LT4012B - Process for the preparation of polipeptides - Google Patents

Description

The present invention relates to the manipulation of genetic materials and, in part, to recombinant techniques for the production of polypeptides having all or part of the parent structural conformation and / or one or more of the biological properties of free erythropoietin.

A. Manipulation with genetic materials

In a broad sense, genetic materials can be defined as chemicals that program and control the production of cellular and viral components and determine cellular and viral responses. The long-chain polymeric compound known as deoxyribonucleic acid (DNA) contains the genetic material of all living cells and viruses except for some viruses programmed with ribonucleic acid (RNA). The structured DNA polymers contain four different nucleotides, each consisting of either purine (adenine and guanine) or pyrimidine (thymine and cytosine) bound to a deoxyribose-sugar to which a phosphate group is attached. Nucleotide binding in the form of a linear polymer occurs by coupling one nucleotide 5'-phosphate with another nucleotide's 3 ¹ -hydroxyl group. Functional DNA is found in the form of stable double-stranded strands consisting of single nucleotide strands (known as deoxyoligonucleotides) formed by a hydrogen bond between purine and pyrimidine bases, that is, a complementary strand existing either between adenine (A) and thymine (A). guanine (G) and cytosine (C). Nucleotides are commonly referred to by the names of their purine or pyrimidine bases, and complementary nucleotides in double-stranded DNA (ie, AT and GC) are referred to as pairs of nitrogenous bases. Ribonucleic acid is a polynucleotide containing more often adenine, guanine, cytosine and uracil (U) than thymine bound to ribose or phosphate group.

In short, DNA programming function is usually accomplished by the process of transcribing specific nucleotide sequences (genes) into relatively unstable polymers of information RNA (iRNA). In turn, iRNA becomes a matrix for the formation of structural, regulatory, and catalytic proteins from amino acids. This process of iRNA translation involves the functioning of small RNA strands (tRNAs). which transport and align individual amino acids along the strands of the iRNA, allowing nucleotides to form the required amino acid sequence. The iRNA information obtained from the DNA, which enables the tRNA to be delivered and the orientation of any of the twenty amino acids to express the polypeptide, is in the form of triplet codons (three nucleotide bases of particular order). In a sense, protein formation is the programmed expression of the final genetic information (determined by the nucleotide sequence of the gene).

DNA promoter sequences typically occur earlier than a gene in a DNA polymer and prepare the site for initiation of transcription into RNA. DNA regulatory sequences are also more often upstream (i.e., earlier) than the gene in this DNA polymer that binds proteins that determine the frequency (or rate) of transcriptional initiation. Collectively referred to as “promoter / regulatory or regulatory DNA sequences, these sequences, which occur before a particular gene (or group of genes) in a functional DNA polymer, determine whether transcription and, ultimately, gene expression will occur. DNA sequences that accompany a gene in a DNA polymer and provide a command to terminate transcription into an iRNA are referred to as transcription terminator sequences.

Microbiological technology of the last decade has focused on attempts to produce industrial and pharmaceutical materials using microorganisms that either do not have pre-genetically encoded information embedded in their DNA desired product or (mammalian cells in culture) do not express detectable levels of the chromosomal gene in the usual way. Simply put, the gene that determines the structure of the desired polypeptide product is either isolated from the donor microorganism or chemically synthesized and stably introduced into another microorganism (preferably self-regulating single-cell microorganism), such as bacteria, yeast or mammalian cells in culture. Existing equipment for gene expression in transformed or transfected host microbial cells works by utilizing exogenous DNA as an transcription matrix of iRNA, which is then translated into an endless sequence of amino acid residues.

This field of technology is often found in patent and literary publications relating to recombinant RNA methodologies, -. for the isolation, synthesis, purification and amplification of genetic material (used for transformation of selected host microorganisms). U.S. Pat. No. 4,237,224 to Cohen et al., For example, is directed to the transformation of single-cell host microorganisms with hybrid viral or annular plasmid DNA combining selected exogenous DNA sequences. Cohen et al. The patent procedures for the first time utilize the production of a transformation vector based on enzymatically cleaved viral or circular plasmid DNA, which is required to form linear DNA strands. Selected foreign ("exogenous" or heterologous) DNA strands, usually having the coding sequences of the desired product, are obtained in a linear fashion using similar enzymes. Linear viral or plasmid DNA is raised with foreign DNA in the presence of stitching enzymes capable of carrying out the repair process, and hybrid vectors are formed by having a selected exogenous DNA segment wrapped in a viral or circular DNA plasmid.

Transformation of compatible host single-cell microorganisms with a hybrid vector results in multiple generation of exogenous copies of DNA in the host cell population. In some cases, the desired result is simply amplification of the foreign DNA, and the grown product contains DNA. More commonly, the purpose of transformation is expression realized by the exogenous DNA of host cells as large-scale commercially important protein fragments or polypeptide encoded by foreign DNA. See also, for example, U.S. Patents No. 4264731 (issued to Shine), No. 4273875 (issued to Manis), No. 4293652 (issued to Cohen), and European Patent Application No. 093619, published October 9, 1983.

The development of specific DNA sequences prior to splicing into DNA vectors is accomplished by a number of techniques, which are largely dependent on the degree of foreignness of the donor to the designed host and the size of the polypeptide formed by the host. Fear of over-simplification, it can be argued that there are three main alternatives: 1) "isolation" of the double-stranded DNA sequence from the donor genomic DNA; 2) chemical synthesis of the DNA sequence generating the code of the polypeptide of interest; 3) in vitro DNA sequence synthesis. using enzymatic reverse transcription of iRNAs isolated from donor cells. The aforementioned techniques involving the use of DNA-iRNA complement formation are commonly referred to as cDNA techniques.

Obtaining DNA sequences is often the method of choice when the full amino acid sequence of the desired polypeptide is known. Methods for obtaining DNA are described in the co-pending U.S. Patent No. 4,834,451 (filed April 15, 1983 and, respectively, PCT US 83/0085, published November 24, 1983, under No. WO83 / 04053), for example, providing tools for such highly anticipated results. achievement as:

(1) prediction of alternating codons commonly observed in the host microorganism selected for expression (eg, prediction of the best codon in yeast or E. coli); 2) avoidance of untranslated intronic sequences (typically found in mammalian genomic DNA sequences and their RNA arrays) that are readily processed by host cells; 3) avoidance of unwanted polypeptide sequences (usually encoded by genomic DNA and cDNA sequences but often difficult to distinguish, using bacterial or yeast host cells, from the polypeptide of interest); 4) predicting easy insertion of DNA into suitable expression vectors in templates with desired promoter / regulatory and terminator sequences; 5) predicting easy assembly of genes encoding polypeptide fragments and analogs of desired polypeptides.

When the complete amino acid residue sequence of the desired polypeptide is not known, direct DNA sequencing is not possible. Isolation of DNA sequences encoding polypeptides by the cDNA method becomes a method of selection, despite the potential difficulties of fitting expression vectors (capable of providing a high level of microbial expression as mentioned above). Standard methods for isolating cDNA sequences include plasmid cDNA libraries resulting from reverse transcription of cDNAs, often found in donor cells selected for high levels of gene expression (e.g., cDNA libraries derived from cells expressing relatively high levels of growth hormone products). When large portions of the amino acid sequence of polypeptides are known, then labeled probe single-stranded DNA sequences that duplicate the sequence presumed in the target cDNA can be used in the methods described in Weissman et al., U.S. Patent No. 4,394,443. The use of long oligonucleotide hybridization probes is discussed in Wallace et al., Nucl. Acids Res., 6, 3543-3557 (1979), Reyes et al. P.N.A.S. (USA), 79, pp.3270-3274 (1982) and Iaye et al., Nucl. Acids Res., 11, 2325-2335 (1983). See also U.S. Patent No. 4,358,335 to Falkow et al. and descriptive methodologies for DNA / DNA hybridization (during diagnostics); European Patent Applications Nos. 0070685 and 0070687, assigned to light emitting tags in single-stranded polynucleotide probes; work: Davis et al. Manual for Genetic Engineering anoanced Bacteriae Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. p.55-58 and p.174-176 (1980) for hybridization techniques and epidemic diseases and New England Nuclear (Boston, Mass), Gene. Screen Hybridization Transfer membrane materials.

One of the most important recent advances in techniques for recombinant clone screening hybridization is the use of labeled hybrid oligonucleotide probes, each of which is a complete complement of a specific DNA sequence comprising a heterogeneous mixture of single-stranded DNA and RNA. These methodologies have been found to be particularly useful for the detection of cDNA clones obtained from sources that generate very small amounts of the polypeptide iRNA sequences of interest. In short, the use of stringent hybridization conditions (to avoid nonspecific fusions) sometimes clonautoradiographic visualization at the end of a single mixture probe that is in its full complement. Mostly see: Wallace et al., Nuc. Acids, Res., 9, pp. 879-897 (1981); Suggs et al., P.N.A.S. (USA), 78, pp. 6613-6617 (1981); Choo et al., Nature, 299, p. 178180 (1982); Kurachi et al., P.N.A.S. (USA), 79, p.64616464 (1982); Ohkubo et al., P.N.A.S. (USA), 80, pp. 2196-2200 (1983); Kornblihtt et al., P.N.A.S. (USA), 80, p.32183222 (1983).

In general, work: Wallace et al. (1981) mixed probe techniques have been extended by various authors to the point where reliable results were reported for the isolation of cDNA clones using hybrid 32-membered oligonucleotide probes to enable specific cDNA hybridization.

Ί a pool of 16 bases (16-men) of uniformly fluctuating DNA sequences, together with a unit of 11-men for two-way positive confirmation of the cDNA of interest. See the work in Singer-Sam et al., P.N.A.S. (USA), 80, p.802806 (1983).

The use of genomic DNA isolates from the three methods mentioned above is the least common for the development of specific sequences used in recombinant techniques. This is particularly true for recombinant methodologies for the microbial expression of mammalian polypeptides, and is largely determined by the complexity of mammalian genomic DNA. Thus, although reliable methodologies exist for the development of human and other mammalian genomic DNA [see, for example, Lown et al., 15, pp.1157-1174 (1978), for methodologies for generating a human genomic library (commonly referred to as Maniatis library). ; work by Karn et al.,

P.N.A.S. (USA), 77, pp. 5172-5176 (1980), which deals with the human genomic library by an alternative restriction method of endonuclease fragmentation; Blatnes et al., Science, 196, p.161-169 (1977), describing the structure of a bovine genomic library], have undergone some relatively successful attempts using hybridization in isolated genomic DNA techniques without the prospective prediction of amino acids or DNA sequences. An example is Fiddes et al. J. Mol. and App. Genetics, 1, pp. 3-18 (1981), which reports a successfully isolated gene encoding an alpha subunit of human pituitary glycoprotein hormones using a maximally long probe comprising 621 complete pairs of nitrogenous bases (cDNA sequences previously isolated to the alpha subunit). ). In another work, Das et al., P.N.A.S. (USA), 80, pp. 1531-1535 (1983) reported the isolation of human genomic clones in a HLA-DR human using 175 pairs of nitrogen-based synthetic oligonucleotides. And finally, in the work of Anderson et al., P.N.A.S. (USA), 80, pp. 6838-6842 (1983) reported the isolation of a bovine genomic clone pancreatin trypsin inhibitor (BPTI) using a single probe having a length of 86 nitrogen bases and assembled according to a known amino acid sequence of BPTI. The authors note the poor prospects for isolation of iRNAs suitable for synthesis of the cDNA library, since apparently low levels of iRNAs in primary sources, parotid gland and lung tissue, are observed. Thereafter, the hope is expressed that the probe of the genomic library using a mixture of labeled probes will be successful and it is stated: Mixed sequence oligodeoxynucleotide probes were mainly used to isolate protein sequences of unknown sequence from cDNA libraries. Such probes are typically a mixture of 8-32 oligonucleotides having a length of 14-17 nucleotides and are representative of each of the sampled small length (5-6 residues) amino acid sequence codon combinations. Under stringent hybridization conditions, where the poor situation for mismatched nitrogen residue probes is created, these mixtures are capable of localizing gene-specific sequences in low-complexity clonal libraries. Nevertheless, due to their short length and heterogeneity, hybrid probes often lose the specificity needed to probe complex sequences such as the mammalian genome. As a result, this approach becomes impractical for the isolation of mammalian protein genes when the corresponding iRNAs are unavailable. ”(No references).

Thus, there remains a need in the art to develop methods for rapid and efficient isolation of cDNA clones, especially when little is known about amino acid sequences and enriched iRNA tissue sources are not readily available for cDNA library assembly. Such improved methods would be particularly useful if they could be used to isolate mammalian genomic clones when information on the amino acid sequences of the polypeptide (encoded by the gene being searched for) was incomplete.

B. Erythropoietin as the dominant polypeptide

Erythropoiesis (production of red blood cells) occurs throughout human life to make up for the disruption of cells. Erythropoiesis is a highly regulated physiological mechanism that allows the blood to produce a sufficient number of red blood cells, which are needed for proper oxygenation in the body, but not so high that the cells interfere with blood flow. The production of red blood cells occurs in the bone marrow under the control of the hormone erythropoietin.

Erythropoietin, an acidic glycoprotein with a molecular weight of about 34,000 Daltons, can be found in three forms: ^, fi> and asial. and [2> forms differ slightly in carbohydrate components, but have the same potency, biological activity and molecular weight. The form of asial is the form of ck and β> without terminal carbohydrate (sialic acid). Plasma concentrations of erythropoietin are very low when the body is healthy and the tissues receive sufficient oxygenation from the red blood cells. This normally low concentration is sufficient for normal red blood cell turnover, which is usually killed by oxidation.

Plasma erythropoietin levels increase with hypoxia, resulting in a reduction of blood oxygenation. Hypoxia can be due to the loss of high blood volume during bleeding, the erosion of red blood cells due to radiation, decreased oxygen uptake or prolonged unconsciousness, and various forms of anemia.

In hypoxia-exposed tissues, erythropeptin increases the production of red blood cells by stimulating the conversion of primary hematopoietic cells to pro-erythroblasts in the bone marrow, where it gradually matures, synthesizes hemoglobin, and is released into the bloodstream as red blood cells. Erythropoietin blood flow is reduced when red blood cells exceed the levels needed to provide oxygen to the tissues.

See mainly the following works: Test et al., Exp. Hematol, 8 (Supp.8), 144-152 (1980); Tong et al., 1981, J. Biol Chem. 256 (24): 12666-12672; Goldvasser, J., Cell Plupiol, 110 (Supp.I), 133-135 (1982); Finch, Blood., 60 (6), 12411246 (1982); Sytowski et al., Exp. Hemat., 8 (Supp.8), 5264 (1980); Naughton, Cenn, Clin. Lab. Sec., 13 (5), 432438 (1983); Weiss et al., Am. J., Vet Res, 44 (10), 18321835 (1983); Lappin et al., Exp. Hematol, 11 (7), 661-666 (1983); Murphy et al., Act Hematologica Japonica 46 (7), 13801396 (1983); Dessypris et al., Brit. J · Haematol, 56, 295306 (11984); and Enumananael et al., Am. J.Physiol, 247 {1 pt

2), 168-76 (1984).

Because erythropoietin is important for the production of red blood cells, the hormone has potentially beneficial uses both in diagnostics and in the treatment of blood disorders associated with low or insufficient red blood cell counts. See the following works: Penathur-Das et al., Blood, 63 (5), 1168-1171 (1984) and Haddy, Asn. Yuor. Ped. Hematol./Oncol., 4, 191-196 (1982) on erythropoietin in the treatment of diseases, and Eschbach et al., J. Clin. Invest., 74 (2), 434-441 (1984), which describes a therapeutic regimen for uremic oestrus based on an in vivo response to infusion of plasma enriched with erythropoietin, and proposes a dose of 10 U EPO / kg / day that corrects for this type of , associated with chronic renal failure, anemia for 15-40 days. on the track. See also works at Crane, Henry Ford, Hosp. Med. J., 31 (3), 177-181 (1983). Recently, it has been estimated that the presence of sufficient amounts of erythropoietin in the US alone will cure 1600,000 people a year. See, e.g., Morrison, "Bioprocessing in Space on Overviev", p. 557-571 in World Biotech. Report 1984, Volume 2: USA, Online Publications, New York, N.Y. (1984). Recent studies have provided the basis for the efficacy of proposed erythropoietin therapy in a variety of hematologic disorders, disorders and conditions: Vedaoto et al., 1984, Acta Haematol., 71, 211-213 (beta-thalassemia); Oichinsky et al., J. Pediatr., 105 (1), 15-21 (1984) (cystic fibrosis); Cotes et al., Brit. J. Obstet. Gyneacol., 94 (4), 304311 (1983) (pregnancy, menstrual disorders); Haga et al., Act. Pediatr. Scand., 72, 827-831 (1983) (early anemia of prematurity); Claus-Walker et al., Arch. Phys. Med. Rehabil., 65, 370-374 (1984) (spinal cord injury); Dunn et al., Eur. J. Appl. Physiol., 52, 178-182 (1984) (space flight); Miller et al., Brit. J. Haematol., 52, 545-590 (1982) (acute blood loss); Udupe et al., J. Lab. Clin. Med., 103 (4), 574-580 and 581-588 (1984); Lipschitz et al., Blood, 63 (3), 502-509 (1983) (aging); Dainiah et al., Cancer, 51 (6), 1101-1106 (1983) and Schvvartz et al., Otolaryngol., 109, 269-272 (1983) (various diseases of noeplastic conditions accompanied by abnormal erythropoiesis).

Previous attempts to obtain erythropoietin with good plasma or urinary yield have been relatively unsuccessful. Small amounts of contaminated and unstable extracts containing erythropoietin are usually obtained with sophisticated and demanding laboratory techniques.

the method of isolation from diseased patients is described in Miyake et al., J. August 10) pp.5558-5564. This includes ion exchange chromatography, gel filtration, adsorbent activity 70400

U.S. Patent No. 3033758 describes a method for partially purifying erythropoietin from plasma of lambs when a low amount of an impure erythropoietin-containing erythropoietin is obtained. Initial tests have shown the release of unstable, biologically inactive hormone erythropoietin from urine. U.S. Patent No. 3,865,801 describes a method for stabilizing the biological activity of an urinary substance containing erythropoietin. Erythropoietin is obtained with 90% erythropoietin activity and stability.

Another erythropoietin in aplastic anemia, urinary Biol. Chem., 252, 15 (1977, Seven-Step Ethanol Deposition, Chromatography. Erythropoietin is obtained.

V / mg protein at 21% yield.

U.S. Patent No. 4,379,740 to Takezavvra et al., Describes methods of "obtaining an erythropoietin product" from urine in healthy individuals with painful cations. It is stated that low molecular weight products "have no inhibitory effect on erythropoietin".

In British Patent Application No. 2085887, Sigimoto et al. A method for obtaining hybrid lymphoblastoid human cells is described on May 6, 2005 and the production level of erythropoietin is reported to be 3-420 units / ml of cell suspension (distributed after culture in host-mammalian concentrations).

At the highest fitting, must be achieved, speeds of 40 to 4000 units / ¹⁰⁶ cells per 48 hours. in vitro culture with subsequent cell translocation to proliferation systems in vivo. (See the corresponding equivalent U.S. Patent No. 4,375,713).

There have been many suggestions for the elimination of erythropoietin from tissue sources, but its yield has been very low. See, e.g., Jekman et al., Exp. Hematol., 11 (7): 581-588 (1983); Tambaurin et al., PNAS (USA), 80, 6269-6273 (1983); Katsuoka et al., Gann, 74, 534-541 (1983); Hagiwara at 10 ⁷ cells / ml which is a product of production, erythropoietin et al., Blood, 63 (4), 828-835 (1984); and Choppin et al., Blood, 64 (2), 341-347 (1984).

Other isolation techniques used to obtain purified erythropoietin included immunoassays. The polyclonal, a derivative of a plasma antibody directed against erythropoietin, develops by injection into human or erythropoietin rats or rabbits. The injected human erythropoietin will be recognized as an antigenic foreign body in the animal's immune system and will trigger the production of antibodies against the antigen.

Different cells respond to antigenic stimulation to produce and circulate antibodies in the bloodstream that are slightly different from those produced by other reactive cells. When an animal's blood is extracted, its activity in the plasma remains. And although crude plasma or antibody preparations purified as the plasma fraction of immunoglobulin G can subsequently be used to detect complexation with human erythropoietin, it is not convenient. This plasma antibody, made up of a variety of different antibodies produced by individual cells, is polyclonal in nature and forms complexes in its constituent extracts in a manner different from single-unit erythropoietin.

