform and precipitated with ethanol. The cohesive ends of the molecules were filled using DNA polymerase I (the Klen fragment) in a reaction supplemented with 250 µM of each deoxynucleoside triphosphate. The reaction was carried out for 20 minutes at 14 ° C and at that time the DNA was extracted twice with phenol-chloroform and then precipitated with ethanol. The resuspended DNA was then completely digested with Clal and subjected to electrophoresis on a 6 percent acrylamide gel. The vector fragment was eluted from the gel, extracted with phenol-chloroform and precipitated with ethanol.

the six N-terminal amino acids of the 3-phosphoglycerate enzyme purified from humans are as follows x

- 2- 3- 4- 5- 6 SER - LEU - SER - H5M - LTS - LEU One of the translation readout frames generated from the DNA sequence of the 141 bp Sau3A-to-Sau3 restriction fragment (containing the internal HincII site; see PGK the reattraction * map in Fig. 11) produces the following amino acid sequence.

you? / - '-'.— Λ. t

V —V. ,

MET - SER - LEU - SER - SER - LYS - LEU

After separation of the initiator methionine, it can be seen that the PGK N-tenain amino acid sequence has such amino acid homology that 5 of the 6 amino acids are the same as the N-terminal amino acid sequence of human PGK.

This sequencing result suggested that the start of the yeast PGK structural gene was encoded by DNA in the restriction Sau1A fragment pB1 at 141 bp. Earlier work (20) suggested that DNA sequences specifying PGK mRNAs may be located in this region of the Hind III fragment. Further sequencing of the 141 bp Sau3A fragment further the DNA sequence of the PGK promoter (Figure 12).

A synthetic oligonucleotide with sequence 5′ATTTGTTGTAAA3 ′ was synthesized by standard procedures (Crea et al.,

Nucleic Acids Res. 8, 2331 (1980)). 100 ng of this example was labeled at the 5 'end using 10 units of T4 polynucleotide kinase in a 20 μl reaction which also 32 contained 200 yUCi / gamma-P / ATP. This labeled primer solution was used in a repair-primer reaction intended to be the first phase of a multiphase process that is to place an EcoRI restriction site in PGK 5 * flange DNA that precedes the sequence of the PGK structural gene.

100 µg of pB1 (20) was completely digested with HaeHI and then chromatographed on a 6% polyacrylamide gel. The highest band on the ethidium stained gel (containing the PGK promoter region) was isolated by electroelution as described above. This 1200 bp HaeHI piece of DNA was bound with HincII and then chromatographed on a 6 percent acrylamide gel. 650 bp tape isolated by electroelution [

Isolation is 5 I g of DNA. This 650 bp Haelll-for-HincII piece of DNA was resuspended in 20 ^ ul E ^ O, then mixed with 20 µl of phosphorylated primer solution as described above.

This mixture was 1x extracted with phenol-chloroform and then precipitated with ethanol. The dried DNA was resuspended in 50 [mu] L H &lt; 0 &gt; O and then heated in the bath with boiling water for seven minutes. This solution was then rapidly cooled in a dry ice-ethanol bath (10-20 seconds) and then transferred to an ice-water bath. To this solution was added 50 µl of solution containing 10 µl of 10Χ DNA polymerase I buffer (Boehringer Mannheim), 10 µl of solution previously made 2.5mM with each deoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP), 25 1 H ₂ O and 5 units of DNA polymerase I,

Maple Fragment. This 100 µl reaction was incubated at 37 ° C for 4 h. The solution was then 1x extracted with phenol-chloroform, precipitated with ethanol, dried by lyophilization and then extensively limited with 10 further units of Sau3A. This solution was then chromatographed on a 5 percent acrylamide gel. A band corresponding to 39 bp in size is cut off from the gel and then isolated by electro-elution as described above. This 39 bp tape has one blunt end and one Sau3A adhesive end. This fragment was cloned into a modified pFIF trp 69 vector (5). 10 I g pFIF trp 69 was linearized with Xbalm extracted once with phenol chloroform and then precipitated with ethanol. The Xbal adhesive end was filled using DNA polymerase I Klenov fragment in a 50 ml reaction containing 250 µm of each nucleofeid triphosphate. This DNA was excised with BamHI and then chromatographed

