RU2129613C1 - Method of human erythropoietin preparing - Google Patents

Abstract

FIELD: genetic engineering. SUBSTANCE: invention relates to human erythropoietin gene cloning. Mammalian cells transformed by the recombinant vector are cultured in suitable medium. Vector has DNA sequences given in tables 3 and 4. DNA sequence can be introduced into vector containing DNA of bovine papilloma virus. Mammalian cells are lines COS, CHO, C127 or 3T3. Cultural acid can contain fetal serum. Obtained erythropoietin is separated from cells and medium. Method ensures to obtain the cloned human erythropoietin genes exhibiting highly levels of expression. EFFECT: high levels of expression of the cloned erythropoietin genes. 6 cl, 11 tbl, 12 dwg, 15 ex

Description

 The invention relates to the cloning of human erythropoietin genes that provide unusually high levels of expression, expression of these genes and the production of active human erythropoietin in vitro.

 Erythropoietin (further EPO) is a circulating glycoprotein that stimulates the formation of red blood cells in higher organisms. See Carnot et al., Compt. Rend. 143: 384/1906 /. As such, EPO is sometimes referred to as a factor stimulating erythropoiesis.

 The life span of human red blood cells is about 120 days. Thus, about 1/120 of the total number of red blood cells is destroyed daily in the reticuloendothelial system. However, a relatively constant number of red blood cells is produced daily to maintain red blood cell levels all the time / Guyton, Textbook of Medical Physiology pp. 56-60, W. B. Saunders Co., Philadelphia / 1976 //.

 Red blood cells are produced by maturation and differentiation of erythroblasts in the bone marrow, and EPO is a factor that acts on less differentiated cells and induces their differentiation into red blood cells / Guyton, above /.

 EPO is a promising therapeutic agent for the clinical treatment of anemia and, in particular, renal anemia. Unfortunately, the use of EPO is not yet generally accepted in practical therapy due to its low availability.

 When using EPO as a therapeutic agent, it is necessary to consider the problems of antigenicity, and therefore it is preferable that EPO be obtained from raw materials of human origin. For example, you can use human blood or urine from patients suffering from aplastic anemia or similar diseases in which large amounts of EPO are released. These starting materials, however, exist in limited quantities. See, e.g., White et al., Rec. Progr. Horm. Res. 16-219 (1960); Espada et al., Biochem. Med. 3: 475 (1970); Fischer, Phurm. Rev. 24: 459 (1972) and Gordon, Vitam. Horm (N.Y.), 31: 105 (1973).

 The production of EPO products, as a rule, is carried out through the concentration and purification of urine of patients who find high levels of EPO, such as patients with aplastic anemia or similar diseases. See, for example, US patents NN 439780, 4303650 and 3865801, the disclosure of which is incorporated herein by reference. The limited supply of such urine is an obstacle to the practical use of EPO, and it is therefore highly desirable to obtain EPO from the urine of healthy people. The difficulty in using urine from healthy people is the low content of EPO in it compared with the urine of patients suffering from anemia. In addition, the urine of healthy people contains a sufficiently high concentration of certain inhibitory factors that act against erythropoiesis, therefore, a satisfactory therapeutic effect from EPO can be obtained only after subsequent significant cleaning.

 EPO can also be isolated from the blood plasma of a sheep; separation of EPO from such a blood plasma ensures the creation of sufficiently potent and stable water-soluble drugs. See, Goldwasser, Control Cellular Dif. Development. Part A, pp. 487-494, Alan Pziss, Inc., New York (1981), which is incorporated herein by reference. Sheep EPO, however, can be antigenic in humans.

 Thus, although EPO is a desirable therapeutic agent, the traditional methods of isolation and purification used in working with natural sources of raw materials are insufficient for the mass production of this compound.

 Sugimoto et al. In US Pat. No. 4,377,513 describe one method for mass production of EPO comprising in vivo propagation of human lymphoblastoid cells, including Nomalva, BALL-1, NALL-1, TALL-1 and JBL.