The present invention has been of interest in recent developments in the cultivation of intact cell cultures that are capable of producing a unit type of antibody that is specifically immunologically active against the unit antigenic determinant of a selected antigen. See work: Chisholm, High Technology, Vol 3, No.1, pp.57-63 (1983). Attempts have been made to use cell fusion and hybridization techniques for the development of "monoclonal" antibodies to erythropoietin and for the isolation and quantification of human erythropoietin. An example is a report in the form of an annotation at work: Leo-Huang, Abstract No.1463, Fed. Proc., 41, p.520 (1982), which refers to the successful generation of a mouse-mouse hybridoma cell line capable of secreting human erythropoietin monoclonal antibodies. Another example would be a step-by-step description of the preparation and use of a monoclonal anti-poetic antibody in Weiss et al., P.N.A.S. (USA), 79, 5465-5469 (1982). See also works: Sasaki, Biomed.

Biochim. Act, 11/11/12. p. 202-206 (1983); Yanagawa et al., Blood, 64 (2), 357-364 (1984); Yanagawa et al., J. Biol. Chem.

259 (5), 2707-2710 (1984) and U.S. Patent No. 4,456,624.

Also of interest in the present invention are reports on the immunological activity of synthetic peptides, which essentially duplicate the amino acid sequence retained in freely found proteins, glycoproteins, and nucleoproteins. Specifically, polypeptides with relatively lower molecular weights are shown to be involved in immune responses, with time-dependent immune responses to physiologically relevant proteins (such as viral antigens, polypeptide hormones, etc.). Such polypeptide immune responses include the provocation of specific antibodies in immunologically active animals. See, for example, Lerner et al., Cell, 23,309-310 (1981); Ross et al., Nature, 294, 654656 (1981); Walter et al., PNAS (USA), 77.5197-5200 (1980); Learner et al., PNAS (USA), 78, 3403-3407 (1981); Walter et al., PNAS (USA), 78, 4882-4886 (1981); Wangetal, PNAS (USA), 78.7412-7416 (1981); Breen et al., Cell, 28, 477487 (1982); Niggetac et al .; PNAS (USA) 79,53225326 (1982); Baron et al., Cell, 28, 395-404 (1982); Dresman et al., Nature 295: 158-160 (1982); Lerner, Scientific American, 248, no.2, 66-74 (1983). See also work: Kaiser et al., Science, 223, p. 249-255 (1984) concerning the immunological and biological activity of synthetic peptides, which approximate secondary peptide hormone structures but may have different primary structural conformations. Of course, these studies concern the amino acid sequences of proteins other than erythropoietin, since no substantial information has been published on the latter. United States Patent Application No. 4,637,224 to J.Egrie, 1983; February 4, 1984, August 22 European Patent Application No. 0116446 describes a hybridoma mouse-mouse cell line (ATCC ΝΓ.ΗΒ20209) that produces a highly specific monoclonal anti-erythropoietin antibody, incidentally, to an immuno-inactive polypeptide having the following amino acid sequence: NH ₂ -Ala-Pro-Pro -Arg-Leu -Thr-Cys-Asp-Ser-Arg-Val-Leu-Glu-ArgTyr-Leu-Leu-Glu-Ala-Lys-COOH.

A polypeptide sequence is a sequence that binds to the first twenty amino acid residues of a mature human erythropoietin isolated according to the procedure described in Meyake et al., J. Biol. Chem., 252, 5558-5564 (1974), which was subjected to amino acid analysis by gas phase sequencer (Applied Biosystems) according to the procedure: Hevvik M., J. Biol. Chem., 256, 79907997 (1981). See also work: Sue et al., Proc. Nat. Acad. Sci. (USA), 80, 3651-3655 (1983) describing the development of polyclonal antibodies (against a synthetic 26-amino acid amino acid sequence basis) and the work of: Sytowski et al., J. Immun. Methods, 69, 181-186 (1984).

Although monoclonal and polyclonal antibodies, as described above, contain very useful substances for immunoassays for the discovery and quantification of erythropoietin and may be useful in the purification of erythropoietin, it appears unlikely that these large-scale secretory substances can provide sufficient amounts of erythropoietin of mammalian origin. for further analysis, clinical trials, and possible widespread therapeutic use in the treatment of, for example, chronic kidney disease, where erythropoietin production is no longer guaranteed by damaged tissue. Therefore, recombinant techniques for the large-scale microbial synthesis of the compound are expected to be the most promising for the full characterization of mammalian erythropoietin and for the production of large amounts (possibly for diagnostic and clinical use).

Although considerable efforts have been made to isolate the DNA sequences encoding erythropoietin in humans and other mammals, no attempt has been made. This is due to the scarcity of tissues (particularly human) and the presence of iRNA rich enough to assemble a cDNA library from which DNA sequences encoding erythropoietin can be isolated (using known methodologies). In addition, little is known about the uninterrupted amino acid residue sequences of erythropoietin, which cannot, for example, result in the installation of long polynucleotide probes suitable for reliable selection of DNA / DNA-hybridized cDNAs and especially DNA genomic libraries. The twenty amino acid sequence used for the aforementioned monoclonal antibody generated by A.T.C.C. No. HB8209, did not allow the construction of an unambiguous sixty-base oligonucleotide probe as described by Anderson et al., Supra. The genetic material encoding human erythropoietin is estimated to represent less than 0.00005% of the total human genomic DNA that must be in the genomic library.

The most successful attempts known to date to construct DNA sequences by recombinant methods suitable for the microbial expression of isolated amounts of erythropoietin in mammals have by no means succeeded. For example, see Farber et al., Exp. Hematol, 11, Supp. 14, Abstract, 101 (1983), which reports the extraction of iRNA from baboon kidney tissues treated with phenylhydrazine and injection of iRNA into Xenopus Laevis oocytes with a relatively transient mixture of translational products' ¹ that have expressed erythropoietin biological properties. More recently, Farber et al., Blood, 62, no.5, Sapp. No.1, Abstract 392, p.122a (1983) reported the translation of human renal iRNA in frog oocytes in vitro. The resulting translation product mixture was evaluated to contain approximately 220 mg of the translation product having activity per ml of erythropoietin iRNA introduced. And while exogenous levels of exogenous translation of exogenous iRNAs encoding erythropoietin have been found to be completely low (even compared to previously published levels of baboon's iRNA translation into the target product), the results suggest that the human kidney is an expression domain of erythropoietin - a library from which the desired gene can be extracted. [See also Farber, Clin. Res., 31 (4), 769A (1983).]

Since filing U.S. Patent Nos. 561024 and 582185, only one report has been published on the cloning and expression of what is recognized as human erythropoietin cDNA into E. coli. In short, a number of cDNA clones were introduced into E. coli plasmids, and the β2-lactamase fusion products were recognized as immunoreactive (with a monoclonal antibody) to an unidentified epitope on human erythropoietin. See Lee-Huang, P. Nat. Acad. Sci. (USA), 81, pp.2708-2712 (1984).

SHORT TERM

For the first time, novel purified and isolated polypeptide products are proposed which have all or part of the conformation (ie, a continuous amino acid residue sequence) of free erythropoietin and its allelic variants and one or more of its biological properties (eg, immunological and in vivo biological activity). ). These polypeptides are also uniquely characterized by being a product of expression of exogenous DNA sequences obtained by genomic or cDNA cloning or genetic engineering of a prokaryotic or eukaryotic host (e.g., bacterial, yeast, or mammalian cells in culture). Microbial expression products in vertebrate (eg, mammalian and avian) cells may differ from human proteins and other contaminants and may be associated with erythropoietin in its natural mammalian cellular environment or in media such as blood plasma or urine. The products of typical yeast (e.g., Saccaromyces arevisial) or prokaryotic (e.g., E.Coli) host cells do not bind to any mammalian protein. Depending on the host selected, the proposed polypeptides may be glycosylated or non-glycosylated with mammalian or other eukaryotic carbohydrates. The proposed polypeptides may also have an amino acid residue (at position 1) of the parent methionine.

The proposed novel glycoprotein products include those having a primary structural conformation sufficiently duplicating the conformation of free-form (e.g., human) erythropoietin to adopt one or more of the latter's biological properties, and those that differ from the free-form (eg, human). a carbohydrate composition of erythropoietin. The vertebrate cells (e.g., COS-I and CHO) of the present invention, as determined by radioimmunoassay, are capable of continuous proliferation in vitro and when cultured can produce above 1000 V (preferably above 5000 V, preferably above 1000-5000) in their growth medium. o

V) erythropoietin over 48 hours per 10 cells.

Also provided are synthetic polypeptides that completely or partially duplicate the continuous amino acid residues of erythropoietin. These sequences, by distinguishing primary, secondary or tertiary structural and conformational characteristics, may have biological activity and / or immunological properties with free erythropoietin and may be used as biologically active or immunological substitutes for erythropoietin in therapeutic and immunological processes. Accordingly, monoclonal and polyclonal antibodies (produced by standard techniques) that are immunoreactive to such peptides and, preferably, immunoreactive to free-form erythropoietin are proposed.

The present invention is illustrated by cloned DNA sequences of human and monkey species bases, and polypeptide sequences derived therefrom and, respectively, representing the parent structural conformation of human and monkey species bases.

Novel, biologically functional viral and circular plasmid DNA vectors comprising the DNA sequences of the invention are also proposed and stably transformed or transfected with host microbial (e.g., bacterial, yeast, and mammalian) organisms. Thus, novel methods of producing useful polypeptides are proposed comprising culturing such transformed or transfected microbial hosts under conditions facilitating large-scale expression of exogenous vector DNA sequences and isolation of desired polypeptides from media, cellular lysates or cell membrane fractions.

The isolation and purification of microbial polypeptides provided by the present invention may be accomplished by conventional techniques involving, for example, preparative chromatographic and immunological separations containing monoclonal and / or polyclonal antibody preparations.

Having elucidated the amino acid residue sequence of erythropoietin, the present inventor proposes a complete and / or partial structure of DNA sequences encoding erythropoietin with favorable characteristics such as preparation of cleavage sites for codons selected for "desirable" host-mammalian expression, restriction endonuclease enzymes, starter, terminal, or intermediate DNA sequences that help to construct easily expressed vectors. Thus, construction (as well as development by specific cDNA and genomic DNA mutagenesis) of DNA sequences encoding microbial expression of polypeptide analogs or erythropoietin derivatives that differ from the free form in one or more amino acid residues (i.e., analogs with less total residues) is proposed. EPO, and / or substitution analogs wherein one or more residues are substituted with other and / or additional analogs wherein one or more amino acid residues are attached to the back or middle portion of the polypeptide); and which have some or all of the properties of freely available forms.

The proposed novel DNA sequences encompass all sequences suitable for expression in host prokaryotic or eukaryotic cells of polypeptide products which have at least a portion of the primary structural conformation and one or more of the biological properties of erythropoietin, which include: (a) the DNA sequences shown in Tables 5 and 6; complementary threads;

b) DNA sequences which are hybridized (as illustrated herein, or under more stringent hybridization conditions) to DNA sequences or fragments thereof as defined in (a); (c) DNA sequences which must hybridize to the DNA sequences defined in (a) or (b) as a result of the degeneracy of the genetic code. In particular, it includes (b) the genomic DNA sequences encoding the allele variants of monkey and human erythropoietin and / or encoding erythropoietin of other mammals. In particular, it includes the DNA sequences encoding EPO, EPO fragments and EPO analogs assembled in part (c), the DNA sequences of which may include codons to aid in the transcription of the RNA information in invertebrates.

The present invention encompasses that class of polypeptides encoded by the complement of DNA into the upper portion of genomic DNA shown in Table 6, i.e. complementary inverted proteins as described in Tpamontano et al., Nucleic Acids Research, 12, pp. 5048-5059 (1984).

The invention also encompasses pharmaceutical compositions containing effective amounts of the polypeptide products of the present invention together with suitable diluents, stimulants and / or carriers to provide eriptropoietin therapy, in particular for the treatment of anemic conditions and, most importantly, for the treatment of chronic renal failure.

Proposed polypeptide products may be "labeled" by covalent association with a detectable marker (e.g., 19-labeled I) to identify and quantify erythropoietin in solid tissue or fluid samples such as blood or urine. DNA products can also be labeled with detectable markers (eg, radiolabel and non-isotope tags such as biotin) and used in DNA hybridization processes to localize the position of the erythropoietin gene and / or the position of any related genetic family on a human, monkey, or other mammalian chromosomal map. They can also be used to identify lesions at the DNA level of the erythropoietic gene, and to be used as a genetic marker to identify neighboring genes and their lesions.

As described in detail above, the present invention provides substantial improvements in detection methods for specific single-stranded polynucleotides of unknown sequence present in a heterogeneous cellular or viral sample (comprising repeat single-stranded polynucleotides) in which:

(a) obtains a mixture of probes of labeled single-stranded polynucleotides with uniformly variable base sequences, and each of the indicated probes is potentially specifically complementary to a base sequence that is presumably unique to the lower polynucleotide;

b) binding the sample to a rigid substrate;

c) treating the substrate (having a sample bound thereto) to prevent further polynucleotide binding;

d) transiently contacting the treated substrate (containing the sample bound thereto) with said labeled probe mixture under conditions which promote hybridization only between fully complementary polynucleotides;

e) detecting the specific polynucleotide by the presence of a hybridization reaction between it and the fully complementary probe (contained in said mixture of labeled probes). This is observed by an increase in the amount of labeled material in the substrate with a specific polynucleotide compared to the amount of labeled material in the substrate without this nucleotide.

These methodologies are particularly effective in situations that require the use of 64, 128, 256, 512, 1024 or more

Methods for monkey monkeys have been steadily improved by coding from anemic mixed polynucleotide probes with 17 to 20 bases in DNA / DNA, RNA / RNA or DNA / RNA hybridizations.

infra, the foregoing enabled the identification of cDNA clones, erythropoietin, within the library (obtained by renal cellular iRNAs). Specifically, a mixture of 128 variable 20-probes based on amino acid sequence information derived from alternating fractions of human erythropoietin was used in colony hybridization techniques to identify seven positive erythropoietin cDNA clones in 200,000 colonies. Even more, the practice of using the improved methodologies of the present invention has enabled the rapid isolation of three positive clones from the screening threshold of 1,500,000 sterile spots of the human genomic library. This was done using the aforementioned mixture of 128 20-probes in combination with a second set of 128 17-m probes based on amino acid analysis of the continuous human erythropoietin sequence.

The foregoing illustrative techniques are the first known example of using hybrid repeat oligonucleotide probes in DNA / DNA hybridization processes for the isolation of mammalian genomic clones and the first known example of using a mixture of more than 32 oligonucleotide probes for the isolation of cDNA clones.

Many aspects and advantages of the invention will become apparent to those skilled in the art upon reading the following detailed description of the invention, which exemplifies the practical use of the invention in the best embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, isolated and characterized DNA sequences have been isolated that partially or fully encode the human and monkey erythropoietin polypeptide sequence (hereinafter EPO). In addition, human and monkey DNA have become the subject of eukaryotic and prokaryotic expression, producing secreted amounts of polypeptides having the biological (e.g., immune) properties of free EPO and the biological activity of EPO in vivo and in vitro.

Monkey species-based DNA was isolated from a cDNA library assembled with iRNA derived from chemically induced monkey kidney tissue, which serum (as determined by immunoassay) had higher EPO levels than normal monkey serum. Isolation of the required cDNA clones containing EPO-encoding DNA was accomplished using DNA / DNA hybridization colonies and a set of 128 mixed, radiolabeled 20-mer oligonucleotide probes, and included rapid screening of 200,000 colonies. The calculation of the oligonucleotide probes was based on the amino acid sequence information obtained from enzymatic fragmentation and sequencing of a small sample of human EPO.

Human DNA was isolated from the genomic human DNA library. Isolation of clones containing EPO-encoding DNA was accomplished using DNA / DNA hybridization spots and the aforementioned 128 mixed 20-mer oligonucleotide probes and a second set of 128 radiolabeled 17-mer probes based on amino acid sequences (derived from a different enzymatic human EPO fragment). .

Positive colonies and spots were screened for dideoxy clonal DNA sequencing using a set of 16 sequences within a pool of 20 probes, and the nucleotide sequence of selected clones analyzed with the final deduction of the primary structural conformation of the EPO polypeptides encoding them. The deduced polypeptide sequences exhibited a high degree of homology to each other as well as to the partial sequence generated by amino acid analysis of human EPO fragments.

The selected positive monkey cDNA clone and the selected positive human genomic clone were transferred to a shuttle DNA vector, which was amylated into E. coli and used for transfection in mammalian cell culture. The culture growth of the transfected host-host cells was reflected in preparations floating on the culture medium and containing up to 3000 U EPO per ml of culture medium.

Other examples are provided to illustrate the invention, and in particular to emphasize the methodologies previously realized for the identification of EPO which encode monkey cDNA clones and human genomic clones, and immunological confirmation of EPO expression in such systems.

More specifically, Example 1 is for the determination of the amino acid sequence of human EPO fragments and, based on the results of this sequencing, for the assembly of radiolabeled probe mixtures. Example 2 focuses on the techniques used to identify monkey positive cDNA clones and provides information on treatment of animals and preliminary analysis of their serum radioimmunoassay (RIA). Example 3 is for obtaining a cDNA library, screening hybridization colonies and screening for positive clones, DNA sequencing of a positive cDNA clone, as well as generating information on the primary structural conformation (amino acid sequence) of monkey EPO polypeptides. Example 4 addresses the methodologies used to identify human positive genomic clones and thus provides information on the source of the genomic library, staining hybridization techniques, and screening of positive clones. Example 5 is for DNA sequencing of a positive genomic clone and for generating information on the amino acid sequence of human EPO polypeptides (including comparison with the monkey EPO sequence). Example 6 is for methods for constructing a vector comprising EPO-encoding DNA (derived from a monkey-positive cDNA clone) for use in vector transfection of COS-I cells and for culture growth of transfected cells. Example 7 is for methods for constructing a vector comprising the use of an EPO-encoding DNA (derived from a human positive genomic clone) vector for transfection of COS-I cells and for culture growth of transfected cells. Example 8 is for immunoassay procedures carried out in supernatant liquid media obtained during culture of transfected cells (according to Examples 6 and 7). Example 9 is for in vitro and in vivo biological activity of microbially expressed EPO (Examples 6 and 7). Example 10 is devoted to the development of monkey EPO cDNA and human genomic DNA and mammalian host expression systems, including Chinese Hamster Ovary (CHO) cells, and the immunological and biological activity of the products of these expression systems, as well as the characterization of such products. Example 11 is for the generation of genes encoding human EPO, EPO analogs having genes for the expression of desirable codons in E. coli and yeast host cells, as well as systems for such expression.

Example 11 belongs to the immunological and biological activity profiles in the products of the systems of Example 11.

EXAMPLE

A. Determination of the amino acid sequence of the human EPO fragment

Human EPO is excreted from the urine, which is digested with trypsin and obtained and excreted by 17 individual fragments. Their quantities are approximately 100-150 picomoles.

The fragments are numbered freely and their amino acid sequence analyzed using a gas sequencer (Applied Biosystems). The resulting amino acid sequence is shown in Table 1, where the alphabetic codes used and X represent an unspecified residue.

Result of sequence analysis

TABLE

Enzyme no

T4a T4b T9 T13 T16

T18

T21 T25 T26a T26b

T27

T128 T130

T31

T33

T35 T38

A-P-P-R

G-K-L-K

A-L-G-A-O-K

V-L-E-R

A-V-S-G-L-R

L-F-R

K-L-F-R

Y-L-L-E-A-K

L-I-C-D-S-R

L-Y-T-G-E-A-C-R

T-I-T-A-D-T-F-R

E-A-I-S-P-P-D-A-A-M-A-A-P-L-R Ε-Α-Ε-Χ-I-T-T-G-X-A-E-H-X-S-LN-E-X-I-T-V-P

V-Y-S-N-F-L-R

S-L-T-T-L-L-R

V-N-F-Y-A-VV-K

G-Q-A-L-L-V-X-S-S-Q-P-WE-P-L-O-L-H-V-D-K

B. Calculation and Installation of Oligonucleotide Probe Mixtures

The amino acid sequences in Table 1 are examined in the context of genetic code degeneration to explore the potential of hybrid probe techniques for DNA / DNA hybridization in cDNA and / or DNA genomic libraries. This analysis demonstrates the existence of a sequence of 7 amino acid residues (Val-Asn-Phe-Typ-Ala-Trp-Lys) within fragment No.T-35, which can be uniquely characterized as one of the 128 possible DNA sequences comprising 20 nitrogen bases. pairs. Therefore, a first set of 128 20-mer oligonucleotides is synthesized using standard phosphoamidine techniques (see, e.g., Beaucage et al., Tetrahedron Letters, 22, p. 1859-1862 (1981)) using a solid support and according to the sequence in Table 2. .