- 39 dreams on a 6 percent acrylamide gel. The vector fragment was isolated from the gel by electroelution and then resuspended in 20 yUl H ₂ O. 20 ng of this vector was subjected to ligation with a 20 ng 39 bp fragment made above for 4 hours at room temperature. One-fifth of the ligation mix was used to transform E. coli strain 294 to ampicillin resistance (on LB + 20 µg / ml amp. Plates).

Plasmids from transformants were examined by a rapid test procedure (44). One plasmid, pPGK-39, was selected for sequence analysis. 20 ^ of this plasmid was digested with

Xbal, the stanozemp is ethanol and then treated with 1000 units of bacterial alkaline phosphatase at 68 ° C for 45 minutes. The DNA was extracted 3x with phenol-chloroform, then precipitated with ethanol. The dephosphorylated ends were then labeled in a 20 µl reaction containing 200 µCi 32 / gamma -P / ATP and 10 units of polynucleotide kinase.

The plasmid was cut with Shali and chromatographed on a 6 percent acrylamide gel.

The band of the labeled insert was isolated from the gel and sequenced by a chemical degradation process (52). The DNA sequence at the 3'end of this promotional piece was as expected.

2. The construction of the 312 bp Pvul-for-EcoRI PGK promoter fragment ^ ug pPGK-39 (Fig. 13) was simultaneously digested with Sall and Xbal (5 units each) 1 then subjected to electrophoresis on a 6 percent gel. A 390 bp band containing a 39 bp promoter piece is isolated by electroelution. The resuspended DNA was bounded by Sau3A and then subjected to ^c · 'J *.

. · ... * · electrophoresis on β percent acrylamide gel. 39 bp PGK promoter band was isolated by electroelution.

This DNA contained the 39 bp 5 'end of the PGK promoter on the Sau3A-for-XbaI fragment.

yig pB1 is bounded with Pvul and KpnI and then electrophoresed on a 6 percent acrylamide gel. The .8 kbp strip of DNA is isolated by electroelution, then confined with Sa * 3A and subjected to electrophoresis on a 6 percent acrylamide gel. The 265 bp band from the PGK promoter (Fig. 11) is isolated by electroelution.

This DNA is then subjected to ligation with the 39 bp promoter fragment from the above paragraph for two hours at room temperature. The ligation mix was bounded with Xbal and Pvul and then subjected to electrophoresis on a 6 percent acrylamide gel. The 312 bp Xba-for-PvuI restriction fragment was isolated by electroelution, then added to a ligation mix containing 200 ng of pBR322 (41) (previously isolated missing Pvul-for-PstI restriction fragment) and 200 ng of XbaI-for-PstI LeIF A cDNA gene previously isolated from 20 [mu] g of pLeIF trp A 25. This three-factor ligation mix is used to transform E. coli strain 294 to tetracycline resistance. The transformant colonies are miniscule (44) and one of the colonies, pPGK-300, is isolated and has 304 bp PGK 5′-flange DNA fused to the LeIF A gene in the pBR322 base vector. The 5 'end of the LeIF A gene has the following sequence:

5'-CTAGAATTC-3 '. Thus, the condensation of the Xbal site from the PGK promoter fragment into this sequence allows it to be added to the Xbal site of the EcoRI site. Thus, pPGK contains a portion of the PGK promoter that is isolated in the Pvul-to-EcoRI fragment.