TABLE

Residue - Vai - Asn Phe Typ Ala Trp Lys

3 *

CAA TT AAG AT

TA A A G

C

CGA ACC TT T C c

5 '

Further analysis reveals that a sequence of 6 amino acid residues (Gln-PnO-TnpGlu-PrO-Leu) exists within the framework of fragment No.T-38, which can be used to obtain a pool of 128 mixed oligonucleotide 17-probes as shown in Table 3.

TABLE

Remnant - Gln

Pno Tnp Glu Pro Leu

GTT

C

GGA ACC CTT GGA GA

C

Oligonucleotide probes at the 5 'end are labeled with gamma -32p-ATP, 7500-8000 Ci (mmol / JCN) using polynucleotide kinase T ₄ (NEN).

EXAMPLE

A. Monkey Processing Methods and RIA-Analysis

Female Macaca fasicularias female Cynomologus (2.5-3 kg, 1.5-2 years) is treated with subcutaneous injections of phenylhydrazine chiorhydrate (pH 7.0) at a dose of 12.5 mg / kg on days 1.3 and 5. Hematocrit is monitored prior to each injection. On day 7 (or as soon as hematocrit falls below 25% of baseline), ketamine chlorohydrate (25 mg / kg) is injected and serum and kidneys are immediately frozen in liquid nitrogen and stored at -70 ° C.

B. RIA - Analysis

For the quantification of EPO in samples, radioimmunoassays are used according to the following procedures:

The erythropoietin standard or unknown sample is incubated with antiserum at 37 ° C for two hours.

The sample tubes are then chilled with ice and added '1

Erythropoietin I and incubated for at least 15 hours at 0 ° C. Each test tube contains 500 µl of incubation mixture containing 125 dilutions: 50 µl immune serum, 10,000 cp Ieritropoietin, 5 µl trasilol and 0-250 µl EPO standard (or unknown), as well as PBS containing 0.1% BSA , to fill the remaining volume. The serum used contains the second sample of rabbit blood, immunized with 1% pure human urine erythropoietin. The final dilution of antiserum 125 is adjusted so that the amount of I-entropoietin bound to the antibody does not exceed 10-20% of the total amount. In general, this corresponds to a final dilution of antiserum from 1: 50000 to 1: 100000.

- Erythropoietin bound to antibody is added with 150 µl of Staph A. After incubation for 40 minutes, the samples are centrifuged and the separated pellet is washed twice with 0.75 ml of 10 mM Tris-HCl, pH 8.2 containing 0.15 M NaCl, 2 mM 125

EDTA and 0.05% Triton Χ-100. Determine the percentage of I-erythropoietin binding by measuring the gamma radiation intensity of the washed sediment. For correction of nonspecific deposition, the amounts of pre-immune sera bound are subtracted from the results. Erythropoietin in unknown samples is determined versus standard curve.

The monkey serum used in this procedure was obtained according to the method described in Part A. Normal serum levels are estimated to be approximately 36 mV / ml, whereas the serum of treated monkeys ranges from 1000 to 1700 mV / ml.

EXAMPLE

Installation of A.Beždionis cDNA library

The reference DNA is isolated from normal and anemic monkey kidneys using guanidine thiocyanate according to the method described by Chirgvvin et al., Biochemistry, 18, p.5294 (1979), and the poly / A / iRNA is purified by double passage through an oligo / dT / -cellulose as described on pages 197-198. Maniatis et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Springs, Harbor, N.Y., 1982). The cDNA library is assembled according to slightly modified basic procedures described in Okayma et al., Mol. and Cell Biol., 2, pp. 161-170 (1982). The key moments of the currently accepted methodologies are: 1) pVC8 is used as a backbone vector, cut with Pst1 and then generated with a 60-80 base tail using oligo dT; 2) HindiI cleavage is used to unlock the oligo dT tail from one end of the vector; 3) synthesis of the first filament and tail attachment using an oligo are performed according to published methodology; 4) BamHI digestion is used to uncouple the oligo dCode from one end of the vector; 5) The conversion of RNA to DNA is accomplished in the presence of two binding units (GATCTAAAGACCGTCCCCCCCCC and ACGGTCTTTA) with triple molar excess compared to the oligo dG-tailed vector.

B. Colonial hybridization techniques for monkey cDNA library screening

9000 colonies of transformed E.Poli are evenly spread on 10x10 cm plates with nutrient medium containing 50 mcg / ml ampicillin. Genesoreen filters (New England Nuclear Catalog - 1972) are pre-wetted in a BHJ-CAM dish (37 g / l bactoextract brain / heart, 2 g / l Casamic acids and 15 g / l agar containing 500 mcg / ml chloramphenicol) and used in the dishes nourishing existing colonies. Colonies were grown in the same medium for 12 hours or more to amplify the number of plasmid colonies. Amplified colonies (flanks) are treated by placing filters over two 3 mm thick Watman slices soaked in the following solutions:

1) 50 mM glucose, 25 mM Tris-HCl (pH8.0), 10 mM

EDTA (pH8.0) - 5 minutes

2) 0.5 M NaOH for 10 minutes

3) 1.0 M Tris-HCl (pH 7.5) for 3 minutes.

The filters were subsequently dried under vacuum at 80 ° C for 2 hours. The filters are then digested with proteinase K by treatment with 50 µg / ml protease enzyme buffer K [0.1 M Tris-HCl (pH8.0) - 0.15 M NaCl, 10 mM EDTA (pH8.2) 0.2 % SDS]. Specifically, apply 5 ml of the solution to each filter and continue decomposition at 55 ° C for 30 minutes and then remove the solution.

The filters are then treated with 4 ml pre-hybridization buffer (5xSSPE 0.5%, SDS 100mg / ml SS, E. coli DNA 5xBFP).

The pre-hybridization is carried out at 55 ° C for 4 hours or more and then the solution is removed.

The hybridization process is carried out as follows. To each filter was added 3 ml hybridization buffer (5xSSPE 0.5%, SDS 100 mkg / ml SS, yeast tRNA) containing 0.025 picomoles of each of the 128 probe sequences in Table 2 (complete mixture labeled EPY), and the filters were added. Store at 48 ° C for 20 hours. This temperature is 2 ° lower than the dissociation temperature (Td) of any of the probes.

At the end of hybridization, the filters were washed three times for 10 minutes with 6xSSC-0.1% SDS solution at room temperature, and then 2-3 times with 6xSSC-0.1% SDS solution at hybridization temperature (48 ° C).

Among the 200,000 colony filters sown, seven positive clones are found by autoradiography.

Initial sequence analysis of one of the putative monkey cDNA clones (designated clone 83) is performed using a modified screening technique as described by Wallace et al., Gene, 16, p.2126 (1981). Briefly, DNA-plasmid from monkey cDNA clone 83 is linearized by digestion with EcoRI and denatured by heating in boiling water. The nucleotide sequence is determined by the dideoxy method described by Sanger et al., P.N.A.S. (USA), 74, pp.5463-5467 (1977). An EPV-mix probe set of 16 sequences is used to initiate sequence reactions.

C. Sequencing of monkey EPO cDNA

Analysis of the nucleotide sequences of clone 83 is performed according to the methods described in Messing, Methods in Enzymology, 101, pp. 20-78 (1983). Table 4 shows the results of the pre-restriction map analysis of the 83 cloned fragment of EcoRI / HindIII clone containing approximately 1600 paramyazotic bases. The nucleotide sequence is determined by finding the sequences of individual restriction fragments in order to pair the overlapping fragments. For example, the sequence information overlap presented in the restriction fragment nucleotide analysis is designated CII3 (Jan 3A at - III / SmaI at 324), and the reverse order sequence is designated C73 (Al at -424 / BstFII at -203).

TABLE

Area of restriction enzyme identification

EcoRI

Jan3A

Sroa I

BstEII

Sma I

KpnI

RsaI

Alui

PstI

For beer

Alui

PstI

PvuII

Alui

Alui

Alui

Rsa I

PstI

A] ui

U ui

Because I

SsujA

Alui

Alui

Alui

PstI

RsaI

Alui

HindlII

Alui

HindlII

Approximate Find (s) .1

111

180

203

324

371

372,424,426,430 4,666,56,601

604

605,762,786,792,807,841,927,946

1014 1072 1115 1223 1301 1343 1384 14 49 1450 1585

Sequencing of approximately 1342 nitrogenous bases (ranging from 3 'to EcoRI and HindIII) and analysis of all available scanning systems assist in the development of DNA and the amino acid sequence information in Table 5. In the table, the putative primary amino acid residue of the mature EPO amino acid residue (as verified by the twenty amino acid residue sequence correlation in the above assay) is represented by the numeral 41. Presence of the deciding methionine-ATC codon (designated 27) as the downstream primary amino acid residue of the primary amine. residues labeled for the amino acid sequence of the mature protein are an indicator of the fact that EPO is initially expressed in the cytoplasm in the form of a precursor comprising a crucial region of 27 amino acid residues, which is cleaved before the mature EPO enters the bloodstream. Areas of potential glycosidation at the polypeptide are indicated by asterisks. In the translational domain, the calculated molecular weight is 21,117 Daltons and the molecular weight of the 165 polypeptide residues forming the monkey's mature EPO is 18,236 Daltons.

n no o n d <. n r n n n tn O k- 33 t- * —H 01 ra n n 33 01 n * < n c n ►- n c 3> n —1 c n “H n V) -i n 33 33 O T) -I n O n 3> O < O H n n n years n 33 n —I ω cna d. while μ  n o n 33 n n n n -) tn —I —1 n n n r d -1 n n ra Vi 3 » n t— i -c ra n —1 n Cl> 1 o n> 4 n'c i— -I c n. n n 33 n n n o n r- n x> n r n n n 13 | - —I ra ημ + ra n n n : i> c n c n ai ►- n c n o • H n n n s> tn -i r n T3 n i ~ n 33 ' n C) ra ra n μ —I ra n H 33 O 1-1 o c i> o n c n n n '33 33 s> - n n o n το ή tn d d 'n d * < 33 Ι- n i-i n 3> X 1 n n n years Ο c 33 o —1 fl —T ra m 33 n n σ si n n n tn O 33 n 33 n r H n d ra n ί- n fi ή ra n n n n n n ο ω Oid n c d f- · n n n < n -H -v r " 33 r n r ~ n < n n ra 33 «<M ra —1.01 n < n - d c η ω o n c n> -. -4 Oh —1 CJ o> - n n 33 £> d d 33 f-i -i tn i -1 -H 33 Ui .33 f ^- * -t f- · no ►— n x n n —T □ d c n ra n> i o 33 i— d n n. m -j '33 o n O T3 -i n n r ~ - n n 33 f- d 1— ci * < —1 round Cl Cl 33 n O C n you —1 Ui n c 33 f n n C n n 33 X> n cn d 33 n -a n n > Uh - * 33 f- 33 W n> -i -t ή n n —1 □ C) C n-a —I a Cls n -1 (Λ · n n 33> —1 > 33  33 tons n r ~ n d -t t- · 33 ω * 'n ra. —I ra n ό n d n ra -H □ n <1 n c n-fi n d —1 o Ή n 3> —1 d < n 33 n d 33 - 1 Ο ΣΓ t · —1.01 n> -i »- d t— n 33 i n n. Π 1-1 o n> - 33ia a nx ΠΗΜ n n n o o o -1 n n < s> -1 n < no ^- n n —T 31st o cr —H 01 —I ra -4 —1 ' n n O f d m n i— · n c n fi n - 0X3 —1 Cl n -a 33 X n r- n td n n Π> 1 ra ra n <1 33 n 33 o d rr n c 3 »o n n

B) ra

N <

PN <

H ·

O

P ra * to te) te)

O <+

P

P to

I— *

H ·

P

O

H · e_i.

P isj (-3 te) f

you) ·

TABLE (cont'd)

> .o O «X P o —I et CJ o O O P O u <X ro p a 'o > o LTl Ι- □ o Ο 3 o P CC b- ai p L2 O _J o j_ »U □ o ω p OJ P X et '—1 o CTO ro o U. O p o • tx «X et O V> CJ 3 O x qj p _J «X _l o GO •> O P O p o p p Oh o c re o c o IAHU P «X et O o o P P ao > * ec P O i- ί- P P α. 'o P o xp re p o_ p > o c o O et V) <x r-l et et «X O O Mon p Fri d ro p ro p > o > o v> et air p > ret Ό —i o _J <x <x o u O c o JZ o <-l <t p <x Oh o 0.0 c o 10 et r-To «X- et O ύ o

O P 3P ro o P o OJ Ι- P o D-O Ο o et O C O . > O O «X r-l d ρ o Fri d O O. o o o o P o o p ί- c o a, o ο ω o p d tn P —I tn et o o u p ai o re o CJ o r — I 1— P o tn p P et «X o c u ro o et al ui et —L o P O d <x d to O O ro o • U) d 3 O r-> o >> d OJ P d u et -J <-J • □ o ai- ro o ω p to d p o —J P d O et O r-10 p CJ Oh CTO CO 1—: 0) P * P P. o > o X d p et O ro o to u □ P P O —I d OJ P et O X o -I o C O o o □ o r-l d a> p OJ P O O _J o _l o > -O c o P P r-10 r — t Ct X o Oh o Oh o P <x_ CTO o o P o P O OJ P X o et O _J o p o: 3 O o o o OJ o OJ Ι- O \ P o P P Ο o CL O P <t -H O □ o P o ro p P et OJ o > o O O et ro p OJ O eno —1 o X P P o et O CL P et O

0? o P P P er OJ CJ X P 0. P P o C P X o to et P et et et O) et p O P O oo o et O tn p 3 O P o OJ P >. <x _l o P P O et P o P O to P a. o > o ro p σ> <χ p o P o et O et O ro p OJ O P o X P d o □ L P P o 0 3 0 0) o <S OJ P tn p P -J o re o to et P o >> et et O -J et ro o to (j P o > -O et O O p G P OJ o to «X X P d a CL P o d P P P o X o n. o P et 3 o GO OJ P UJ et o o et O P o ro p 4) O P u tn p et O (U o P P P P X O p et P <t

AGGATGGGGGCTGGCCTCTGGTTCTCGTGGGGTCCAAGCTT

HindlII

o -4 o —1 O J> —4 a P .o o o r -i -t - ( -4 J> Ci Ci Ci —4 O 4- * —4 fB n Ci n —4 O Ci -4 Ci O O < O C -i o she -4 O J> O Ci o o 33 33 -4 —1 n O ' 33 n a j> n j> - · 1 33 Ci n n o J> n —4 > cn a n o -4 Ci o *>, n O n O 0X3 o he o Π. Ci j> n j> J » n —4 o —4 Ci n —4 -4 O —4 O She J> H- Ci Ci t— n Ci -4 Ci n -4 o Ci n Ci> 1.0 \ C3 M Vi 33 Ci Ci —4 Ci O She -4 n she ta vi She »<o 33 She J> J> Ci She n n 33 O o n O n Ci j> a O —4 O J> Γ ~ o n C3 n n She She a J » O TJ 33 »< 33 33 o j> j> Ci Ci j » O She α m o Ci CT —4 Ci She —4 —4 Ci n Ci n o Ci o O C3 Ci o ci r " o H she j> —4 o -t O -1 o -4 Π) O j> o n Ci She Ci H o She Ci C a She —1 C3 Ci ' O She α Ci o n She n n J> Ci Ci j> Ci o she c ~ —4 She Ci she I> —1 J » n -4 -4 J3 * < J> -4 Ci o J> —4 -4 "4 O o Ci (Λ Ci Ή —1 j » J » -4 'n n J> o o J> —4 Ci —4 J> —4 o n Ci O Γ ” a n Ci Ci —4 J> ϋ ϋ n —4 —4 rounds o Ci Ci She -4 She J> a she o o c -H o -4 -4 O O O —4 o o O n O O J » -4 Ci Ci —4 She —4 -4 O n Ci She -4 Ci She n O n J3 * < J » j> Ci o C) J> —4 n Ci o O> 1 Ci C3 She -4 n O n she Ci -4 o o She O She Ci —4 o Ci O 3i —I —4 —1 tn a Ci She n o She O o see O -4 33 & Ci Ci Ci J> H Ci a> -i o -4 Ci o J = Ci a n o a -4 Ci She n T> She -4 a She Ci Ci 33 O Ci J> j » . Ci O O a o Ci 4— · • H O Ci n n H a J> a J » ο * < Ci She Ci -4 O Ci Ci O j> -4 o C3 n —4 O She Ci 33 -4 O Ci Ci · C3 n -4 J> -4 ' o a -4 n o She I—> J> -4 -4 Ci -4 J » n n Ci She Ci c Ci —T Ci Ci —A She She —4 j> o -4 Ci 33 -4 Ci —4 n O 'Oh n O 33 O Ci n. n Ci n —4 J> Ci τ » O> - * u 33 jj C3 J · n -4 O a o n ω o Ci n D O J> a o Ci o She Ci —4 She o Ci 33 n a -4 —4 O t— 33 Ci O She n n £ 7) n —4 o Ο * <Ui O -t She Ci n n Ci n n o Ci tn a n 33 Ci o She jj a a —4 o o n n —4 -4 She a she n -t J> 33 —4 —T o Ci -4 J> a Ci j> n C3 1 Ci -4 33 -4 —4 J> a j> o 33 o <a She Ci 33 Ci 33 n Τ » Ci n O 33 33 -4 O 33 Ci Ci —4 O She's 33 n n J> Ci O o She She She H O Ί 33 33 —4 —4 —4 n a She She J> she is n o —4 ~ 4 Ci -4 Ci n n O She

TABLE (cont'd)

The polypeptide sequence of Table 5 can be readily analyzed for particularly hydrophilic regions and / or secondary conformational characteristics (which are indicative of potentially highly immunogenic regions) by the methods proposed by Hopp et al., P.N.A.S. (USA), 78, p. Kute et al., J. Mol. Biol., 157, p. 105-132 (1982) and / or Chou et al.

Biochem., 13, p. 222-245 (1974) and Advances in Enzymology, 47, pp. 45-47 (1976). Computerization of the assay by Hopp et al., Is possible using a program labeled PEP Reference Section 6.7 at Intelligenetics, Ine., 124 University Avenue, Palo Alto, California.

EXAMPLE

A. human genomic library

The human fetal liver genomic library (containing the AhC4A phage) obtained according to the methodologies of Lown et al., Cell, 18, p. 533-543 (1979), is preserved for hybridization assays.

B. Stain hybridization techniques for human genomic library seeding

Phage particles are lysed and DNA is fixed on filters (50,000 spots per filter) according to the methods described in Woo, Methods in Enzimology, 68, pp. 389-395 (1979), with the exception that Gene Screen Plasma Filters (New England Nuclear Catalog no. .NET976) and NZYAM plates (NaCl - 5 g / l, MgCl ₂ .6H ₂ 0 - 2 g / l, NZAminA - 10 g / l, yeast extract - 5 g / l, casino acids - 2 g / l, maltose) - 2 g / l, agar - 15 g / l).

The air-dried filters are heated for 1 hour at 80 ° C and then decomposed with proteinase K as described in Example 3B. Preliminary hybridization was performed at 55 ° C using 1 M NaCl - 1% SDS buffer for 4 hours or more and then the buffer was removed. Hybridization and bottom hybridization washes were performed as in Example 3B. Both the 128 20M probe mixture (EPO labeled) and the 128 17M probe mixture (Table 3) in Table 3 are used

EPQ). Hybridization takes place at 48 ° C with the EPV probe mixture and 46 ° C with the EPO probe mixture, i.e. 4 ⁰ below the calculated Td for the mixture components. For re-hybridization, hybridized probes are removed by boiling in 1xSSC - 0.1% SDS for 2 minutes. Filters autoradiography discovers three positive clones (active with both probe mixtures) among 150,000 phage spots sown. Screening of positive clones for EPO-coding is accomplished by DNA sequencing and electron micrographic imaging of the heteroduplex formation with Example 3 monkey cDNA. This technique also proves the presence of repeat introns in the DNA sequence.

EXAMPLE

The nucleic sequences of the positive clones (designated hE1) are analyzed and the results obtained to date are presented in Table 6.

AAGCTTCTGGGCTTCCAGACCCAGCTACTTTGCGGAACTCAGCAACCCAGGCATCTCTGAGTCTCCGCCCA

o ω . ω ω. ω u X ω u ο ω ·  V- V— u X u ω ω «X ω ω ω <τ w Ο v— <χ ω ω. ω ω ο 04 —ι et CJ ω ω <χ ω ω et ι χ ω et et ω ω; ω ω ω U ω CX «X ω ω ν— —Ι ο u ω ω ω ν— ω «X η ν- ω ω V— ω ω et ω > Ο o et ω ω ω ω ω u Ο ι- et ο <χ ω V— V— ω · ω ω ω ω —Ι ω U «X ω ο 1— ω ω ω ω O ω V— ω ω ω ω CJ ω ω ω ω 1— ι- j_> ω «X ω ω ω ω ω ν- 04 ω ν— u ω ω ω ω ω ω 1 X et u ω ο ω ω V— ω «X ω ω et ω ω et v— «X ν— ω ω V— ω ω <x ω 'ω ω ω ω ω «X CJ ω ω ω ω ω ω ω et «X V— ω ω X ω ω ω ω ω ω et ω ω ω LJ ο ω <χ ω ω ω ω ω ω ω ω ω ω «X ω k— ω ω - ω ω ω ω ω ι- 'ω «X ω ω ω ω et ι- ω ω ω ω ω V— et ω ο ω ω V— ω ω ω ω ο ω V— ω ω ω ω ω ω <χ ω ω «X ω ω <x «X <χ ω ω ω ω ω ω ω u V— ω ω ω ω ω 1— ' ω ω ο V— ω ω ω ω ω et ω ω ω ω ω et ω ω ω ω et ω ω ω et ω ω et ω ω et <Χ X ω ω ω ο ο .ω. ω ω ω ω ω ο ω ω <χ ω et V— ω ω ω ω ω ω ω ω et ω ω ω ω ω ω ω ω ι— V— ω ω et ω ω V— ω ω ω et ω ω ω ω V— ω ω ω <χ ω ω ω ω ω ω V— ω et ω ω ω ω ω ω ω ω «X V— «X V— ω ω ω ω V— ω ω ω «X ω ω ω ω et ω «X ω ω ω ω V— et ω ω V— et ω ω ω ω ω ω ω ω ω V— ω ω «X h- . ω ω ω ω V— ω ω ω ω ω ω ω ω ω ω ω · ω ω et V— V— ω ω ο ω V— ω ω ω ω ω ω ω ω ω V- ω ο ω ω ω ω ω ω ω ω ω «X et. ω ω ω ω ω ω et ω ω ω ω ω ω V— ω ω et ο ω ω ω «X ω et V— et ω ω ω ω ω ω ω ω ω et et ω ο ω ω et et ω ω ω V— ν— ω et ω et V— ω ω ω ω V— ω ω ω ω ω V— ω «X ω ω ω «X ω ω ω ω ω ν- ω ω «X ω V— et ω ν- ω «X et · et ω ω ω ω ω

GTGAGTACTCGCGGGCTGGGCGCTCCCGGCGGCCGGGTTCCTGTTTGAGCGGGGATTTAGCGCCCCGGCT

3 «

CCAGGAACCTGGCACTTGGTTTGGGGTGGAGΤΓGGGAAGCTAGACACTGCCCCCCTACATAAGAATAAGTC

33 -1 CT 33 er r CT CT —1. CT CT 3s o rr rv 2> ω —I ro 33 Ή • H 2> CT —1 n h σ \ cro CT c CT CT n CT I » —1 CT CT CT - < CT CT n 33 cn -i tn 1 CT CT 23rd -H CT CT -H CT Π) O ro > - · -S n CT CT CT O CT K! CT> i Q. CT CT 23rd CT Ή CT 33 CT 33 CT CT CT 23rd O CT 33 CT r CT CT. n CT CT 23rd 33 n n h • h ro -1 23rd n CT 23rd CT O 33 (0 o CT c 33 CT  23rd —1 CT 23rd O -d 33 CT -1 CT CT O CT < CT D CT CT 23rd CT —I CT O —10) CT H —S CT CT CT CT -H —1 CT i— -i a CT -t . 23rd CT ' CT CT —I -H CT —1 CT CT CT n ct r n r -I CT CT 33 CT CT n CD -h ro CT n 33 CT CT -H ct CT c · CT c - < CT 2> CT CT CT n 33 33 -H 33 CT CT Ϊ3 CT CT CT CT CT -1 33 CT • CT CT o 33 t— ' CT t— CT 33 CT CT —1 CT CT c CT << 33 CT —1, 23rd —1 13th 33 CT —1 CT CT n 33 33 CT (- CT 1 CT CT -1 —1 2> 33 CT rl —I re 33 k— M CT 33 CT —1 23rd —1 CTia CT c 33 C VI 23rd CT CT CT CT —T 33 23rd CT CT CT O -d -t CT u -1 CT CT CT CT CT 23rd o 33 * < CT. H CT < CT CT —1 CT CT 33. CT rj Ϊ »o - <ω —1 CT —F CT CT D CT ' '33 CT 33 CT 33 CT! ~ CT < CT Ό —1 CT -H CT CT CT —I CT —1 0> CT H CT CT CT —1 CT 33 CT C CT t- · —10 CT 23rd CT CT 33 —I CT “H n 33 Π -i Γ n r CT 33 1 —1 - CT CT —L CT —T —T re -t ro CT i— M CT —4 -H —1 —1 ct CT c CT C CT 03 O CT CT CT CT —1 33 • 23 33 CT CT CT n CT CT CT CT —1 -1 CT CT 23rd CT —H CT 23 Ι- CT (-> 1 CT 23rd -H CT CT CT n Ο c CTx h- CT Ώ CT CT —1 33 23rd —T CT: CT Ή —4 2> o CT 23 CT 33 CT r ~ n. —1 CT ' CT CT 33 CT H- CT Ι- + —I ro 23rd —1 —H CT CT n O 03 Ο 0) h- » CT C CT n CT CT 23rd CT CT CT M 2> CT CT 23 r ~. CT T3 —1 —1 CT 33 CT 23rd CT o 23 - <M CT <-J n <-i —1 —T CT 33 CT -H CT tn O 33 O CT o -1 CT -1 .33. CT -1 CT CT -i 23rd CT CT CT CT CT Ό CT r ~ - ( -H CT CT CT 2 » 23 Ι- CT 1 —I ro CT ' -H —1 CT 33 CT Ο C 33 O —I c CT CT CT —4 23rd CT CT CT 23rd CT CT CT CT 2> CT 33 er r- CT CT CT CT CT u CT H- n 1-1 -i ro CT 33 —1 CT CT 33 CT 03 cna CT c CT CT CT CT CT n CT 33 —1 -1 CT -1 CT CT CT f " n r · CT CT CT CT 2> o 23 H- . -i ro -i ro CT CT - ( -t CT o CTC CT C CT c · CT 2 » CT —4 CT Ή 1 CT 23rd CT CT CT n 33 23 , 33M - <tn —1 CT i-1 23rd CT CT 2> ω * '-) 1- er ro CT CT l-l. CT CT CT -S □ π ro CT H . 23rd 2> • CT CT 33 CT CT cn CT —1 CT 23 l-l -h π ct r · -1 CT « CT CT 23rd - (t- · CT v; —I ro CT. > 33 l_J CT n - ct re —I in CT c —4 CT 23rd CT

TABLE (cont'd)

TGGTGGCCCCAAACCATACCTGAAACTAGGCAAGGAGCAAAGCCAGCAGATCCTACGCCTGTGGGCCAGGG

□ et P P · t— Ι- p o et - («I . > o: 1— Ο o et et O CJ 1- ι-  et o o O O O o o Ι- O ra p- ω o ί- et o Ο et r — l o C Ι- ο O «T et O et C Ο. l · - P- P · O k- O et et ι- 1- o m t- CP et O ι- et o >> C3 (Λ et CJ et ο et et CJ t- et et et O o et O CJ et Ι- et Ι- r — 1 Ρ » CJ O Ι- et Ι- ι — 1 CJ ra i— 1— O εί that, Ο O CJ > L3 P- et et et o 1— O O P- o H CJ V) et Ι- O et P- I— r. o > <x Ο et O H et 1— <t _J et 1— O O et O P · O O O o P O Ι- 1— 1— et et Z. U Ο et et et O I— «t o et O et O p · et et O O CJ 0.0 p * et O <t O et ΙΛ et P- Ι- O P- O O et CJ 1— Ο o o O t— P- O o Ι- a ι- O et I— et o Ο o ι- P O P- et p- 1— Ι- σ: a. o I— et et et Ο CJ 1— O O O o CJ r-10 1— O et O o Ι- re i— 1— O O o et Ο > CJ P- o Ι- o et ρ- P- o Ο o O CJ o u ι- Ι- o o 1— o ί- ό · t: o Ι- P- o et  P- ο h- et Ο et et O 1— CJ o O O et P- CJ OJ O Ι- P- O O et CJ r-l U- Ι- et o P- P- o u-l et Ο et u- O et o et O ct et O o C 1— O et et 1- et o • r W et Ι- O P ct O Ι- et et Ο O <t O O Ο O Ι- O O <x J ChJ m J o et Ο et P- o - <a m — et et ί- O o O CJ o o • et ο O et l_ O u o P- O C P * 4-1 p u- et et o V) et OJ Ί— et U Ι- o ; j et et. X et O Ι- Ο Ι- o Ο o Ο t '.i J CJ CTO 1— o - et o c .: OJ 1— ' •. P O 1— o et P- c.? -J 1— et et 1— et O et a: 1— O O O o P o . in o et et et O ι- OJ CJ • O O O Ι- ι- cn et _J et Ι- O et Ο ο Ο O O o o ω o OO o J CJ P- Ι- CJ >> CJ Uh, O o O Ι- Ο et O Ρ- i- ί- et o Ι- et CJ O Ι- Ο CC et ω o ο ra o o Ο et O O • H et m —i o o o O P- O X O et O 1- o O O

TTTGAGGCTGCAGTGAGCTGTGATCACACCACTGCACTCCAGCCTCAGTGACAGAGTGAGGCCCTGTCTCA i

Cl . O n i> t- Oh o o < o < O O 35 -1 3 » o> 1 H- 33 4- · —4.01 -4 01 KZl O 33 • 35 -1 X » O (Q O d □ * '· O h- ΠΗ £ Λ o O 35 —T n -1 -4 35 —4 a O J> o r * o r Oh o o O 35 n d O H- -4 ra -4 ra O t— ' o I » o -4 —T -4 (U cp c o c o- < —4 —4 35 D o o —4 3 » O Ή o r · o X O 3a o o 33 O 35 —T —T re 3a H · Oh i-l 33! -> O 33 X> -4 O o c —4.01 O (O o o o -4 O C) n o -4 35 o —4 o d O < o o o o d O 35 O 23rd d t— -4 ω O F- 33 ί- o 33 35 u £> 33 o ί- o-c ο- o 33 -4 35 Cl O O -4 35 o O 33 ο 33 o o O 33 o O 35 33 O h- 33 (Λ 3a l-> O 1 - ¹ O \ 33 3a o C3 O a —4X3 O □ oh oh a O -4 35 —I . · o -4 35 x> r ^- ►— 33 n o r— 33 Γ O 23 o < o O 35 23 · < > - ' • O s> ►— 33> < O ►— ¹ -4 01 33 33 35 o w Ch 33 o □ VI 33 (Λ O 03 33 H- O -4 35 O 33 —4 H o o O o O 33 o r o O O O 35 σ \ 33 · -> 33 —1 O> - -4 ra <33 33 i— O 33 35 33 C 3a O o « O C o 33 C O 23rd -H n 3> —4 O o S O U -K O o < -4 r ^- O < O 33 35 tsj o> - o —4 —T ω —4 rounds -4 a 23rd 33 O i-3 n years -t '33 O 1— * O C Oh t-i O O Cl n O ..... 33 -4 Cl ft 3 => -t o O 33 cn H— o < -4 —i —T O o S · —I 1- * o 33 o re o -4 01 O> 4 O —4 -H Oh TO —T O -4 H o O> - ΟΧ3 O -4 Cl 33 O o 3a —1 c4- -4 (Λ h- —1 o O O 3> 33 O O -4 -4 35 * ra o re -1 o O h- 3a 1/1 X- 33 Ι- o -4 —I he O -i o O 23rd o- < Oh o Ο o 33 O o h * · n n O O n J3 O X -4 23rd n r ~ —I cn O O O 33 3 » O M -4 o -) ra o ra O 1- —4 -4 35 CQ -H O O —4 —I c -4 i-1 o- < O 13th —4 n —4 O O 35 Oh TO n n O 33 —I cn Oh t ~ 13th O o Oh i-l —4 -4 n * -i o ra —4 rounds o —4 35 33 O -4 O 0 (0 Oh i-l o c -4 —4 - < -4 O O O —4 O 3 => O -1 33 cn o o O 33 o -4 Cl LD w -4 -4 o ra 33 ί- O Ι- o O 35 -tO O O O H ο D Ο 01 33 · ' —T —4 -4 n O -4 —4 n 33 O n O Γ O X o r- Ϊ3 —4 3> n ί- o n -4 ra Oh i-1 -4 ra O O —4 ο ω o -4 o c O O o c —4 o -H 33 -4 O —1 n O 33 o -4 33 -4 —4 -4 or ^- O o 35 n> -> -4 O o rr O M —T re < 33 13th -H n ω d -4 O 1-1 ΟΧ3 o c o O o —4 a O -4 o O -i cn 23rd -4 33 -4 O O -4 cn O —4 35 o re d 33 O ΖΓ 33 H- o re O —1 n I »n o 33 -4 1-1 O C O> 4 o o -4 o O —4 o O O 3 » 33 33 o r * O X o o O —4 35 O h- O 33 —4 rounds O 1-1 KO 33 1— -4 O O -4 o n O o c Oh o a 33 C 3a o —4 -4 O O -4 n O 33 O O or ^- o r- O 33 -4 35 O> - · n O -4 ra —T ra O 1— O O -1 O -4 33 -4 c o c -4 O o —I

40A

TABLE (continued)

Lt O JLCJ d ω ω ω ω o ω d ω ω —I CJ . CJ ω d o d o d d in n CJ CJ d ι- · ω π- H- d ο o CJ ω Ι- d 1— ω d π- n u Lt d CJ d Ο ω 1— ω d · ω X JZ CJ CJ ω I— ω d ι— o d 1- H- t - d Ι- d d ο CJ ω ω Ο o Ο d ω π- d d r-l U cncJ Ι- o ο ο o ι- ω ω ra n Lt CJ Ι- n d d CJ ω ω d > o d d ο d d ω d d d o o ω π- d o CJ d π CJld in F- ω ω ο o Ι- o π Lt O >> CJ Π ω ι— ο F— Ο o d d ω CJ · - d π- d ο 1— 1— o π CJ ω o d 1— ω f— ω ω ω J ro cj d ι— d ω CJ o o ω JS Π VO r (t d ω o I- o d o d o. t- -1 d cj CJ ·, Ω 1- ω ω d Ι- CJ CJ ο CJ d d ω Ο CJ □ u □ CJ d d F- d ω o o Π OJ h— —F days CJ ω ω d d o o CJ _l U CJ CJ Ι- ω d t— CJ n * o π- Ο ο d π- o o ω 1— O ΙΛ d i > -O o ω ω ι- d o ω d d> -d .t CJ CJ ο o ω ω d d Ι- —I _i d CJ CJ Ι- d d o ω n ω Ο Ο ω o d o π- d H- CJ) o Lt d CJ π- d ω F- ω o O Π o SZ CJ d ω O o d d π- H- d ω F-d o ο d d CJ d ι- n- CJ d ω O F- o ω π- ω ω Π u d ω ω d F- d  ω ι- JZ t- > -d CJ π Ι- o 1— o ω ο D. I- 1- 1- CJ ω Ο d d o ω d i- Π π - ι— ω d 1— ω d U H- □ CJ d ω ω H- d ω ο ω JC CJ OJ F— Π ο d H- d ω d ω 1d To -1 CJ d ο d d ω d ο π- CJ ο o ω o o d o O. CJ cn CJ CJ . .ο CJ o π- I— d o ΙΛ no > -d o d d d ω d ω • o «X U _J d o Ο o d ω CJ ο o π- ω d o ω o ο F- re n □ CJ ω π ω CJ d d π- cf —T CJ 0) Π- ω ο ω d ω ω ο ω d CJ ω o d ω . o ω CJ d ο n- u ω o o o o π- c? Lt H ω cj ω ω ω d ω o ι- L- JZ ω > -d Ι- ω o o π- o ω ¹ c? In— ςχ _i d. Ο d d · d ω d ω o π- d ω ω d ω ω o OJ U > -d ω o 1- ω ω 1- π- Π-— —T 1— —T CJ π π ω ω I— '-J. ω ω > no CJ CJ o ω π o ω ω d L- o ω d o o ω ω CJ Lt et o CTO d π ω o d. d ο CJ JZ CJ IA Lt CJ ω  - ω d d o 1— ο o h- d d u ω d o d 1— ω ο CJ ω n O ω ω Η- κ- ω o d J CJ d ω d 1— ω d d ω d Lt CJ oh i— CL O ο d n- ω ω ω o o d cj -J CJ O Π ω ω ω ω ω ο CJ n- ω o d d ω ο CJ d O □ CJ OJ CJ VO CJld ω d o π- ο CJ n r \ ω n SZ Ι- VO Π CJ ω ω d π- ω X d —1 J (J Ο. H- -4d ω ω ω d Π- d F— d π ω o o Ι- ω d ω o d C Η- αω ω Ι- o CJ Ο . π CJ d Lt CJ σ) no V »no ω Ο o o π- ω X ω CL CJ d d d o ω d d 1— ω ι— d d

In Table 1, the primary continuous DNA sequence maps to the upper 620 base chain such that it appears to be an untranslated sequence followed immediately by the transposed portion of the human EPO gene. Specifically, the sequence consists of a 5-terminal gene that leads to a portion of the translated DNA encoding the first 4 amino acids (-27 to -24) in the "leader" sequence ("pre-sequence"). The four pairs of nitrogenous bases in the sequence preceding the latter encode the leader start, but are not yet uniquely identified and are therefore denoted by "X". This is followed by an intron consisting of approximately 639 pairs of nitrogenous bases (439 base pairs are identified and the other 200 base pairs are designated "I.S."), immediately followed by a glutamine codon depicted as the -23 residue of the translated peptide. The following exon sequence encodes the amino acid residue via an adenine residue (designated as the + 1 amino acid residue of the mature EPO gene) to a codon that expresses threonine at the +26 position, followed by a second intron consisting of 256 bases, specifically and designated. The sequence behind this exon exon sequence for amino acid residues 27-55 extends to a third intron consisting of 612 pairs of nitrogenous bases. The following exon encodes the residues of 56-115 human EPO. It is followed by a fourth intron of 134 pairs of nitrogenous bases, which is extended by an exon encoding residues 116-166 and a "stop" codon (TGA). Finally, Table 6 identifies the sequence of 568 pairs of nitrogenous bases consisting of the 3 'human EPO gene of the untranslated region. The order of the two nitrogen base pairs (“X”) is not unambiguously determined.

the table allows the identification of the parent structure of the mature human EPO conformation (amino acid sequence), which consists of 166 identified amino acid residues (with an established molecular weight = 18399). The table also shows the DNA sequence encoding the leader sequence of the 27 residues along with the DNA sequence 5 'and 3', which may be significant for the promoter / operator function performed by the gene operator. Segments for potential glycosidation of the human EPO polypeptide are marked with asterisks in the table. It should be noted that in Table 6, the specific amino acid sequence probably represents the free-form allele form of human erythropoietin. The rationale for such a statement is the results of continuous testing of human urinary erythropoietin isolates, which allows for information that a significant portion of the erythropoietin molecules contain methionine in residue 126 as opposed to serine, as shown in the table.

the table illustrates the degree of homology between the human and monkey polypeptide sequences. In the top continuous line of the table, the alphanumeric notations are used to deduce and translate human EPO sequences starting with 27 residues. The bottom continuous line depicts the monkey EPO deduced polypeptide sequence, starting with the number 27 representing the residue. Asterisks are used to denote sequence homology. It should be noted that human and monkey EPO deduced polypeptide sequences reveal an additional lysine (k) residue at position 116 (human). Reference to Table 6 means that this residue is at the end of an imaginary iRNA and genomic chain compound. The presence of a lysine residue in the human polypeptide chain is verified by infra-sequencing a human cDNA clone (derived from iRNA) isolated from COS-I cells transformed with human genomic DNA in Example 7.