'J. * * '

Γ. . · 'Γ ι._.:.::

I ..,., '' ... f. j - - * - ···. - - 'a. · .-- - ·, ·· -' V * V. · ... ·,. '

3. Construction of the 1500 bp EcoRI-for-EcoRI PGK promoter fragment yug pB1 was digested with Pvul and EcoRI and chromatographed on a 6 percent acrylamide gel. 1.3 kb Pvul-for-EcoRI DNA strand from PGK 5'-flange DNA was isolated by electroelution and 10 p. g pPGK-300 was digested with EcoRI and Pvul and the promoter fragment of 312 bp was isolated by electroelution after electrophoresis of the digest mixer on a 6 percent acrylamide gel. 5µg of pFRL4 was digested with EcoRI, precipitated with ethanol and then treated with bacterial alkaline phosphatase at 68 ° C for 45 minutes. After three extractions of phenol / chloroform DNA, ethanol precipitation and resuspension in 20 ml Η ₂ θί 200 ng vector ligation with 100 ng 312 bp EcoRI-for-PvuI DNA from pPGK-300 and 100 ng EcoRI-for-Pvu DNA from pBl. The ligation mix was used to transform E. coli strain 294 to ampicillin resistance. One of the transformants obtained was pPGK-1500.

This plasmid contains a 1500 bp PGK promoter fragment as a single EcoRI-for-EcoRI or HindIII-for-EcoRI piece of DNA.

yug pPGK-1500 was completely digested with Clal and EcoRI and then the digester mix was subjected to dextrophoresis on a 6 percent acrylamide gel. A 900 bp fragment containing the PGK promoter was isolated by electroelution. 10 p. g pIFN-gamma trp 48 was completely digested with EcoRI and HincII and subjected to electrophoresis on a 6 percent acrylamide gel. A 938 bp band containing "DNA directly expressable IFN-gamma was isolated from the gel by electroelution.

The yeast expression vector was constructed in a three-factor reaction by joint ligation of the PGK promoter frag-

mint (on a Clal-for-EcoRI piece), omitted pFRM-31, and IFN-gamma cDNA isolated above. The ligation reaction was incubated at 14 ° C for 12 hours. The ligation mix was then used to transform E. coli strain 294 to ampicillin resistance. Transformants were analyzed for the presence of a properly engineered expression plasmid, pPGK-IFNgam. (Figure 16). Plasmid! containing the expression system used. are for. transformation of the yeast RH218 spheroplast strain for tryptophan prototropy into tryptophan-free agar. This recombinant yeast was then analyzed for the presence of immune interferon.

The yeast extracts were made as follows: 10 ml of the cultures were cultured in ΥΝΒ + CAA until Α $ _β θ ^ 1-2 was reached, collected by centrifugation and then resuspended in 500 µL of 1 PBS buffer (20 raM NaH ₂ PO ₄ . pH = 7.4, 150 mM NaCl). An equal volume of glass beads (0.45-0.5 mm) was added and the mixture was then vortexed for 2 '. The extracts were spun for 30 seconds at 14,000 rpm and the supernatant separated. The interferon activity in the supernatant was determined to be 16,000 units / ml based on comparison with the IFN-alpha standard using a CPE inhibition assay.

M. Construction of the pSVgama69 vector of steel culture

The 342 base pair Hindlll-PvuII fragment comprising the SV40 source was translated into the fragment bound to the EcoR1 restriction site. The HindIII site can be translated by the addition of a synthetic oligomer (5 showingAGCTGAATTC) and the PvuII site is translated by blunt-ended ligation in an EcoR1 site that is filled using polymerase 1 (Klen fragment). The resulting EcoR1 fragment was inserted into the EcoR1 site of pML-1 (28).

The plasmid with the SV40 late promoter oriented R from the amp gene was further modified by separating the EcoRI site R of the nearest amp gem pML-1 (27). «

The Hpal-Bglll fragment of a cloned HBV DNA of 1023 base pairs (60) was isolated and the Hpal site of hepatitis B virus (HBV) was converted to an EcoRI site with a synthetic oligomer (5 'dGCGAATTCGC). This EcoRI-Bglll bound fragment was directly cloned into the EcoRI-BamHI site of the plasmid described above carrying the SV40 source.