Monkey AISLPDAA5AAPLRTITADTFCKLFRVYSNFLRGKLKLYTGEACRRGOR

r tri Φ tri Φ N> r o o N < O Ρ » 09 09 P · 03 N < ri C N < i P · to 0) P · ca O O P ri Φ-

® ·

4 3a < 4 < 4 t-t ro z 4 z 4 tn α X 4 X * 0 - < 4 - < Vi 4 X 33 4 33 O 4 o X 4 S. 4 33 X 4 X • 4 33 X * X 4 cn z 4 z 4 33 m 4 m 4 33 < # < 4 X no d 4 d 4 r ~ o o 4 £ 3 0 \ 4 33 o 4 £ 3 o 4 —1 33 4 33 4 l-l < 4 < 4 —S m 4 m 4 S » < 4 < 4 o Ό. 4 rt * -1 £ 3 4 O 4 x t— ¹ d 4 O X £> i- 4 (~ 4 X O 33 4 Ϊ » ♦ r r ^- 4 Γ J ♦ x f " 4 r o ♦ X (71 4 (71 ★ < m 4 m 4 - < 33 4 33 ♦ cn < 4 < . · 4 z i- 4 r ^- 4 X t— X # - X 4 l ~ (71 o 4 d 4 X o o 4 £ 3 4 n 33 4 X * X < r o 4 r ~ f " 4 (- 4 X Ϊ3 < 4 Z 4 z 4 - < cn 4 cn * —T (71 4 (71 4 n 1— O 4 £ 3 4 m o X 4 X 4 33 o X rt 4 n m 4 m \ 0 * X X 4 X o -H r ~ 4 r * d 'Ό • £ 3 4 o r 4 r- 4 X X 4 X z < σ 4 σ X 4 X 4 33 h- · 1-1 < o (71 4 (71 o d 4 C3 f " 4 l ~ X 4 X cn 4 cn 1-1 i " -1 4 -i - ( 4 -H Γ 4 i " P> r 4 r ^- h ^-1 X 4 X O 33 4 33 r * r ~ Cl 4 d 33 • 33 £ 3 4 £ 3

I X

Z # z d 4 d < 4 < X 4 X m 4 IX n 4 d 1 X 4 X ΙΌ 33 4 33 O a 4 a: c <j < r 4 f- g 4 a O r 4 f- 09 r 4 f— P Γ- 4 i- ri ΙΟ 4 co a Γ 4 | - 1 < | - 1— * P · cn 4 co.o 4 r 4 (- X 4 X σ ' Γ 4 Γ- Φ d 4 Ο N4 1- 4 (- P * X 4 X N < < 4 < P · X Ι- O d 4 Ο + ri 33 4 33 H- ' φ · X 4 X ω X 4 X X 4 X X r 4 f- o 1— ( 4 1 - Vol H d 4 d P · σ 4 o X cn 4 co i— Φ X 4 X o XI < 4 < e + ι- 4 r " P · ιχ 4 m P · X 4 X , P - < 4 -c ι- 4. | - M π 4 | - m 4 m o 33 4 33 M X 4 X o X m 4 m P 33 4 33 J-J rx 4 ΓΠ z 4 z 05 < t-1 P · - ( 4 - ( ri p. z ( d 4 d S Γ3 4 d C4 P co 33 O to m 4 rx co X n 4 d (71 4 co r ~ 4 (- z 4 Z ' m 4 IX z 4 z 1-1 4 l-l X> - ( 4 -ι α < 4 < X 4 X O 4 σ -t 4 - (

TABLE

EXAMPLE

For primary microbiological synthesis assays, the selected expression system for the isolated amounts of the polypeptide erythropoietin material encoded by the monkey cDNA obtained as described in Example 3 is a mammalian host cell (e.g., COS-I cell, ATCC No..CPL- 1650). The indicated cells carry out transfection with the shuttle vector which has the ability to spontaneously replicate in E. coli (thanks to the presence of DNA derivatives from pBP322 plasmids) and in mammalian hosts (thanks to the presence of SV-40 DNA derivatives).

More specifically, the expression vector is constructed according to the following procedures. The 83 plasmid clone obtained in Example 3 is amplified into E. coli and, by digestion with EcoRI and HindIII, has isolated approximately 1.4 kB of the monkey DNA encoding erythropoietin. The 4.0 kb HindIII / SalI fragment was isolated from pBR322 plasmids separately. About 30 pairs of the nitrogen base (bp) linking EcoRI / SalI fragment were obtained from R13 DNA (Labs R and I) from DNAs with M13mp10 repeats. The indicated linker consisted sequentially of the EcoRI adhesive end followed by the recognition zones SStI, SmaI, BamHI and Xbal and the self-adhesive SalI end. The above three fragments are stitched together with the resulting approximately 5.4 kb intermediate plasmid (pERs) EPO-DNA, which on the one hand is restricted by the use of endonucleosis recognition by the block of useful regions. The PERs plasmid is then transformed with HindIII and SalI to produce erythropoietin from DNA and EcoRI into the SalI binding linkage (M13mp10).

The above 1.4 kb DNA fragment is stitched into the BamHI / SalI region of plasmid pBP322, which is about 4.0 kb in length, and other HindIII / BabI1 linking fragments of the M13mp10 fragment. The M13mp10 linking fragment is the HindIII-adhesive end followed by the recognition fragments PstI, SalI, XbaI and BamHI. The stitching product again yields a useful intermediate plasmid pBR-EPO containing EPO-DNA, which is flanked on both sides by the restriction region sequences.

features that are capable of autoduplication expression

Selected EPO-DNA for expression in COS-I (pDSVL.1) cells is pre-engineered for

E.Coli screening and autoduplication. The above characteristics are achieved thanks to the initiation of replication and the DNA gene sequence resistant to the action of ampicillin, which is located in the region of pBP322 plasmid nucleotide 2448 and 4362 approximation. The indicated sequence is structurally modified by the introduction of '' linking regions, which provide recognition of HIndlII as soon as 2448 nucleotides flank it before it can incorporate the specified vector. In addition to vector selection, useful properties of EPO-DNA include COS-I cells and the presence of a queue that acts to amplify the virus in mammalian cells. The above characteristics are achieved thanks to the start of the replicative DNA sequence and the late sequence of the DNA gene, which is a viral enhancer involved in a 342-base pair that constructs the SV40 genome nucleotides S170 to 270. A unique Bounding Region (BamHI) is formed on the vector to the viral enhancer sequence due to the presence of the standard amplifier sequence using the linker sequence commercially available (Collaborative Research). In addition, a sequence of 23 nitrogen pairs (as a derivative of nucleotide numbers 2553 to 2770 of the SV40 virus) containing a polyadenylation signal of the viral iRNA late gene (commonly known as a transcription terminator) was introduced into the vector. The indicated fragment is located in the vector in the appropriate order relative to the late gene virus enhancer through the unique BamHI region. The specified vector also represents another mammalian gene located in an immaterial position with respect to possible gene transcription, which will be located within the specified unique region between the viral enhancer and the terminator. [The indicated mammalian gene contains approximately 2500 bp of dihydrofolia-reductose (DHFR) - mouse minigene isolated from pMG-I, Gresser plasmid, etc., P.N.A.S. (USA), 79, p.6522-6526, 1982] And again, most components of the active plasmid pDSVLI consist of 2448-4362 nucleotides from pBR322 together with 5 / A1-270 (342 bp) and 2353-2770 (273 bp). nucleotides of viral DNA containing SV40 virus.

According to the previously described procedures, e.g., Maniatis et al., The erythropoietin-encoding DNA is isolated from the pBP-ERO plasmid in the form of the BamHI fragment and sewn into the pDSVLI plasmid cut in BamHI. Two of the resulting clonal vectors (duplicated H and L vectors) are analyzed by the restriction fragment (see Figure 2 for an illustration of pDSVL1-M _E E) to confirm the correct insertion of the EPO gene. We left the misaligned EPO-gene vectors as regulators of negative sizes in transfection experiments for the determination of EPO expression levels in hosts transformed with vectors with the correct EPO-DNA orientation.

We combine the M, L, X, G vectors with DNA carrier (mouse liver and spleen DNA). 60 mm plates were used for transfection using calcium phosphate micron precipitation methods. Double 60 mm plates were transfected with DNA carrier as an "imitation" of negative size transformation control. After 5 days, the new culture medium was assayed for the presence of polypeptides which possess the immunological properties of natural erythropoietin.

EXAMPLE Isolated from lamb gene, boiled

A. Initial EPO expression system using COS-I cells

Selected primary levels of microbial synthesis are exposed to isolated amounts of the polypeptide human erythropoietin encoded by the DNA genome EPO clones, and the system comprises expression of host mammalian cell traits (e.g., COS-I cells, A.T.C.C. No..CL-1650). Initially, the human EPO gene is subcloned (cleaved) into a "shuttle" vector that has the ability to autonomously replicate in both host E.coli (thanks to DNA derived from pBR322) and the COS-I lineage in a mammalian cell (thanks to the presence of SV40 virus-derived DNA). ). The "shuttle vector" containing the EPO polypeptide material is produced in transfected cells and secreted into the cell culture medium. More specifically, the trait expression vector is constructed according to the methodologies outlined below. DNA hEl clone containing

BamHI and HindIII genomic by EPObinding endonuclease and isolating! 5.6 DNA fragment found to contain the complete EPO gene. The indicated DNA fragment was mixed and ligated with the bacterial pVCB plasmid (Bethesda Research Laboratories, Ine.); which is similarly processed to generate the intermediate plasmid puC8-EVE, thereby generating a suitable source of the organic fragment.

Selected vectors for expression of EPO-DNA are pre-generated in the COS-I cell. The plasmid pSV4SEt, which contains the DNA sequence, offers the possibility of selection and autoduplication in E. coli. These characteristics are achieved thanks to the initiation of replication and the sequence of the DNA gene that is resistant to ampicillin, which is located in the region approximating nucleotides 2442 to 4332 of the bacterial plasmid pBR322. The given sequence changes structure due to the introduction of a linking strand which provides the recognition region as soon as it touches nucleotide 2443. The pSV4SEt plasmid can also autoduplicate in COS-I cells. The above characteristics are achieved thanks to the use of the 342 bp fragment, which has the origin of viral replication with the help of SV40 virus (nucleotides 5171 to 370). The above moiety is modified by introducing a linker that provides an EcoRI recognition region that is adjacent to 270 nucleotides, and by introducing a specific linker that delivers a region close to nucleotide 5171. This vector contains a 1061 bp fragment (nucleotide number 1711 to 2772) of the SV40 virus with an additional linker that provides the SalI recognition region at nucleotide 2772.

Within the given fragment is a unique recognition BamHI sequence. In conclusion, the pV4SEt plasmid contains unique recognition regions BamHI and HindIII that allow for the introduction of the human EPO gene. The indicated sequences allow replication and selective selection in E. coli and replication in COS-I cells.

To introduce the EPO gene into the pSV4SEt plasmid, the p40-HuE plasmid is digested with BamHI and HindIII to the limiting endonuclease, and the 5.6 kb EPO encoding the DNA fragment is isolated. pSV4SEt plasmid m.p. digested with BamHI and Hindlll, and the main portion of the fragment consisting of 2513 pairs of bases is isolated (retaining all necessary functions). The indicated fragments are mixed and stitched to form the final vector pSVgHu EPO '(see Figure 3). This vector is diluted in E. coli and the IIIKt vector is isolated. A restriction enzyme analysis is performed to confirm that the EPO gene has been introduced.

The DNA pSVgHu EPO plasmid is used to express the features of the EPO-polypeptide substance in COS-I cells. In detail, the DNA pSVgHu EPO plasmids bind to the DNA carrier and transported to COS-I cell triplicate 60 mm plates. For control of the DNA carrier, one transfected into COS-I cells.

The culture medium of the cells is selected 5-7 hours later. and is tested for the presence of polypeptides that have immunological properties specific for free human EPO.

B. Second expression system of EPO traits in COS-I cells

Another expression system is used to obtain improved products of human erythropoietin polypeptide material encoded by the human genomic DNA clone forming EPO in COS-I cells (A.T.C.C. No. CRL-1650).

In the previous system, the EPO traits in COS-I cells are expressed using an enhancer present there, which is in the BamHI region of the restriction fragment, according to HindIII, respectively, and is 5.6 kb long. In another structure, the EPO gene changes upon expression of traits using the late viral SV40 enhancer. More specifically, a restriction fragment of the human genomic EPO, i.e. the cloned 5.6 kb BamHI corresponding HindIII is modified by this procedure. As described previously, the pVC8-HuE plasmid is cleaved in the presence of BamHI and the limiting endonuclease BStEII. The restriction endonuclease BStEII cleaves the 5.6 kb EPO gene at a site 44 pairs of bases 5 'to the original ATG encoding the peptide precursor and approximately 680 pairs of bases 3' to the HindIII restriction site. This is followed by a fragment containing about 4900 pairs of bases. A DNA fragment with a synthetic linker consisting of SalI and BStEII adhesive ends and an internal BamHI recognition region is synthesized and purified. Both of these fragments are fused and linked to plasmid pB322, which is cleaved with SalI and BamHI to give the intermediate plasmid pBRgHe. The human EPO genomic gene can be isolated from it as a fragment of Barnis with 4900 pairs of bases, which contains the complete gene structure with a unitary ATG 4.4 bp 3 'corresponding BamHI region adjacent to the end of the coded amino group.

The resulting fragment is isolated and introduced as a BamHI fragment into the plasmid pDSVLI-cleaved expression vector BamHI (see Example 6). The resulting plasmid pSVLgHu EPO, as shown in Fig. 4, is used to highlight the features of the EPO polypeptide from COS-I cells as described in Examples 6 and 7a.

EXAMPLE

Culture medium grown from 6 transfected COS-I cell cultures in Example 6 was analyzed by radioimmunoassay according to the procedure outlined in Example 2, Part B. Each sample was analyzed at an aliquot of 250, 125, 50, 25 μl. Layers of growing cells with the help of putatively transfected vectors with the wrong orientation of EPO genes had negative immunoreactivity. For each sample of two fluid layers derived from COS-I cells transfected with vectors (H and L) with the correct orientation of EPO DNA, the percentage of R5I-EP0 inhibition coupled to the antibody ranges from 72 to 88%, which fits all values into the supernatant. part of the standard curve. Therefore, the true concentration of erythropoietin in floating culture layers cannot be accurately estimated. Small calculations of 300 mv / ml were made, but the calculations were made with the largest aliquot sample (250 mkl).

The culture fluid sample obtained in Example 6 and the 57-day culture fluids described in Example 7a were analyzed by radioimmunoassay to compare the activity of monkey and human recombinant EPO with the free human EPO standard. The results obtained are plotted in FIG.

In short, the results obtained unexpectedly showed that recombinant monkey EPO competed substantially for antibody bound to human anti-EPO, which could not be completely inhibited in the assays.

The maximum percentage inhibition values for recombinant human erythropoietin are still close to standard values. The parallel type of the dose-effect curve shows the immunological identity of the sequences (epitopes). Prior to calculating monkey EPO concentrations in culture fluids, reassessments were made at high values of the indicated dilution. They have been found to range from 2.91 to 9.12 U / ml. The calculated levels of human EPO production were determined in 392 U / ml of sample cultured for 5 days and 567 mV / ml in sample cultured for 7 days, respectively. Monkey EPO production levels calculated in Example 7B expression systems were comparable or better.

EXAMPLE

The culture fluids obtained in Examples 6 and 7 were analyzed in vitro to determine EPO activity according to Goldvasser et al., Endocrinology, 97, p.2. 315-313 (1975). Monkey EPO values in the culture fluids tested ranged from 3.2 to 4.3 U / ml. Human EPO culture fluids were also active in the resulting calculations in an in vitro assay, and furthermore, the activity reported can be neutralized by treatment with anti-EPO antibody. Culture fluids of recombinant monkey EPO in Example 6 were also assayed for quantitative in vitro bioactivity. according to the general methodology of Cotes et al., Nature, 191, p. 1065-1067 (1967) and Hammand et al., Ann. N.Y. Acad. Sci. 149, p. 516-527 (1968). The indicated activity levels ranged from 0.94 to 1.24 U / ml.

EXAMPLE

In the foregoing examples, recombinant human or monkey EPO material is derived from vectors used for transfection into COS-I cells. The indicated vectors replicate in COS-I cells due to the presence of SV40 T antigen within the cell, starting with replication in the indicated vectors.

Although the resulting vectors reproduce the good properties of EPO in COS-I cells, these properties are transient (7-14 days) due to possible loss of the vector. In addition, only a small percentage of the reproduced COS-I becomes productively transfected with the indicated vectors. This example describes trait expression systems using Chinese hamster ovary DHFR cells (CHO) and a selectable marker, DHFR.

For consideration of related expression systems, see U.S. Pat. No. 4,399,216; 117058 1/7059 and 117060 issued August 29. In 1984.

CHO in DHFP cells (Dux-Bll) CHO KI, Urlaub et al., Proc. Nat. Acad. Sci. (USA), vol.77, p. 4461 (1980), due to mutations in structural genes, lack of enzyme dihydrofolate reductase (DHFR) and therefore necessitating glycine, hypoxanthine and thymidine in culture media.

Plasmid pDSVL-MKE (Example 6) pDSLV-gH ₄ EP0 (Example 7B) was co-transfected into DHFR CHO cells grown in medium containing hypoxanthine, thymidine and glycine in 60 mm culture plates. Plasmid pSVgH ₄ EP0 (Example 7a) was mixed with plasmid pMG ₂ , which contains a mouse gene based on dihydrofolate reductase, cloned into the bacterial plasmid pRR 322 (according to Gasser et al., Supra). The plasmid-DNA mixture is transfected into DNA-CHO cells (cells that take one plasmid will usually also take the second plasmid). After 3 days, the resulting cells are dispersed by trypsinization in several 100-mm culture plates in medium lacking hypoxanthine and thymidine. Only cells that are transformed to a stable state under the influence of the DHFR gene show the survival of the EPO gene in that medium. After 7-21 days, colonies of living cells become visible. Specified colonies of transformants, following dispersion by trypsinization, can continue to proliferate in media lacking hypoxanthine and thymidine to form new cell strains (e.g., CHO pDSVL-MKEPO, CHO pSVgH ₄ EP0, CHO pDSVL-9B ₄ EP0).

Culture fluids from the above cell strains are assayed by radioimmunoassay for monkey or human recombinant EPO. Strain medium CHO pDSVL-MKEPO contains EPO with the same immune properties as EPO derived from COS-I cells transfected with plasmid pDSVL-MKEPO. A 65-hour culture fluid sample has a monkey EPO of 0.6 v / ml.

Culture fluids derived from CHO pSVgH ₄ EP0 and CHO pDSVLgH ₄ EP0 have recombinant human EPO having the same immunological properties as EPO derived from COS-I cells transfected with pSVgH ₄ EP0 or pDSVL-gH ₄ EPO. A 3-day culture fluid sample derived from CHO pSVgH ₄ EP0 contains 2.99 U / ml human EPO, and a sample maintained 5.5 days obtained from CHO pDSVL-gH ₄ EP0 contains 18.2 U / ml human EPO as shown by radioimmunoassay results,

The quality of EPO derived from the above can be enhanced by amplifying genes, creating new cell strains with greater efficiency. The enzyme dihydrofolate reductase (DHFR), which is a product encoded by the DHFR gene, can be inhibited by methotrexate (ΜΤΧ). Cells that reproduce in media lacking hypoxanthine and thymidine are inhibited or killed by methotrexate. Under certain conditions (eg, minimal methotrexate concentration), cell resistance and their ability to grow on methotrexate (ΜΤΧ) can be obtained. These cells have been shown to become resistant ΜΤΧ due to the enhancement of a number of their DHFR genes, resulting in increased enzyme production. Live cells, in turn, can be treated with elevated concentrations of methotrexate, resulting in increased levels of DHFR genes in cell strains. "Passing genes" (eg, EPO) carry the expression vector along with or transform it with the DHFR gene, as it is often found, to increase its number of reproductions.

An example of a practical implementation of an amplification system is the CHD pDSVL-MKE cell strain treated with methotrexate at increasing concentrations (0 nM, 30 nM, 100 nM). Samples cultured for 65 hours at each amplification step contain erythropoietin in amounts of 0.60, 2.45 and 6.10 U / ml, respectively, as determined by radioimmunoassay. CHO pDSVL-9H ₄ EP0 cell strains were treated with methotrexate at increasing concentrations in the series: 30 nM, 50 nM, 100 nM, 200 nM, 1 mkM and 5 mkM. Sample containing 3 days supernatant fluid treated with 100 nM methotrexate (ΜΤΧ) contains 3089 ± 129 U / ml human erythropoietin in a radioimmunoassay. Samples containing 48 hours culture medium treated with methotrexate at 100 nM and 1 mKM contained 466 and 1352 U / ml, respectively, in human erythropoietin radioimmunoassay (mean value 3 times for the specimens). According to the methodology given 60

O mm in a Petri dish containing 5 ml of medium and inoculated 1x10 cells. After 24 hours, the medium is removed and replaced with a new medium (5 ml) containing no high serum DMEM with the addition of 0.1 M non-essential amino acids and L-glutamine. Erythropoietin accumulates in serum-free media for 48 hours. The medium is then assayed by radioimmunoassay and the cells are trypsinized and then counted. The mean values for erythropoietin in radioimmunoassay in the samples are approximately 467 and 1352 U / ml for cells grown at concentrations of 100 nM and 1 mMM methotrexate. The actual amount of erythropoietin in the Petri dish was 2335 and 6750 V. The average cells in the dish were 1.94 * 10® and 3.12 * 10®, respectively.

Thus, the effective production rate under the given conditions is 1264 and 2167 V / 10® cells within 48 hours.

The cell cultures described above are genetically diverse products. Standard screening methods are used to isolate the genetically dead clones that have the highest production potential, (see Section A, Part 2, June 1, 1984, US Biological Preparations Science). screening unit, Center for Medicines and Biopreparations, Food and Drug Board).

The productivity of the CHO cell lines producing erythropoietin described above may be increased using appropriate cell culture techniques. Serum is usually required for the growth of mammalian cells in culture media. The method of obtaining erythropoietin from CHO cells in serum-free medium facilitates the rectification of erythropoietin from the culture medium. Serious amounts of erythropoietin in serum-free media, sufficient for production, can be obtained as described below.

For CHO pDSVL-gH ₄ EP0 lines, cells grown under standard cell culture conditions were seeded with cell culture vials. The indicated cells reproduce in vials as a suspension cell line inoculated with 50:50 DHEM in high-glucose medium and F12Ham supplemented with 5% cow embryo serum, L-glutamine ¹ , penicillin, streptomycin 0.05. mM of a mixture of non-essential amino acids and the corresponding concentration of methotrexate.

Suspended cell cultures have the potential to rapidly replicate erythropoietin, which is derived from CHO cells. CHO cells, grown in suspension, are used to seed cells in rotating vessels. Initial 7 2 culture density 1.5 * 10 viable cells in a 850 cm designated vessel containing 200 ml medium.

days, cells are cultured to monolayer formation with relatively dense cell lines. The cell culture medium used in this phase is analogous to the medium used for cell culture in suspension. At the end of the 3-day cell growth period, serum-containing medium is separated and replaced with 100 ml serum-free medium. This medium contains a 50:50 mixture of high-glucose DHEM and F12Ham, supplemented with 0.05 mM of non-essential amino acids and L-glutamine. Rotating dishes with prepared medium are placed in the incubator for 2-3 hours. The medium is then removed and replaced with 100 ml of new serum-free medium. After 1-3 hours of incubation in serum-free medium, the concentration of serum protein contamination decreases. The rotating dishes are then placed in the incubator for 7 days. During this period, erythropoietin accumulates in serum-free culture media. The old medium is then removed and replaced with the new serum-free medium required for the second production cycle. A practical example of the realization of the given system, stored in serum-free medium for 7 days, contained 3892-409 U / ml erythropoietin in a radioimmunoassay. Based on cell density calculations of about 0.91.8 * ^{10 5} cells per cm ² , each rotating dish of 850 cm ² area contains between 0.75 and 1.5 * 10 ⁸ cells. The rate of erythropoietin production over 7 days in 100 ml culture is then between 750 and 1470 U / 10 ⁶ cells in 48 hours.

The supernatant fluids from the cell line CHO pDSVL-MKEPO amplified with methotrexate at 10 nM were assayed by radioimmunoassay for erythropoietin activity. in vitro and in vivo. A radioimmunoassayed sample prepared in concentrated medium contains 41.2 + 1.4 U / ml erythropoietin; Biological activity was determined at 41.2 + 0.064 U / ml in vitro and 42.5 + 5 U / ml in vivo. In the sequencing of polypeptide products of amino acid residues, erythropoietin products of the dominant type have 3 residues in the leading sequence, which are linked to putative amino groups terminating in alanine. It is currently unclear whether this is the result of improper processing of the CHO cell polypeptide nucleus or whether the structure of the human and monkey erythropoietins is different at the amino terminus.

The supernatant fluid prepared from the CHO cell line pDSVL-gH ₄ EP0 was tested in 3 assays. By radioimmunoassay

5.5 U / ml in the recombinant human erythropoietin assay: 15.8 + 4.6 U / ml, in the in vivo assay: 16.8 ± 3.0 U / ml.

Prepared from CHO cell line pDSVL-gH ₄ EP0 and incrementally amplified with 100 nM methotrexate was assayed in 3 assays. Radioimmunoassay for 3 days contains 3089 + 129 U / ml recombinant human erythropoietin; 2589 + 71.5 U / ml in the in vitro study and 2040 + 160 U / ml in the in vivo study. The acetic acid sequence demonstrates consistency with the data in Table 4.

The conditioned cell medium prepared from CHO cells and transfected with the pDSV-MKE plasmid pDSV-MKE in 10 nM methotrexate is combined and dialyzed against methotrexate over several days, resulting in a culture with erythropoietin activity of approximately 221 ± 5.1 U / ml (erythropoietin - conditioned media). EPO-CCM). The following experiment was performed to evaluate the effect of EPO-CCM on the hematocrit of a single particle in the Balb / c line. Conditioned cell media prepared from untransfected CHO (CCM) cells and EPO-CCM was regulated using standard buffer for reticulocytes (PB). The conditioned cell medium is used for the control group of animals (3 mice) and for the experimental groups (2 mice group) 2 dosage levels of EPO-CCM are used: 4 units for injection and 44 units for injection. During the 5-week course, 7 mice were injected 3 times per week into the abdomen. In the control group, after the 8th injection, the mean hematocrit was 50.5%, in the 4-unit EPO-CCM group 55.1% and in the 44-unit 67.9%.

Pretty pure mammalian cell expression products can be readily isolated from the culture medium using HPLC (C ₄ ) with a reference gradient, preferably at pH7.

A preliminary attempt was made to determine the properties of recombinant glycoprotein products derived from conditioned COS-I cells and erythropoietin (from human urine) CHO gene expression medium by both Western blot and SDS-PAGE analyzes. The aforementioned studies showed that erythropoietin produced by CHO cells has slightly higher molecular weight than COS-I cell expression products, which in turn are slightly higher than human urine extract. To some degree, all products are heterogeneous. Neuraminidase, an enzyme used to remove sialic acid, causes recombinant cellular COS-I and CHO products to have approximately the same molecular weight and (both) higher molecular weight than human urine extract containing azial groups. Endogenous glucosidase enzyme treatment (EC 3.2.1) allows the recombinant CHO cell product and the urine extract product (for complete carbohydrate removal) to produce nearly homogeneous products of almost identical molecular weights.

Purified erythropoietin from human urine and recombinant CHO cells from erythropoietin according to the present invention are analyzed for carbohydrate composition according to the method (Zedcen et al., Methods in Enzymology, vol. 83, p.139191 (1982)). modified by the hydrolysis method described in Nesser et al., Anai. Biochem., Vol. 142, pp. 58-67 (1984). Experimentally determined carbohydrate content of urine isolate (expressed in molar ratios) is as follows: hexose - 1.73, acetylglucosamine - 1, non - amino acid acetyl - 0.93, fucose - 0, acetylgalactosamine - 0.

Corresponding values of recombinant products (cell line CHO pDVSL-gH and EPO derivatives) (after 3 days in culture medium supplemented with 100 nM methotrexate) have the following values: Hexose 15.09, N-acetylglucosamine 1, Non-amino acid acetyl 0.998, Fucose 0 , Nacetylgalactosamine - 0. The results are in agreement with the above assays (Western blot and SDS-PAGE).

The primary structural conformation of the glycoprotein products of the present invention mimics the free-form erythropoietin to a sufficient degree to obtain one or more of its biological properties, but their carbohydrate content varies.

EXAMPLE

The proposed example covers the complete cycle of erythropoietin production by fitting the nucleotide bases of two structural genes encoding the human erythropoietin sequence presented in Table 6 and having expression codons in E. coli and yeast cells (S. Cerevisiai).

This example further describes the construction of genes encoding erythropoietin analogs. Briefly, the protocols used are described in Alton et al., Supra. (WO 83/04053). These genes are engineered for the initial assembly of complex diploid oligonucleotide components, which are in turn assembled in three discrete sections (blocks). These blocks are assembled for amplification and, when removed from the amplification system, can be mounted sequentially or through a fragment of complex structure-ligation to a suitable expression vector.

Tables 8-14 show the scheme and construction of a gene encoding an erythropoietin translational product that has no upstream sequence or sequence leader but has an initial methionine residue at position 1. Further, the gene introduced into the major part of the E. coli cell prefers codons, and said construct is called the ECEPO gene.

TABLE TABLE ECEPO oligonucleotides

1. AATTCTAGAAACCATGAGGGTAATAAAATA

2. CCATTATTTTATTACCCTCATGGTTTCTAG

3. ATGGCTCCGCCGCGTCTGATCTGCGAC ii. CTCGAGTCGCAGATCAGACGCGGCGGAG

5. TCGAGAGTTCTGGAACGTTACCTGCTG

6. ClICCAGCAGGTAACGTTCCAGAACT

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

9. ACCACTGGTTGTGCTGAACACTGTTC

10. CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACCGTAATGTTTTCGTT

TABLE

Part 1 of ECEPO

Xba I

EcoRI - 1 t 3 aattciag aaacsatgag ggtaataaaa taćtggctcc GČCGCGTCTG

GATC TTTGGTACTC CCATTAT1TT RETURNGG CGGCGCAGAC 2 4

ATClGCGAck CGAGAGTTCT TAGACGCTGA GCTCfrCAAGA

TGAAAACATC

ACTTTTGTAG

S t

CCACTGGTt

GGTG [INCCAA

GGAACGTTAC

CCTTGCAATG

G1GCTGAACA

CACGACTTGT

CTGCTgGuage

GACGACCTTČj ctgttcĮtttg

GACAAGAAAČ]

CTAAAGAAGC

GATTTCTTCG

IEA

AACGAAAACA

TTGCTTTTGT

KpnI BamHI TTACGGTACC G AATGCCATGG CCTAG

TABLE TABLE ECEPO oligonucleotides

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

4. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

6. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAGGGTCIGGCACTGCTGAGCG

8. GCCTCGCTCAGCAGIGCCAGACCCTG

9. AGGCTGTACTGCGTGGCCAGGC'A

10. GCAGTGCCTGGCCACGCAGTACft

11. CTGCTGGTAAACTCCTCTCAGCCGT

12. TTCCCACGGCT-GAGAGGAGTTTACCA

13. GGGAACCGCTGCAGCTGCATGTtGAC

14. GCTTTGTCAACATGCAGCTGCAGCGG

15. AAAGCAGTATCTGGCCTGAGATCTG

16. GATCCAGATCTCAGGCCAGATACT w

P

Email &

H

P rH

P

O

Ph (3

O

H ra ra • H

P

S) • O

OJ

►- {  X ε co «I ω o no t— d CJ CJ ωΐα 1— (1 CJ CJ CJ CJ CJ CJ μ— d CJ CJ jjJcj »- < CJ CJ no μ- CJ CJ i- d ♦ - < μ- d 1— d d μ— CJ CJ r— < α CJ CJ CJ CJ CJ CJ CJ σ μ— d CJ CJ h- d CJ CJ cn - Ι- d 1— CJ CJ CJ CJ CJ Ο CJ CJ d μ- d ί- d t- μ- d CJ CJ CJ ο

et H-

CJ CJ CJ α μ- d μ- d CJ CJ d μ- ® | α α α α h * d CJ α μ- d α α | i— d <? l CJ ο α α α CJ Ό CJ CJ CJ ο α α ιη | α α —ι CJ CJ μ- d μ- d ρ | ρ d CJ CJ α α α α οι | α α d P μ- d d ρ F "to ρ d α u —Ild μ- d ι— b— d ρ | α α r-1 | d ρ μ- d CJ CJ μ— d α CJ μ- d α α H- d p α α ΰ, * - CJ CJ α α α α α d μ- α α μ- F- _| α u d ι— α u α α d μ- £ =. d Η * d α α d ι— 1- d d μ- d rj ~ * - CJ o π- d _LiJ α α α ω O d μ- d μ- CJ CJ d μ- α α α α d b— d p d ι- α α μ— d CJ CJ α α α α μ- d CJ CJ Ρ d d μ- α α d P ρ d α α ρ d CJ CJ OJ | ω α α α d μ- d P d μ- α α ° ι α Ο CJ CJ α α α α -Ί α α d Ι- α α σ ^ μ- d μ- d d ρ α α α α CJ Ο d μ- α α α α

u

CJ

CJ u

CJ να:

o

CJ

CJ

C c t - · μα:> old o

UI d

α α α ρ. d μ- <τ d ρ μ- d r \ | a α - < m | d μ- Ό | α α -4 Ια α α α d μ- μ- d μ- d μ- d α α α α α α α α α α μ- <Ζ— α α μ- d α α α ι- β: α α d α α α α d Ρ

TABLE

ECEEO 3 divides

1. GATCCAGATCTCTGACTACTCTGC

2. ACGCAGCAGAGTAGTCAGAGATCTG

5. TGCGTGC.TCTGGGTGCACAGAAAGAGG ii. GATAGCCTCTTTCTGTGCACCCAGAGC

5. CTATCTCTCCGCCGGATGCTGCATCT

6. CAGCAGATGCAGCATCCGGCGGAGA

7. GCTGCACCGCTGCG.TACCATCACT.G

8. ATCAGCAGTGATGGTACGCAGCGGTG

9. CTGATACCTTCCGCAAACTGTTTCG

10. ATACACGAAACAGTTTGCGGAAGGT

11. TGTATACTCTAACHCCTGCGTGGTA

12. CAGTTTACCACG.C.AGGAAGTTAGAGT

13. AACTGAAACTGTATACTGGCGAAGC

1Δ. GGCATGCTTCGCCAGTATACAGTTT

15. ATGCCGTACTGGTGACCGCTAATAG

16. TCGACTATTAGCGGTCACCAGTAC

TABLE

EOETO 3 divides

BaniH I B g 111

GA TCCAGATCTCTG GTCTAGAGAC

1 ACTACTCTGC TGATGAGACG 2 [tgcgtgctct gggtgcacag aaagaggotaota tctctccgcc ACGCAAzGAGA CCCACGTGTC TTTCTCCGAT AGjAGAGGCGG 4 GGATGCTGCA CCTACGACGT 6th L ² L Σ TCTCCTGCAC CGCTGCGTAC CATCACTGpT GATACCTTCC AGACGACpTG GCGACGCATG GTAGTGACGA CTHTGGAAGG θ GCAAACTGTT CGTTTGACAA 10th n, TCGfTGTATAC TCTAACTTCC TGCGTGGTAP ACTGAAACTG AGCA'CAT Ali G AGAT1GAAGG ACGCACCATT IGACtT T TGAC 12 TATACTGGCG ATATGACCGC 1Δ 15 · SslI AAGC ^ TGCCG TACTGGTGAC CGCTAATAG TTCGIACGGJC ATGACCACTG GCGATTATC AGCT 16th

TABLE

ECEPO Gene

Xba I CTAG

AAACCA7GAG

TTTGGTACTC

GGTAATAAAA

CCATTATTTT

-1 1 MetAla

TAATGGCTCC

ATTACCGAGG

GCCGCGTCTG

CGGCGCAGAC

ATCTGCGACT CGAGAGTTCT GGAACGTTAC CTGCTGGAAG CTAAAGAAGC TAGACGCTGA GCTCTCAAGA CCTTGCAATG GACGACCTTC GATTTCTTCG

TGAAAACA7Č ACCACTGGTT GTGCTGAACA CTGITCTTTG AACGAAAACA ACITTTGTAG TGGTGACCAA CACGACTTGT GACAAGAAAC TTGCTTTTGT jr. 7TACGGTACC AGACACOAAG GTTAACTTCT ACCCTTGGAA ACGTATGGAA AAIGCCATGG TCTGTGdTTC CAATTGAAGA TGCGAACCTT TGCATACCTT

GTTGGICAAC aagcagi) tga agtttggcag ggtctggcac tgctgagcga CAACCAGTTG TTCGTCA'ACT TCAAACCGTC CCAGACCGTG ACGACTCGCT ggctgtactg CGTGGCCAGG cactggtggt aaactcctct cagcCGGGGGGGGGGAC

AACCGCTGCA GCTGCATGTT GACAAAGCAG TATCTGGCCT GAGATCTCTG TIGGCGACGT CGACGTACAA CTGTTTCGTC ATAGACCGGA CTCTAGAGAC

ACTACTC1GC TGCGTGCTCT GGGTGCACAG AAAGAGGCTA TCTCTCCGCC TGATGAGACG ACGCACGAGA CCCACGTGTC TTTCTCCGAT AGAGAGGCC-G

GGATGCTGCA TCTGCTGCAC CGCTGCGTAC CATCACTGCT GATACCTTCC CCTACGACGT AGACGACGTG GCGACGCAIG GTAGTGACGA CTATGGAAGG

GCAA'ACTGTT TCGTGTATAC TCTAACTTCC TGCGTGGTAA ACTGAAACTG CG777GACAA AGCACATA7G AGAT7GAAGG ACGCACCATT 7GAC777GAC

Hall

7A7AC7GGCG

A7A7GACCGC

AAGCATGCCG

TTCGTACGG'C

TAC7GG7GAC

ATGACCAC7G

CGC.TAATAG GCGATTATCA GC7

Specifically, Table 8 illustrates the use of oligonucleotides to generate the I-terminal portion of the ECEPO gene's terminal residue, which encodes the terminal human polypeptide residue. Oligonucleotides; two (1 and 2, 3 and 4 and so on) are joined together and then stitched together to form part I of ECEPO as in Table 9. It should be noted that the assembled portion has EcoRI and BamHI adhesive limb ends, furthermore, the EcoRI current of the adhesive end is directed by the restriction enzyme XbaI recognition region, and the KpnI recognition site is directed against the BamHI site of the adhesive end.

the portion can be easily amplified using the phage MI3 phage used to verify the order of the portion. Some difficulties unexpectedly arise in extracting a portion of the XbaIKpnI fragment from DNAPF generated by E. coli. probably due to the recognition region of methylation of KpnI bases in the host zone. Therefore, the DNA phage is isolated and reproduced in bipedal form by in vitro primer extension and subsequently isolated to the required bipartite fragment.

and 3 portions of the ECEPO gene (Tables 9 and 13) are generated in the same manner from oligonucleotides in Tables 10 and 12, respectively. Each part is amplified in the MI3 vector which is used for sequence screening and isolated from the DNA phage. As can be seen from Table 11, the second portion of ECEPO is composed of the BamII and SalI sticky ends and can be isolated from the DNA PF as a BglII / SalI fragment. The three moieties thus obtained can be readily assembled into a DNA primary sequence (Table 14) encoding a complete human EPO polypeptide with a methionine terminal codon (ATG) to initiate the translational start of E. coli. It should be noted that there is a series of nitrogen bases upstream of the primary ATG current, sufficiently duplicating the E. coli of the OMP-f gene, strongly expressed by the fragment sequence-binding ribosome.

Any suitable expression vector can be used for ECEPO transfer. A specific vector for ECEPO gene expression as a temperature sensitive plasmid pCFM536 is the plasmid pCFM414 [A.T.C.C. 40076], as described in co-pending U.S. Patent Application No. 636727, filed April 4, 1984. August 6

More specifically, pCFM536 cleaves with Xbal and HindIII; large fragment is isolated and used for double-sided stitching with the ECEPO gene. Part I / XbAI / KpnI /, 2 / KpnI / Bgl / and 3 / BglII / Salle / are pre-assembled into the MI3 and EPO gene in the correct order from there as a separate fragment XbaI / HindIII. This fragment incorporates a portion of the polysynthetic linkage from the MI3 Mp9 • phage, which constructs SalI and Hindlll fragments therein.

Expression control in the resulting expression plasmid, p536, is effected by the promoter lobe P _L , which can itself control the repressor gene C- ₈₅₇ (such as that produced by the KI2 Htrp chain E. coli).

The produced ECEPO gene can be modified by various methods for encoding erythropoietin analogs such as [AS _n ² , des-Pro ² according to ILe ⁶ ] hEP0 and [His ⁷ ] hEP0 as described above.