An IFN-gamma gene was inserted into the remaining EcoRI site on the Pstl fragment of p69 from 1250 base pairs after conversion of Pst1 ends into EcoRI ends. Clones in which the SV40 late promoter converted to the IFN-gamma structural gene were isolated. The resulting plasmids were then introduced into the tissue of the cell cultures (29) using a DEAE-dextran technique (61) that was modified so that transfection in the presence of DEAE-dextran was left for 8 hours. The substrates were changed every 2-3 days. 200 microliters per day was collected to interfere with interferon bioassay. Typical yields were 50-100 units / ml on samples tested three or four days after transfection.

N. Parolee purification of monkey cell derived immune interferon

For the purpose of producing larger quantities of human IFN-gamma derived from monkey cells, fresh COS-7 monolayers of ten 10 cm plates were transfected with a total of 30 µg of pDLIF3 in 110 ml DEAE-dextran (200 µg / ml DEAE / dextran 500,000 MW). .05 M Tris pH 7.5, in DMEM). After 16 hours at 37 ° the plates were washed twice with DMEM. Then on to each

·. .-Si ..

- 44 plates were added 15 ml of fresh DMEM supplemented with 10 percent f.b.s., 2 mM glutamine, 50 µg / ml penicillin G, and 50 mg / ml streptomycin. The substrates are replaced with serum-free DMEM the next day. Then fresh, serum-free substrates are added every day. The collected substrates were kept at 4 ° while either being tested or bound for CPG. The collected fractions from 3 and 4 days post-transfected samples were found to contain substantially all of the activity.

0.5 g of CPG (Controlled Porous Glass, Electronucleonics, CPG 350, mix size 120/200) was added to 100 ml of steel supernatant and the mixture was stirred for 3 hours at 4 ° C. After brief centrifugation in the centrifuge, the deposited beads are packed in a column and completely washed with 20 mM NaHPO ^ 1 M NaCl,

0.1% by beta-mercaptoethanol, pH 7.2. The activity is then eluted with the same buffer containing 30 percent ethylene glycol and then further eluted with the above buffer containing 50 percent ethylene glycol. These fractions were collected and diluted with 20 mM NaHPO 4, 1 M NaCl pH 7.2 to a final concentration of 10 percent ethylene glycol and applied directly to a 10 ml Con Sepharose (Pharmacia) column. After complete washing with 20 mM NaPO4, 1 M NaCl pH 7.2, the activity was eluted with 20 mM NaPO4 1 M NaCl 0.2 M alpha-methyl-D-mannoside. A significant amount of activity (55 percent) did not bind to this lectin. 45 percent of the activity was eluted with alpha-methyl-D-mannoside.

• it

Pharmaceutical preparations

The compounds of the present invention can be formulated according to known methods for making pharmaceutically useful preparations, wherein human immunological interferon

- 45 ~ ν “ϊ combines the product in admixture with pharmaceutically acceptable carriers. Suitable carriers and their formulation are described in Remington's Pharmaceutical Sciences by E.W. Martin, and this publication is incorporated herein by reference. Such preparations will contain an effective amount of interferon protein herein, together with an appropriate amount of carrier, for the purpose of making pharmaceutically acceptable preparations suitable for effective administration to a patient.

A. Parenteral administration

Human immunological interferon can be administered parenterally to patients requiring antitumor or antiviral treatment and to those exhibiting immunosuppressive conditions. Dose and dose rates may be parallel to those currently used in clinical trials with other g

human interferons, e.g., about (1-40) x 10 units per day, and in the case of evening material purity of 1 percent, / r probably up to, e.g., 50 x 10 units per day. Doses of IFN-gamma can be significantly increased for greater effect due to the essential absence of human proteins that are different from IIN-gamma, whereby these proteins in human-derived materials may induce certain undesirable effects.

As one example of a suitable dosage form for substantially homogeneous parenteral IFN-gamma formulation

apply, an IFN-gamma specific activity of, say, 2 x 10 O / mg can be dissolved in 25 ml. 5 N Albumin Serum (Human) - USP, the solution is passed through a bacteriological filter and the filtered solution is aseptically subdivided into 100 vials, each containing 6 x 10 $ units of pure interferon, which is suitable for parenteral administration. The fleas are preferably stacked in the cold (-20 ° C) - s - ^W ··, ί ^ ϊς, 'ί;

before use.