A. [AS _n ² , des-Pro ² by ILe ⁶ ] hEP0

Plasmid 536 carrying the produced ECEPO gene, Table XIV, cleaves with Hindlll and Xhol as an insert of Xbal into Hindlll. The latter endonuclease cuts the ECEPO gene in a universal recognition fragment containing 6 pairs of nitrogen bases, v 3 collapsing the last codon base encoding Asp with a second

codon base Arg ¹⁰ . Under construction bonding link Xbal / Xhol, having such sequence: +1 2 7th 8th Xbal Me Ala Asn . Cys Asp 5'-CTAG ATG GCT AAT TGC GAC-3 Xhol 8 ' -TAC CGA TTA ACG CTG AGCT-5

The Xbal / Xhol binding site and the Xhol / HindIII fragment of the ECEPO gene are inserted into a large fragment resulting from the cleavage of Xbal and HindIII plasmid pCFM526, a derivative of plasmid pCFM414 (ATCC 40076), as described in co-pending U.S. Patent No. 636727. August 6 Charles P. Mororr, for the generation of a plasmid carrying the DNA sequence encoding E. coli expression in the form of the desired analog of Met ' ¹ .

B. [His ⁷ ] hEP0

Plasmid 536 is cleaved with Hindlll and Xbal as in Part A. Tying! chain Xbal / Xhal is made in the following sequence:

Xbal +1 2 3 4 5 6th 7th 8 9 Xhol Me Alla Pro Pro Arg Leu Ala His Asp 5'-CTAG ATG GCT CCG CCA CGT CTG ATC CAT GAC-3 8 ' TAC CGA GCC GGT GCA GAC AG GTA CGT AGCT-5 Binding chain and ECEPO sequences snippet Xhol / HindIII

then inserted into the pCFM plasmid for generation of the DNA encoding expression of the desired analog of Met ' ¹ form E. coli,

Expression of the gene ·· (SCEPO) containing yeast dominant codons occurs as described in Tables XV-XXI. As with the ECEPO gene, the complete structure contains three sets of oligonucleotide derivatives (Tables 15, 17, and 19) that form duplexes and assemble into sections (Tables 16, 18, and 20). It should be noted that the synthesis is partially facilitated by the use of some suboptimal codons in both EPO and ECEPO structures, i. oligonucleotides 7-12 of Section I were identical for both genes, just as oligonucleotides 1-6 of Section 2 in each gene were identical.

6?

TABLE TABLE SCEPO oligonucleotides

1. AATTCAAGCTTGGATAAAAGAGCT

2. GIGGAGCTCTTTTATCCAAGCTTG

3. CCACCAAGAT.TGATCTGTGACTC

A. TCTCGAGTCACAGATCAATCTTG

5. GAGAGTTTTGGAAAGATACTTGTIG

6. CITCCAACAAGTATCTTTCCAAAAC

7. GAAGCTAAAGAAGCTGAAAACATC

8. GTGGTGATGTTTTCAGCTTCTTTAG

°. ACCACTGGTTGTGCTGAACACTGTTC

10. 'CAAAGAACAGTGTTCAGCACAACCA

11. TTTGAACGAAAACATTACGGTACCG

12. GATCCGGTACC.GTAATGTTTTCGTT

TABLE

SCEPO '1 · Part

EcoRl HindlII TO AATTCA AGCTTGGATA

GT TCGAACCTAT 2

AAAGAGCTbc ACCAAGATTG ATCTGTGACT cbAGAGTTTT TTTCTCGAGG TGjGTTCTAAC TAGACACTGA GCTČljCAAAA

GGAAAGATAC

CCTTTCTATG

GTGCTGAACA CACGACTT G T ttgt.tgĮsaag aa'caacctTc] ctgttcĮtttg

GACAAGAAACj

CTAAAGAAGC

GATTTGTTCG

ON

AACGAAAACA UGCTT T T GT

TGAAAACATC

ACTTTTGTAG

KpnI

TTACGGTACC

AATGCCATGG hCCACTGGTT

TGGTGąACCAA

BamHI

G

CCT AG

6ch

TABLE TABLE SCEPO oligonucleotides

1. AATTCGGTACCAGACACCAAGGT

2. GTTAACCTTGGTGTCTGGTACCG

3. TAACTTCTACGCTTGGAAACGTAT

а. TTCCATACGTTTCCAAGCGTAGAA

5. GGAAGTTGGTCAACAAGCAGTTGAAGT

б. CCAAACTTCAACTGCTTGTTGACCAAC

7. TTGGCAAGGTTTGGCCTTGTTATCTG '8. GCTTCAGATAACAAGGCCAAACCTTG

9. AAGCTGTTTTGAGAGGTCAAGCCT

10. AACAAGGCTTGACCTCTCAAAACA

11. TGTTGGTTAACTCTT-CTCAACCATGGG

12. TGGTTCCCATGGT.TGAGAAGAGTTAACC

13. AACCATTGCAATTGCACGTCGAT

1ύ. CTTTATCGACGTGCAATTGCAA

15. AAAGCCGTCTCTGGTTTGAGATCTG

16. ga · CCAGATCTCAAACCAGAGACGG

TABLE

Part 2 of the SCELO

KpnI

EcoRI 1 TT ^ TTCGGTKCC

GCCATGG

AGACACCAAG

TCTGTGGTTC gt | taacttct caaūg ^ aga agt {ttggcaa

TCAAAČČjjTT

CC TTI GT T GGT ggaačaaGAgca

GATHAAGCCG

CTAirTČpGC

ACGCTTGGAA

TGCGAACCTT

4_

GGTTTGGCCT

CCAAACCGGA

SO

TAACTCTTCT

ATTGAGAAGA acgtatLgaa tgcatacčTtt]

GTTGGTCAAC

CAACCAGTTG aagctgttga

TTCGACAACT

TGTTATCTGh ACAATAGACT

CAACCATGGG

GTTGGTAČCC

AGCTGTTTTG

TčŠĮacaaaac

To {aaccattgca uggiĮaacgt. BglII BamHI

TCTCTGGTTT GAGATCTG AGAGACCAAA CTCTAGACCTA G

......

agaggtcaag

TCTCCAGTTC

ATTGCACGTC

TAACGTGCAG

TABLE TABLE SCEPO oligonucleotides

1. GATCCAGATCTTTGACTACTTTGTT

2. TCTCAACAAAGTAGTCAAAGATCTG

3. GAGAGCTTTGGGTGCTCAAAAGGAAG

4. ATGGCTTCCTTTTGAGCACCCAAAGC

5. CCATTTCCCCACCAGACGCTGCTT

6. GCAGAAGCAGCGTCTGGTGGGGAA

7. CTGCCGCTCCATTGAGAACCATC

6. CAGTGATGGTTCTCAATGGAGCG

9. ACTGCTGATACCTTCAGAAAGTT

10. GAATAACTTTCTGAAGGTATCAG

11. ATTCAGAGTTTACTCCAACTTCT

12. CTCAAGAAGTTGGA'GTAAACTCT

13. TGAGAGGTAAATTGAAGTTGTACAC

14. ACCGGTGTACAACTTCAATTTACCT

15. CGGTGAAGCCTGTAGAACTGGT

16. CTGTCACCAGTTCTACAGGCTTC

17. GACAGATAAGCCCGACTGATAA

18. GTTGTTATCAGTCGGGCTTAT

19. CAACAGTGTAGATGTAACAAAG

- 20. TCGACTTTGTTACATCTACACT

TABLE

Part 3 of SCEPO

ΒβιτιΗΪ BglII 1

GATC'CAGATCTTTG ACTACTTTGT GTCTAGAAAC TGATGAAACA

TCAGAGCTTT

AGTČT ^ GAAA

GGGTGCTCAA

CCCACGAGTT

CATIGAGAAC

GTAACTCTTG

AAGGAAGtCA TTTCCCCACC TTCCTTCGG1 PAGAGGGGTGG catcActgct

GTAGTGAČfSA

GATACCTTCA

CTATGGAAGT

AGACGCTGCT

TCTGCGACGA

GAAAGTThTT

CTTTCAAlAA

TCTGCCGCTC

ACHACGGCGAG

TGCAACTTCT

AGGTTGAAGA [tgagaggtaa attgaagttg

THANK YOUCCATT TAACTICAAC ¹ 14

n

CAGAGTTTAC

ČJrCTCAAATG

12th

AAGCCTGTAG

TTCGGACATC

AACTGGTbAC

TIGACCACTG

AGAIAAGCCC

TŪTATTCGGG gactgataaA:

CTGACTATTG aacagtgtag

Tt ^ tcacatc

Hall

ATGTAACAAA G TACATTC-TTT CAGCT

TABLE

The SCHEP gene

-1 +1

Hindlll fiGČTTGGAlA ArgAle AAAGAGCTCC ACCAAGATTG A1CTGTGACT CGAGAGTTTT ACCTAT ' TTTCTCGAGG TGGTTCTAAC TAGACACTGA GCTCTCAAAA GGAAAGATAC TTGTTGGAAG CTAAAGAAGC TGAAAACATC ACCACTGGTT CCTTTCTATG AACAACCTTC GATTTCTTCG ACTTTTGTAG TGGTGACCAA GTGCTGAACA CTGTTCTTTG AACGAAAACA, TTACGGTACC AGACACCAAG CACGACTTGT GACAAGAAAC TTGCTTTTGT AA7GCCATGG TCTGTGGT1C GTTAACTTCT ACGCT7GGAA ACGTATGGAA GTTGGTCAAC AAGCTGTTGA CAATIGAAGA TGCGAACCTT TGCATACCTT CAACCAG.TTG TTCGACAACT AGTTTGGCAA GGTTTGGCCT TGTTATCTGA AGCTGTTTTG AGAGGTCAAG TCAAACCGTT CCAAACCGGA ACAATAGACT TCGACAAAAC TCTCCAGTTC CCTTGTIGGT TAACTCTTCT CAACCATGGG AACCATTGCA ATTGCACGTC ggaacaacca ATTGAGAAGA GTTGGTACCC TTGGTAACGT TAACGTGCAG GATAAAGCCG TCTCTGGTTT GAGA7CTTTG ACTACTTTGT TGAGAGCTT T CTATTTCGGC AGAGACCAAA CTCTAGAAAC TGATGAAACA ACTCTCGAAA GGGTGCTCAA AAGGAAGCCA TTTCCCCACC AGACGCTGCT TCTGCCGCTC CCCACGAGTT TTCCTTCGGT AAAGGGGTGG TCTGCGACGA AGACGGCGAG CAT7GAGAAC CATCACTGCT GATACCTTCA GAAAGTTATT CAGAGTTTAG GTAACTCTTG GTAGTGACGA CTATGGAAGT CT.TTCAATAA GTCTCAAATG TCCAACTTCT TGAGAGGTAA ATTGAAGTTG TACACCGGTG AAGCCTGTAG AGGTTGAAGA AC7CTCCATT TAACTTCAAC ' ATGTGGCCAC TTCGGACATC AACTGGTGAC AGATAAGCCC GACTGATAAC AACAGTGTAG TTGACCACTG TCTATTCGGG CTGACTATTG TTGTCACATC ATGTAACAAA TACATTGTTT Hall G CAGCT

The assembled sections of SCEPO go to MI3 and sections 1, 2 and 3 are secreted from the phage HindlII / Kpnl / BglII and BglII / Sall.

The preferred now expression system for the SCEPO gene products is an expression system based on the S.Cerevisial factor as described in the simultaneous review of U.S. Patent No. 4,877,753, issued June 1983. April 22 Crant A.Bitter, printed in 1984. October 31 In short, the system comprises a construct in which the DNA encoding the yeast-factor gene leader sequence is located immediately downstream of the coding region of the exogenous gene expressed 5 '. As a result, the transduced gene product has a leader or signal sequence that is processed by an endogenous yeast fragment in the direction of secretion of the remainder of the product. Because the construct allows for codon-initiating ά-factor (ATG) translation, it was not necessary to construct such a codon at position -1 of the SCEPO gene. As can be deduced from Table 21, alanine (+1) encoding the sequence is located after the binding sequence, which allows for an immediate insertion into a plasmid containing DNA for the first 80 residues of the <RTIgt; cfactor </RTI> promoter sequence. The specific dominant structure for expression of the CEPO gene has a four-way assembly, including the above CEPO fragment and the large fragment fragment HindIII / SalI plasmid p 03. The resulting plasmid p C3 / SCEP0 delivers the promoter and leader sequences of the <i-factor as well as the SCEPO gene. heating with BamllII and stitching together the plasmid pYE BamHI resulting in the plasmid expression of pYE / SCEPO.

EXAMPLE

This sample is subject to expression within the recombinant expression system of the products produced by the ECEPO and SCEPO - genes, Example 11.

Calculates for use in host E. coli cells using an expression system. Example 11 was transformed with p536 in E. coli AM7 previously transformed with the appropriate plasmid pMWI, which had taken up the gene C _1g57 . Cell cultures in broth (50 mcg / ml ampicillin, 5 mcg / ml kanamycin, preferably with 10 mM MgSO ₄ ) were maintained at 28 ° C and during the cell growth period in culture to OD ₆ 0 = 0.1, inducing EPO expression by raising the temperature to 42 ° C. Cells grow to about 40 ODs, allowing EPO production (gelled) of about 5 mg / OD 1.

Cells are harvested, lysed, pulverized with a French press (10,000 psi) and treated with lysine and detergent NP-40. By centrifugation (24000g), the resulting precipitate was dissolved in guanidine HCl and continue execution of the one-time cleaning _C4 (Vydac) reverse phase HPLC (EtOH 0-80% 50 mM NH ₄ Ac, pH 4.5). Protein base sequencing shows that the product is more than 95% pure and the products obtained have two different NH ₂ ends, APPR ..... and PP-R .... at a 3: 1 quantitative ratio. This recent observation of hEPO and [de-ala] hEPO products implies that the "treatment" of the amino terminus in host cells serves to remove terminal methionine, and in some cases, parent alanine. The activity of isolates determined by radioimmunoassay is in the range 150000-160000 U / mg; in vitro activity determined to be 30,000-62,000 U / mg; and in vitro activity ranged from 120-720 U / mg (compared to human urine isolate standard of 70,000 U / mg for each assay). The dose-effect curve for the recombinant product in the in vitro assay is significantly different from the human urine EPO standard curve.

The EPO-analog plasmids made in Example 11, Parts A and B, were each transformed into pMWI-transformed E.coli AM7 cells and cultured as described above. Purified isolates are tested both in RIA and in vitro. Values obtained from RIA and in vitro [Asn ² , de-Pro by Ile ⁶ ] expression of hEP0 products are approximately 11000 U / mg and 6000 U / mg protein, respectively, whereas [His ⁷ ] hEP0 assay values are 41000 U / mg protein. This means that the analog products are "active" 1/4 - 1/10 in the same way as in the "maternal" expression product studies.

An expression system designed to utilize the host

In S.Cerevisial cells. pYE / SCEPO transforms plasmid into two different strains, YSDP4 (apep4-3trpl genotype) / and PK81 deposit, respectively.

genotype pep4-3trp ⁱ ). Transformed hosts YSDP4 were grown in SD medium (yeast genetics methods, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY p. 62 (1983)) at 0.5%, pH6.5, 30 ° C. Media harvested when cells were increases to 36 ODs containing EPO products at approximately 244 U / ml / 97 mcg / OD1, as clarified by RIA). Transformed cells RK8I grown to 6.5 OD or 60 OD form media with EPO concentrations of about 80-90 U / ml / 34 mcg / O.Dl as explained by RIA /. Preliminary studies have identified significant heterogeneity in the products obtained by expression systems, probably due to alterations in the glycosidation of the expressed proteins as well as relatively high levels of attached carbohydrates.

Plasmids in PctC3 and pYE cells were submitted to E. coli HBIOI for registration under U.S. Patent Office regulations from September 27, I984. Collections of American Typical Cultures, 12301 Parklovvn Drive, Rockville, Maryland Issue A.T.C.C. 39881 and A.T.C.C.39882 Plasmids in pCFM526 cells in MI03 and in pMVVI cells in MI03 were recorded in a similar fashion on November 21, 1984 as A.T.C.C. 33932, 33934 and 33933 respectively.

The strains YSPDA and RK8P Saecharomyces Cerevisial were registered on November 21, 1984 as A.T.C.C. 20,734 and 20,733 respectively.

From the above illustrative examples, it should be clear that the present invention offers particularly valuable products and methods in many respects.

The proposed polypeptides are clearly useful material, be they microbially or synthetically derived products, the primary, secondary and tertiary conformations of which have become known for the first time with the present invention.

As noted above, recombinantly produced and synthetic products of the invention differentiate the activity of EPO isolates from natural sources in vitro and are therefore designed to be suitable substitutes for EPO isolates in culture media used for culturing erythropoietin cells in culture. Also, since the polypeptide products of the present invention confer activity on natural EPO isolates in vivo, they may be useful in the erythropoietin therapy methodology used in mammals, including man, to produce any or all of the effects of EPO in vivo. for example, stimulation of reticulocyte response, development of ferro-kinetic effects (such as plasma iron turnover and time-to-brain effect), stimulation of erythrocyte mass exchange, hemoglobin C synthesis (see Eschlach et al., supra) and, as noted in Example 10, increase in hematocrit levels in mammals. The group of people receiving the products offered includes patients as a rule those who require blood transfusions, including trauma victims, surgical patients, renal patients, including dialysis patients, patients with a blood disorder causing disorders such as haemophilia, physiological anemia and similar. Minimizing the need for blood transfusions in therapy The use of erythropoietin therapy can be expected to reduce the transmissibility of infectious agents. The products offered by recombinant methods are presumed to be free of pyrogens, natural inhibitory products, and therefore undoubtedly have to provide an enhanced overall effect in therapeutic approaches compared to natural products. Erythropoietin therapy using the proposed products should be beneficial in increasing individual oxygen permeability compared to hypoxic conditions of the external environment and possibly providing beneficial cardiovascular effects.

Parenteral (e.g., intravenous, intramuscular, subcutaneous or intraperitoneal) administration is the preferred route of administration of the polypeptide products provided, and the compositions to be administered must, as usual, contain a therapeutically effective amount of product in combination with suitable diluents, carriers or stimulants. Preliminary pharmacokinetic studies indicate a longer half-life of in vivo monkey EPO products when administered intramuscularly than intravenously. Effective doses are expected to vary considerably with conditions, but therapeutic doses are expected to range from 0.1-7 to 100-7000 U / mcg of active ingredient per kg body weight. Standard diluents, such as human plasma albumin, are contemplated in the pharmaceutical compositions of the present invention in addition to standard carriers, such as saline.

Suitable stimulants in the proposed compositions include compounds independently designated as erythropoietic (1982); Kalmanti, Kidney I Eng. J. Med., 289, 72-80 for stimulatory effects such as testosterone, cell precursor stimulators, insulin-like type, prandlandin, serotonin, cyclic ΑΜΦ-prolactin and triiodothyronine, as well as agents commonly used in the treatment of aplastic anemia, such as as methendene, stanozol and nandrelone [see, e.g., Resegott et al., Panminerva Medica, 23, 243-248 (1981); McGonigle et al., 1984, Kidney I, 26/2, 437-444; Pavlovic-Kantera et al., Exp. Hematol., 8 Supp., 283-291 (1980); Kurtz, FEBS Letters, 14a (I), 105-108 (1982)]. Substances that enhance or synergize with the action of erythropoietin or asial EPO, such as adrenergic antagonists, thyroid hormones, adrogenic and BPA, are also contemplated as stimulants. Dunn, Current Concepts in Erythropoiesis, John Willy & Sons / Chichester, England, 1983; Weiland et al., Blut, 44 (3), 173-175 nt, 22, 383-391 (1982); Shahid, New (1973); Fischer et al., Steroids, 30 (6), 833-845 (1977); Urabe et al., J.Exp. Med., 149, 1614-1325 (1979) and Billat et al., Exp. Hematol., 10 (1), 133-140 (1982)], as well as a class of compounds designated as hepatic, erythropoietic factors (see Naughton et al., Act Haemat, 69, 171-179 (1983)) and "Erythropropic" [as described in Congote et al., Abstract 364, Proceedings of the 7th International Congress of Endocrinology (Qebec City, Qebec, 1-7 July 1984; Congate, Biochem. Biophys. Res. Comun., 115 ( 2), 447-483 (1983) and Congate, Ann. Biochem., 140, 428-433 (1984)] and "erythrogenic" [as described in Rothman et al., J. Surg. Oncol., 20, 105- 108 (1982)] A preliminary screening for the measurement of erythropoietic responses in exhypoxic polycythaemic mice pretreated with dihydrotestosterone or pandrodone followed by erythropoietin yielded questionable results.