;., <· '· -. - ^J

Biotest data

A. Characterization of antiviral activity

For neutralizing antibodies, samples were diluted, if necessary, to a concentration of 500-1000 units / ml with PBS-BSA. Equal sample volumes are incubated for 2-12 hours at 4 degrees with serial dilutions of rabbit anticancer leukocytes, fibroblasts, or immunological interferon antisertims. Anti-IEN-alpha and beta were obtained from the National Institute of Allergy and Infectious Diseases. Anti-IEN gamma was made using authentic IEN gamma (5-20 percent purity) purified from stimulated peripheral blood lymphocytes. Samples were centrifuged for 3 minutes at 12,000 x g for 3 minutes before testing. For pH 2 stability testing, samples were adjusted to pH 2 by addition of 1 N HCl, incubated for 2-12 h at 4 °, and neutralized by addition of 1N NaCH before testing.

To test for the sensitivity of pran sodium dodecyl sulfate (SDS), samples were incubated with an equal volume of 0.2 percent SDS for 2-12 hours at 4 ° before testing.

B. Characterization of the IEN-gamma produced by pcrooche of E.ooli and COS-7

Antivitus activity (Units / ml)

E. coli «3110 / pI5N-gamatrp48

COS - 7 cells. pSVgama69

Treatment HN-alpha IEN-beta IEN-gamma extract supernatant Untreated 375 125 250 250 62.5 pH 2 375 125 <6 <12 <4 0.1% SDS 375 - 4 <8 - Anti-IEN ^ rabbit <8 125 250 250 187 Anti-IEN-fl rabbit 375 <8 187 250 125 Anti-IFN-tTmichi 375 125 <4 <8 <4

This table shows the characteristic behavior of IFN-alpha, beta and gamma standards after various treatments. Interferon activity produced

- 47 with E. coli W3110 / pIFN-gamma trp 48 and with COS-7 / pSVgama69 acidity sensitive, SDS sensitive and

Λ It is neutralized with the help of immune interferon antiserum.

It is not neutralized by antibody to IFN-alpha or beta.

These data confirm that interferon is produced in these IFN-gamma systems and that the cDNA insert of plasmid p69 encodes for IFN-gamma.

Purification

One method by which IFN-gamma can be purified from, e.g., bacteria is described by the following general scheme:

1. Extraction of cells in buffer for high cell dissolution (at about pH 8) by passing through a high pressure homogenizer, cooling the expiration on an ice bath.

2. DNA deposition by adding polyethylene imine with stirring, for example, at 4 ° C.

3. pH precipitation of bacterial proteins, again breaking and leaving IFN-gamma in solution.

4. Separation of solids by centrifugation at 4 ° C.

5. Concentrating the supernatant (after adjusting the pH) by ultracentrifugation.

6. Dialysis of concentrate versus low conductivity buffer.

7. Separation of solids by centrifugation leaving IFN-gamma in solution.

8. Ion exchange chromatography on carboxymethylcellulose, eluting with a gradient of increasing ionic strength.

9. Chromatography on calcium phosphate gel eluting with a gradient of increasing ionic strength.

¹ .W> £ «rv ·,. · - - 47a

10. Carboxymethylcellulose ion exchange chromatography under poor conditions for elution denaturation with a gradient of increasing ionic strength.

11. Separation of Gel Filtration Chromatographs.

The above procedure yields material yields greater than 95% purity.

The immunological interference protein is defined by DNA gene determination and deductive amino acid sequencing - see Figure 5. It will be clear that for this particular interferon described herein, natural allelic variations occur and occur from individual to individual. These variations can be demonstrated by the difference (s) in the total sequence or omissions, substitutions, insertions, inversions, or additions of the amino acid (s) in said sequence.

All such allelic variations are included within the scope of the present invention.

In addition, it is potent for utilizing recombinant DNA technology to produce various human IFN-gamma derivatives, which are differentially modified by single or multiple substitutions, omissions, additions or substitutions of amino acids. All such modifications that give rise to such derivatives of human IFN-gamma are included within the scope of the present invention, as long as the essential, characteristic activity of human IFN-gamma is not affected.

&

With DNA and amino acid sequences of IFN-gamma in cancer (see Figure 5), the most desirable reproduction flow! the present invention will undoubtedly involve the production of a poppy complex gene by synthetic means (see, for example, 26),

- 47b 47b or synthetic deoxynucleotides with which a cDNA source can be probed to isolate genes by standard

Hib hybridization techniques. As the nucleotide sequence is encoded for the required IFN-gamma protein, the means of expression, isolation and purification to obtain very pure IFN-gamma preparations can be provided as described above.

Although some desirable embodiments are described herein, it will be clear that the present invention should not be limited to such embodiments, but rather to the legal scope of the appended claims.

- 47ο · ί

c

Bibliography

1. Goeddel et al., Nature 287, 411 (1980).

2. Goeddel et al., Nature 290, 20 (1981).

3. Yelverton et al., Nucleic Acids Research 9, 731 (1981).

4. Gutterman et a)., Annals of Int. Med. 93, 399 (1980).

5. Goeddel et a), Nucleic Acids Reseach 8, 4057 (1980).

ί 6, - Y ip et a 1. > P ^roc · Nat); Acad. Sci. (USA) 78, 1601. (1981).

7, Taniguchi et al., Proc. Nat). Acad, Sci. (USA) 78, 3469 (1981). '

8, Bloom, Nature 289, 593 (1980).

9, Sonnenfeld et a)., Cel i ul a r Immuno 1. 40, 285 (1978).

10. Fleishmann et al., Infectlon and Immum'ty 26, 248 (1979).

11. Blalock et a.), Cellular Ininiunolo9y 49, 390 (1980).

12. Rudin et a)., Proc. Nat). Acad. Sci. (USA) 77, 5928 (1980) 7.

13.i '> Crane et a)., J Nat) Cancer Inst. 61, 871 (1978).

14. Stinchcomb et al., Nature 282, 39 (1979).

15. Kingsman et a)., Gene 7, 141 (1979).

16. Tschumper et al., Gene 10, 157 (1980).

17. Mortimer et al., Microbio ogical Reviews 44, 519 (198),

18. Miozzari et al., Journal of Bacter1ology 134, 48 (1978).

19. Jones, Genetics 85, 23 (1977).

20. Hitzeman, et al., J. Biol. Chem. 255, 12073 (1980).

21. Hess et a)., J. Adv. Enzyme Regul. T_, 149 (1968).

22. Holland et al., Biochemistry 17, 4900 (1978).

23. Bostlan et al., Proc. Nat). Acad. Sci. (USA) 77, 4504 (1980). :

24. The Molecular Bio) ogy of Yeast (Aug 11-18, 1981), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

25. Chambon, Ann. Rev. Biocherci stry, 44, 613 (1975).

25a. Gluzman, Cel) 23, 175 (1981).

26. Goeddel et al., Nature 231, 544 (1979).

27. Itakura et al., Science 198, 1056 (1977),

28. 'Lus ky et al., Nature 293, 79 (1981).

29. Gluzman et al., Cold Spring Harbor Syrap. Quant. Biol.

,. 293 τΓ97Τυ).

1 r '- ¹⁸ - r 30. F i e rs et ΔΙ- » Nature 273, 113 (1978). 31. Reddy et il · · Science 200, 494 (1978). 32. 8oedtker et al ., Prog. and Nucleic Acids Res. Mol. Biol

Γ9, 253 (17757. '~' ~~

33. Berger et al., 8iochemistry 18, 5143 (1979).

. A vi v e t a 1., away. Natl. Acad. Sci. USA 59, 1408 (1972).