The diagnostic utility of the proposed polypeptides is also wide, including the use of labeled and unlabeled forms in a variety of immunoassay protocols, including RIA, ELISA, and the like, as well as in assays to detect activity in vitro and in vivo. See, e.g., Dum et al., Exp. Hematol., 11 (7): 649-660 (1983); Gibson et al., 1984, Pathology 16: 155-156; Krystal, Exp. Hematol., 11 (7): 649-660 (1983); Saito et al., Jap. J. Med., 23 (1): 61-71 (1984);

or 5th suggested

Nathan et al., New Eng. J. Med. 308 (9): 520-522 (1983); and various reference materials related to the studies mentioned in these works. The proposed polypeptides, including synthetic peptides having sequences of first-expressed EPO residues, also possess particularly valuable purity production of polyclonal antibodies and monoclonal antibody blocks specific for continuous and discontinuous EPO epitopes. As one example, preliminary studies on the amino acid sequence of Table VI, in agreement with Hopp et al., P.N.A.S. (USA), 78, 3824-3828 (1981), and secondary structures according to Chon et al., Ann. Rev. Biochem., 47, 251 (1978), explained that synthetic peptides that duplicate continuous residue sequences at positions 41-57 inclusive, 116-118 inclusive and 144-166 inclusive must probably produce a highly antigenic reaction and produce useful monoclonal and polyclonal antibodies. , immunoreactive with both the synthetic peptide and the whole protein. Such materials are expected to be useful in the detection and related purification of EPO and its bound products.

Three synthetic peptides are taken for illustration:

(1) hEPO, 41-57 V-P-D-T-K-N-F-Y-A-W-K-R-M-E-V-G;

(2) hEPO, 116-128 K-E-A-I-S-P-P-D-A-A-S-A-A;

(3) hEPO, 144-166 V-Y-S-N-F-L-R-G-K-L-K-L-Y-T-G-E-A-C-R-T-G-D-R.

Preliminary immunization studies using the above polypeptides showed a relatively weak positive response to hEPO 41-57, an immeasurable positive response to hEPO 116-128, and a strong positive response to hEPO 144-166 as determined by the ability of rabbit serum antibodies to immunoprecipitate human urine EPO labeled with ¹²⁵ I, isolate. Preliminary in vivo activity studies with the three peptides showed no significant activity alone or in combination.

Although the introduced mammalian EPO amino acid sequences in the illustrative examples essentially determine the primary conformation of the mature EPO, it should be understood that the specific sequence of monkey EPO 165 amino acids and Table 6 of the human EPO 165 amino acid residues do not limit the useful polypeptide. , the number of inventors offered. The present invention encompasses various free-form allelic forms of EPO that have been previously tested in biologically active mammalian polypeptides, such as human interferon. (Compare, for example, human immune interferon containing the arginine residue at position 140 EPO reported in published patent application No. 0077670 and containing glutamine at position 140 as reported in Gray et al., Nature, 295, 503-508 ( 1982) .The two species differ in that they form sequences in mature human interferon).

The allele forms of mature EPO may differ from each other and from the sequences shown in Tables 5 and 6 by length and sequence, and / or by deletions, substitutions, insertions, or amino acid additions to the sequence, along with logically sequential variations in glycosidation ability. As previously noted, the allele form of human EPO appears to have a methionine residue at position 126. It is anticipated that the free-form allele DNA sequences and the EPO-encoding genomic DNA probably also occur, and that the above allele types of polypeptide encoding or commonly used are intended to produce the same polypeptides as indicated.

In addition to the free-form alleles of mature EPO, the present invention also includes other EPO products such as EPO polypeptide analogs and fragments of mature EPO. The above-mentioned and published patent application Alton et al., WO0 / 83/04053 /, readily deduce and produce genes encoding microbial expression of a polypeptide having a primary conformation different from that of the mature EPO identified herein and one or more residues with respect to position (for example, substituents at the rear or intermediate and deletions). Furthermore, DNA modifications and genomic EPO genes can be readily implemented by well-known site-directed mutagenesis techniques and used to generate EPO analogs and derivatives thereof. Such EPO products must have at least one of the biological properties of the EPO, but may be different. By way of example, the EPO products designed by the present invention include those truncated, such as the deletions [ASn, des-Pro to Ile ⁶ ] hEP0, [des-Thr ¹⁶³ to Arg ¹⁶⁶ ] hEP0, and 27-55hEP0. the latter contains residues encoded by the deleted exon; or which are more resistant to hydrolysis (and, as a result, may have a more pronounced or longer duration of action than the free EPO); or which have been replaced by deleting one or more potential domains for glycosidation (which may confer high activity on yeast-derived products); or having one or more cysteine residues deleted or substituted, for example, by histidine or serine (such as the [His ⁷ ] hEP0 analog), and potentially more readily released in the active form from the microbial system; or having one or more tyrosine-substituted phenylalanine residues (such as [Phe ¹⁵ ] hEP0, [Phe ⁴⁹ ] hEP0, [Phe ¹⁴⁵ ] hEP0 analogs) and can bind more or less readily to EPO receptors on target cells. Also included are polypeptide fragments that duplicate only part of the continuous amino acid sequence or secondary conformations within the mature EPO, and may also have a single EPO activity (e.g., erythropoietic activity). Of particular interest in this respect are the potential EPO fragments explained in the overview of the human DNA genomic sequences in Table 6, that is, fragments of the complete continuous EPO sequence defined by intronic sequences, which may form highly expressed domains of biological activity. It should be noted that the absence of in vivo activity for any one or more of the EPO products of the present invention does not in general impair therapeutic utility (see Weiland et al., Supra) or utility in other contexts such as EPO-testing or EPO-antagonism. . Erythropoietin antagonists may be very effective in the treatment of polycythaemia or in cases of EPO overproduction [see, e.g., Adamson, Hosp.Practice, 18/12 /, 4957 (1983) and Hellman et al., Clin.Lab. Haemat., 5, 335342 (1983).

Another feature of the present invention is that the cloned DNA sequences described herein that encode human and monkey EPO polypeptides are valuable for the information they offer in the mammalian erythropoietin amino acid sequences that were previously unavailable despite decades of analytical processing of free-standing product isolates. DNA sequences are also very valuable as products suitable for large-scale microbial synthesis of erythropoietin using various recombinant techniques. In addition, the proposed DNA sequences are useful for the production of novel and suitable vectors for viral and circular plasmid DNA, novel and useful transformed and transfected microbial prokaryotic and eukaryotic host cells (including bacterial and yeast cells, and mammalian cells grown in culture), as well as novel suitable methods for culturing such microbial host cells capable of expressing EPO and EPO products. The DNA sequences of the present invention are also quite suitable material for use as labeled probes for the isolation of EPO and its associated protein encoding DNA sequences and mammalian DNA genomes other than those specifically described in monkeys and humans.

The degree of utilization of DNA sequences in various protein synthesis pathways (such as insect cells) or genetic therapy in humans or other mammals has not yet been determined. The proposed DNA sequences are expected to be useful in the derivation of transgenic mammalian species that can serve as eukaryotic hosts for the production of large amounts of erythropoietin products. See, in particular, the work: Palmiteretal, Science, 222/4625 / 803-814 (1983).

The specific illustrative examples discussed herein do not, of course, limit the scope of the present invention, and many variations and modifications will be apparent to those skilled in the art. Although DNA sequences have illustrative examples, one example includes DNA and genomic DNA sequences, as the application provides information on the amino acid sequence important for DNA sequencing. The invention also provides for the production of a DNA sequence that can be made based on knowledge of the amino acid sequence of EPO. They may encode EPO (as in Example 12) as well as EPO fragments and EPO polypeptide analogs (i.e., EPO products), which may have one or more of the free biological properties of EPO but have no other (or varying degrees of others).

The DNA sequences provided by the present invention must thus include all DNA sequences suitable for expression in a prokaryotic and eukaryotic host cell of a polypeptide product having, as a last resort, part of the parent structural conformation and one or more biological properties of erythropoietin and selected from: shown in Tables 5 and 6; b) DNA sequences which hybridize to DNA sequences defined in (a) or fragments thereof; (c) DNA sequences which, due to degeneracy of the genetic code, must hybridize to the DNA sequences defined in (a) and (b). Therefore, it is noteworthy that the human and monkey allelic EPO gene sequences and other mammalian gene sequences are expected to hybridize to the sequences or fragments in Tables 5 and 6. In addition, due to the degeneracy of the genetic code, the genes CEPO and EOEPO and the produced or mutated sequences into cDNA or genomic DNA encoding various EPO fragments and its analogs must also hybridize to the DNA sequences mentioned above. Such hybridizations can be readily accomplished under the conditions described herein for primary DNA isolation encoding human and monkey DNA, or more stringent conditions if reduced background hybridization is desired.

Although the examples above illustrate the invention in the context of microbial expression of EPO products in mammalian DNA expression inserted into hybrid vectors of bacterial plasmid and genomic viruses, the invention similarly provides more diverse system expressions. In particular, expression systems containing vectors of genomic species belonging to diversity in bacterial, yeast and mammalian cell culture as well as expression systems lacking vectors (such as calcium phosphate transfection of cells) are of particular interest. Therefore, it will be clear that expressions such as in monkey DNA cells in host monkey culture and cells in host human culture do indeed exemplify DNA "exogenous" expression, since the high-level expression of the EPO DNA will not have its source in the host genome. The expression systems of the present invention further contemplate the following steps resulting from the cytoplasmic formation of EPO products and the accumulation of glycosidated and non-glycosidated EPO products in the cytoplasm or membranes of the host cell (e.g., accumulation in bacterial periplasmic spaces), or in relatively rare systems such as the expression systems of Pige reginosa (described by Gray et al., Biotechnology, 2, 161-165 (1984)).

The hybridization methodologies of the present invention, although used above for illustration purposes, have been improved. DNA / DNA Hybridizations, Also Suitable for RNA / RNA and RNA / DNA Screening · Mixed probe techniques, as explained herein, first and foremost, provide a number of enhancements to hybridization processes, enabling quicker and more reliable polynucleotide isolation. These many individual processing enhancements include: improved colony transfer and retention techniques; the use of nylon-based filters such as Gene Soreen and Gene Soreen Pluses to enable re-probing of these filters and reuse of the filter, utilizing novel protease treatment techniques (compare, e.g., Taub et al., Anai Biochem. 126, 222-230 (1982), the use of very low individual concentrations (0.025 picomole order) of large numbers of mixed probes (more than 32, for example), and conducting hybridization and post-hybridization cycles at tight temperature ranges close to (e.g., 4 ° C and preferably 2 ° C). range) at the lowest calculated dissociation temperature for any of the mixed probes used. These improvements are related to obtaining unexpected results. This is fully explained by the fact that mixed probe methodologies containing 4-fold probes as previously reported have always been used successfully, even on DNA screens, by comparatively low-numerical information D NR species have been successfully used to isolate a unique sequence gene by screening a genomic library of 1,500,000 sterile spots. This feat was performed, in essence, in agreement with Anderson et al., Realizing that screening techniques using hybrid probes were ... impractical for mammalian gene separation when the DNA in question is unavailable.

The distinctive features of the invention disclosed in the following description of the invention and / or in the accompanying drawings may, in isolation and in any combination, become the material for practicing the invention in its various forms.

Claims (4)

DEFINITION OF INVENTION A purified and isolated polypeptide having a partial or complete parent structure and one or more biological properties of natural erythropoietin, characterized in that it is a product of expression of a prokaryotic or eukaryotic exogenous DNA sequence. 2. The polypeptide of claim 1, wherein it is contaminated with no mammalian protein. 3. The polypeptide of claim 1, wherein the exogenous DNA sequence is a cDNA sequence. 4. The polypeptide of claim 1, wherein the exogenous DNA sequence is a constructed DNA sequence. 5. The polypeptide of claim 1, wherein the exogenous DNA sequence is a genomic DNA sequence. 6. The polypeptide of claim 1, wherein the exogenous DNA sequence is carried on a circular DNA or self-replicating plasmid of a viral vector. 7. The polypeptide of claim 1, wherein the polypeptide has a partial or complete parent structure of human erythropoietin or any of its natural allelic variants shown in Table 6 and Table 6 is part of this definition. 8. The polypeptide of claim 1, wherein the polypeptide has a partial or complete parent structure of monkey erythropoietin or any of its natural allelic variants shown in Table 5 and Table 5 is part of this definition. 9. The polypeptide of claim 1, characterized in that it has the immunological properties of natural erythropoietin. 10. The polypeptide of claim 1, characterized in that it has in vivo biological activity of natural erythropoietin. 11. The polypeptide of claim 1, characterized in that it has in vitro biological activity of natural erythropoietin. 12. The polypeptide of claim 1, wherein it is covalently linked to a detectable labeled substance. 13. The polypeptide of claim 1, wherein said detectable label is a radiolabel. 14. The DNA sequence, characterized in that it provides expression in a cell of a prokaryotic or eukaryotic host, a polypeptide product having at least a partial natural structure of natural erythropoietin and one or more of its biological properties selected from: (a) the DNA sequences set out in Tables 5 and 6, and Tables 5 and 6 are part of this definition, or complementary strands thereof; b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, due to degeneracy of the genetic code, must be hybridized to the DNA sequences defined in (a) and (b). 15. A prokaryotic or eukaryotic host cell, characterized in that it is a transformed or transfected DNA sequence according to claim 14, which allows the host cell to express said polypeptide product. The polypeptide expression product of the DNA sequence of claim 14 in a prokaryotic or eukaryotic host. 17. Purified and isolated DNA sequences characterized in that they encode, in prokaryotic or eukaryotic host cells, expression of a polypeptide having partial or complete erythropoietin parent structure and one or more of its biological properties. 18. The DNA sequence of claim 17, wherein it is a cDNA sequence. A monkey erythropoietin characterized in that it encodes a DNA sequence according to claim 18. 20. The DNA sequence of claim 19, wherein the DNA comprises a protein encoding the region of Table 5, and Table 5 is part of this definition. 21. The DNA sequence of claim 17, wherein it is a genomic DNA sequence. 22. The human erythropoietin characterized in that it encodes a DNA sequence according to claim 21. 23. The DNA sequence of claim 22, wherein it comprises a protein encoding the region of Table 6, and Table 6 is part of this definition. 24. A DNA sequence characterized in that it is constructed according to claim 14. 25. A DNA sequence characterized in that it is constructed according to claim 24 and has one or more codons required for expression in E. coli cells. 26. A DNA sequence characterized in that it is constructed according to claim 25 and encodes the expression of human erythropoietin. 27. A DNA sequence characterized in that it is constructed according to claim 26 and contains a protein encoding the region shown in Table 14, and Table 14 is part of this definition. 28. A DNA sequence characterized in that it is constructed according to claim 24 and has one or more codons required for expression in yeast cells. 29. A DNA sequence characterized in that it is constructed according to claim 28 and encodes the expression of human erythropoietin. 30. A DNA sequence characterized in that it is constructed according to claim 29 and contains a protein encoding the region shown in Table 21, and Table 21 is part of this definition. 31. The DNA sequence of claim 17, wherein it is covalently linked to a detectable labeled substance. 32. The DNA sequence of claim 31, wherein the detectable tag is a radiolabel. 33. The DNA sequence of claim 31, which is a single-stranded DNA sequence. 34. A DNA sequence which encodes a polypeptide fragment or polypeptide analogue of natural erythropoietin. in that [His ⁷ ] hEP0, it encodes [Asn des-Pro ' 35. A DNA sequence flanking [Phe ¹⁵ ] hEP0, [Phe ⁴⁹ hEP0, [Phe ¹⁴⁵ ] hEPO, according to Ile ⁶ ] hEP0, [des-Ihr ¹⁶³ according to Arg ¹⁶⁶ ] hEP0, or [27-55] hEP0. 36. A DNA sequence characterized in that it is constructed according to claim 34. 37. A biologically functional annular plasmid or viral DNA vector comprising a DNA sequence according to any one of claims 14, 17, 34 or 35. 38. A prokaryotic or eukaryotic host cell characterized in that it is stably transformed or transfected with a DNA vector according to claim 37. A polypeptide product of expression of a DNA sequence according to claim 17 or 34 in a prokaryotic or eukaryotic host cell. 40. A glycoprotein product characterized in that it has a parent structure that duplicates the structure of natural erythropoietin sufficiently to acquire one or more of its biological properties and has a middle carbohydrate composition different from the natural erythropoietin composition. 41. A glycoprotein product characterized in that it has a parent structure that duplicates human erythropoietin natural structure enough to acquire one or more of its biological properties and has a middle carbohydrate composition different from human erythropoietin natural composition. 42.The vertebrate cells, which are capable of continuous growth in vitro and are capable of producing more than 100 units (10 ⁶ cells) of erythropoietin per 48 hours in culture (radioimmunoassay data). 43. vertebrate cell according to claim 42, characterized in that they are able to produce more than 500 units (10 ⁶ cells) within 48 hours of erythropoietin. 44. vertebrate cell according to claim 42, characterized in that they are able to produce more than 1000 units (10 ⁶ cells) within 48 hours of erythropoietin. 45. Vertebrate cells according to claim 42, characterized in that they are mammalian or avian cells. The vertebrate cells of claim 45, wherein they are COS-I or CHO cells. 47. A synthetic polypeptide, characterized in that it has the partial or complete amino acid sequence shown in Table 5 and Table 5 is part of this definition and has one or more of the biological activities of native monkey erythropoietin in vivo and in vitro. 48. A synthetic polypeptide, characterized in that it has the partial or complete amino acid sequence shown in Table 6, and Table 6 is part of this definition, but different from the sequence indicated in general sequence 1-20, and possesses the biological properties of human erythropoietin natural. 49. Synthetic polypeptide, characterized in that it has a partial or complete amino acid sequence shown in Table 6 and Table 6 is part of this definition, a secondary conformation but other than the sequence 1-20 in the general sequence, and is characterized by the biological properties of erythropoietin. 50. A method of producing polypeptides having partial or complete parent structure and one or more biological properties of natural erythropoietin, wherein the prokaryotic or eukaryotic host cells transformed or transfected with the DNA vector of claim 37 are cultured under appropriate nutrition conditions and secreted into desired DNA sequences. expression products in this vector. 51. An antibody substance having immunoreactivity for erythropoietin and a synthetic polypeptide and having a parent structure sufficiently duplicating an unbroken amino acid sequence except for any polypeptide having the complete amino acid sequence. A-P-P-P-L-I-C-D-S-R-V-L-E-R-Y-L-L-E-A-K. is 52. An antibody according to the monoclonal antibody of claim 51. point, divergent that that he is 53. The antibody of 51 polyclonal antibody. point, divergent that that he 54. Antibody according to 51 point, divergent that that he exhibits immunoreactivity against erythropoietin and a synthetic polypeptide and has a sequence selected from the following sequences: V-P-D-T-K-N-F-Y-A-W-K-P-M-E-V-G, K-E-A-I-S-P-P-D-A-A-S-A-A, V-Y-S-N-F-L-R-G-K-L-K-L-Y-T-G-E-A-C-R-T-G-D-R. 55. A pharmaceutical composition comprising an effective amount of a polypeptide according to any one of claims 1, 16, 39, 40 or 41 and a pharmaceutically acceptable diluent, stimulant and / or carrier. 56. The purified and isolated DNA sequence set forth in Tables 5 or 6, and Tables 5 and 6 form part of this definition, or a fragment thereof, or a complementary strand of such a sequence or fragment. 57. A polypeptide product of the expression of the DNA sequence of claim 56 in a prokaryotic or eukaryotic host cell. 58. A method of detecting an unknown sequence-specific single-stranded polynucleotide in a heterogeneous cell sample comprising repeat single-stranded polynucleotides, wherein: a) obtains a mixture of probes of labeled single-stranded polynucleotides with uniformly variable base sequences, each of said probes being potentially specifically complementary to a base sequence which can be expected to be unique to the detectable polynucleotide; b) binds the sample to a solid substrate, c) treating a substrate having a sample bound thereto, thereby attenuating further binding of the polynucleotide thereto, except for the polynucleotides of said sample bound for hybridization purposes; d) contacting the treated substrate (having a sample bound thereto) for a period of time with said mixture of labeled probes under conditions which promote hybridization only between fully complementary polynucleotides, e) detecting a specific polynucleotide by the presence of a hybridization reaction between this nucleotide and a fully complementary probe (contained in said mixture of probes), which is monitored by an increase in the amount of labeled material in the substrate with a specific polynucleotide relative to that of labeled material without this nucleotide; it also includes the use of more than 32 mixed probes and one or more of the following: 1) nylon paper used as a solid substrate; 2) a protease treatment of step (c); (3) 0.025 picomoles of individual labeled probe concentrations are used; 4) one of the hybridization conditions used in step (d) is a stringent temperature range approaching 4 ° to the lowest of the Td values calculated for the probes used.