35. Gurdon et al., J. Molec. Biol. 80, 539 (1975).

36. Stewart, The Interferon System. Springer, New York, p.

13-26 (197ΤΠ

37. Lehrach et a)., Bi oc henii stry 16, 4743 (1977).

38. Lynch et al., Vi rol ogy 98, 251 (1979).

39. Hickens et al., J. 8 ί o T. Chem, 253, 2483 (1978).

40. Chang et a)., Natu re 275, 617 (1978).

41. Bolivar et al., Gene 2, 95 (1977).

42. Grunsteln et al., Proc. Natl. Acad. Chief. U.S.A. 72, 3961 (1975). ~

43. Fritsch et al., Whole 19, 959 (1980).

44. 'Birnboim et al., NUcleic Acids Res'. 7_, 1513 (1979).

45. Kafatos et a 1. Nucleic Acids Res. 7_, 1541 (1979),

46. Clewell et al., Biochemistry 9, 4428 (1970).

47. Taylor et al., Biochim. Biophys. Acta 442, 324 (1976).

48. Smith, Methods Enzymol. 65, 560 (1980).

49. Messing et al., Nucleic Acids Res. 9_, 309 (1981),

50. Hinzler, Hormone! Proteins and Peptides (ed. Li) Academic

Press, New York, p. 1 (1973).

51. Nathan et al., Nature 292, 842 (1981).

52. Maxam et a 1., Methods in Enzymol. 65, 490 (1980).

53. Crea e_t al .., Prog Natl Acad. Sc-i (USA) 7_5, 5765 (1978).

54. Southern, J. Prayer. Biol. 98, 503 (1975).

55. Saliva e_t aj_., Nucleic Acids Res. 3, 2303 (1976).

56. Lawn et al., Science 212, 1159 (1981).

57. Lawn et al., Nucleic Acids Res. 9, 1045 (1981).

58. Miller, £ xperiraents in Molecular Genetics, p. 431-3, Cold

Spring Harbor Lab., Cold Spring Harbor, New York (1972).

59. Beggs, Nature 275, 104 (1978).

i

- 4S .60.

61.

Valenzuela et al., Animal Virus Genetics (ed. Fields, Jaenisch and Fox) p. 57, Academic Press, New York (1980). McCuthan et al., J. Natl. Cancer Inst. 41, 351 (1968)

Claims (13)

Patent claims A polypeptide having human immunointerferon (TFN) properties, comprising the following amino acid sequence: 5 ' FIG TnAOTCAGCTAnAGAAGAGAAAGATCAGTTAAGTCCTTTGGACCTGATCAGCTTGATACAAGAACTACreATrTCAACTTCTTTGGCTTAATTCTCTCGGAAACG ATG MA TAT - io 100 S10 S20 1 10 he gin leu eys ile val leu p.ly ser leu gly CYS TYR CYS CLN ASP PRO TYR VAL LYS CLU ALA CLU ASN chr ser claimed leu ala nhe gin leu eys no val leu pty str leu gly tli uk ti »Ttn Air mu uk ml ι.ι» ι.ι.ιι ai.a vi.ij as ACA AGT TAT ATC TTG GCT ITT CAG CTC TGC ATC GTT TTG GGT TCT CTT GGC TGT TAC TGC CAG GAC CCA TAT GTA ΑΛΑ GAA GCA GAA 150 200 20 30 40 LEU LYS LYS TVS PHE ASN ALA CLY HIS SER ASP VAL ALA ASP ASM CLY TUR LEU PI1E LEU CLY ILE LEU LYS ASN TRP LYS CLU CLU SER CK AAG AAA Ϊ ~ Τ TTT ΑΛΤ GCA. GGT CAT TCA GAT GTA GCG GAT AAT GGA ACT CTT TTC TTA GGC ATT TTG AAG ΑΛΤ TGG AAA GAG GAG AGT 250 50 60 20 ASP AP.C LYS ILE HET CLN SER CLN ILE VAL SER PHE TYR PHE LYS LEU PHE LYS ASN PHE LYS ASP ASP CLN SER ILE CLN LYS SER VAL & £ KA AAA ATA ATG V £ N £ UA 'ATT GTC TCC TIT TAC TTC AAA CTT TTT AAA AAC TTT AAA GAT GAC CAG AGC ATC CAA AAG AGT GTG 300 350 80 90 100 CLU TUR ILE LYS CLU ASP MET ASN VAL LYS PHE PHE ASN SER ASN LYS LYS LYS ARC ASP ASP PHE CLU LYS LEU THR ASN TYR SER VAL GAG ACC ATC AAG GAA GAC ATG AAT GTC AAG TTT TTC AAT AGC AAC AAA AAG AAA CGA GaT GAC TTC GAA AAG CTG ACl ml Irti IUj blft 400 450 110 120 130 THR ASP LEU ASN VAL CLN ARC LYS ALA ILE HIS CLU LEU ILE CLN VAL KKT ALA CLU LEU SER PRO ALA ΑΙ.Λ I.YS TUR CI.Y ACT GAC TTG V -.- T GTC CAA CGC AAA GCA ATA CAT GAA CTC ATC CAA GTG ATG GCT GAA CTG TCG CCA GCA GCT ΑΛΑ NA GGG 500 550 LYS AAG ARC I.YS Λ'A vCn «n 160 146 STOP ARC SER CLN ΧΞΤ LEU P11E ARC CLY ARC ARC ALA SER CLN _ AGG AGT CAG ATG CTG TTT CGA GGT CGA Λ6Α GCA TCC CAG ΤΑΛ TGGTTGTCCTGCCTIZMTATnGAAnTrAAATCTAAATCTATnATTAATATnA / VLATTA 6U0 650 TTTATATGGGGAATATATTTTTAGACTCATCAATCAAATAAGTATTTATAATAGCAACTTTTGTGTAATGAAAATGAATATCTATTAATATATGTATTATTTATAATTCCTATATCCTG 7U0 75U TGACTGTCTCACTTAATCCTTTGTTTTCTGACTAATTAGGCAAGGCTATGTGATTACAAGGCTTTATCTCAGGGGCCAACTAGGCAGCCAACCTAAGCAAGATCCCATGGTTGTGTGTT 800 850 900 TATTTCACTTGATGATACAATGAACACTTATAAGTGAAGTGATACTATCCAGTTACTGCCGGTTTGAAAATATGCCTGCAATCTGAGCCAGTGCTTTAATGGCATGTCAGACAGAACTT 950 · 1000 GAATGTGTCAGGTG-ACCCTGATGAAAACAT / iGCATCTCAGGAGATTTCATGCCTGGTGCnCCMATATTGITGACAACTGTGACTGTACCCAAATGGAAMjTAACTCATTTGnAAAA 1050 uoo TTATCAATATCTAATATATATGAATAAAGTGTAAGTTCACAACTAAAAAAAAAAAAAAAAAAAAA H 50 120U 3 ' 0184M 51. The polypeptide of claim 1, which is lacking at the N-terminal end of the shown sequence 1-146, tripetide CYS-TYR-CYS. 3. The polypeptide of claim 1, further comprising methionine bound at the N-terminal. the end of the first amino acid of intferon. The polypeptide of claim 1, wherein ^? it additionally contains a cleavable conjugate or signaling protein, which is N- bound. the terminal end of the first amino acid of interferon. A polypeptide according to any one of claims 1> -3 and 4, which is not bound to the natural glycolysis of Jonr. A DNA isolate with one for the polypeptide according to claims 1-4, encoding a DNA sequence. A replicating expression carrier, which is capable of expressing a polypeptide according to any one of claims 1-4 in a microorganism or cell culture. t 8. A plasmid selected from the group consisting of pIFN-gamma-trp 48, pSV-gamma-69 and pPGK-IFN-gamma. A microorganism or cell culture transforated with an expression carrier according to claims 7 and 8. Cell culture according to claim 9, obtained by transformation of COS-7-line.is monkey kidney fibroblasts. A microorganism according to claim 9, obtained by transformation of pure E. coli culture. A microorganism according to claim 9, obtained by transformation of a pure yeast culture. 52. A method for producing a polypeptide with human immunointerferon (IFN-gamma) properties according to one of claims 1-4, characterized in that the transformed roichroananism or transformed cell culture according to claims 9-12 expresses the gene encoding the polypeptide according to one of the requirements