 A description of the production of EPO using methods of genetic engineering by other authors has appeared in the specialized literature. However, neither suitable disclosure nor the chemical nature of the product has been published. In contrast, the present application provides a suitable disclosure of mass production of proteins exhibiting the biological properties of human EPO. Using these techniques, it is also possible to obtain proteins that may be chemically different from authentic human EPO and yet exhibit similar / and in some cases improved / properties. For convenience, all such proteins exhibiting the biological properties of human EPO will be referred to below as EPO, regardless of whether they are chemically identical to it or not.

 The present invention relates to the cloning of a gene that provides unusually high levels of expression of human EPO, and mass production of in vitro active human erythropoietin. Suitable expression vectors for producing EPO, expression systems, purification schemes, and related processes are also described.

 As described in more detail below, EPO was obtained in partially purified form, subjected to further purification to homogeneity and digested with trypsin to form specific fragments. These fragments were purified and sequenced. EPO oligonucleotides were then constructed based on these sequences. These oligonucleotides were used to screen the human genomic library from which the EPO gene was isolated.

 The EPO gene was tested based on its DNA sequence, which combined many sequenced fragments of tryptic protein. A fragment of the genomic clone was then used to demonstrate by hybridization that EPO mRNA is found in human fetal / at 20 weeks of age / mRNA. A human fetal liver cDNA library was obtained and screened. Three clones of EPO cDNA were obtained (after screening> 750,000 recombinants). Two of these three clones were determined by evaluating the complete coding sequence and the significant 5 '- 3' untranslated sequence as having full length. These cDNAs were expressed as in transformed monkey SV-40 virus cells / COS-1 cell line; Gluzman, Cell 23: 175-182 / 1981 //, as well as in Chinese hamster ovary cells / CHO cell line; Urlaub, G. and Chasin, L. A. Proc. Natl. Acad Sci. USA 77: 4216-4280 / 1980 //. EPO derived from COS-1 cells is a biologically active in vitro and in vivo EPO. EPO derived from CHO cells is also biologically active in vitro and in vivo.

 The EPO cDNA clone has an interesting open reading frame of 14-15 amino acids (AK) with an initiator and terminator of 20-30 nucleotides up in the direction of the coding region. A typical sample of E. coli transformed with the cloned EPO gene was deposited by the American Type Culture Collection, Rockville, Maryland, where it is available under accession number ATCC 40153.

Table 1 is an exon sequence of 87 base pairs of the human EPO gene;
FIG. 1 illustrates the detection of EPO mRNA in human fetal liver mRNA;
table 2 represents the amino acid sequence of the EPO protein derived from the nucleotide sequence of λ-HEPOF L13;
table 3 represents the nucleotide sequence of EPO cDNA in λ-HEPOF L13 / shown schematically in FIG. 2 / and the amino acid sequence derived from it;
FIG. 3 illustrates the relative positions of DNA inserts of four independent genomic clones of human EPO;
FIG. 4 is a map of the apparent intron and exon structure of the human EPO gene;
Table 4 illustrates the DNA sequence of the EPO gene shown in FIG. 4B;
FIG. 5A, 5B and 5C illustrate the construction of vector 91023 / B /;
FIG. 6 depicts SDS polyacrylamide gel analysis of EPO obtained in COS-1 cells compared to native EPO;
table 5 illustrates the nucleotide and amino acid sequence of the clone EPO λ-HEPOF L6;
table 6 illustrates the nucleotide and amino acid sequence of the clone EPO λ-HEPOF L8;
table 7 illustrates the nucleotide and amino acid sequence of the clone EPO λ-HEPOF L13;
FIG. 8 is a schematic representation of the plasmid pdBPY-MMTheo / 342-12 /. All tables are presented at the end of the description.

 The present invention relates to the cloning of EPO genes and the production of EPO by in vitro expression of these genes.

 Patent and scientific literature is replete with methods that are suitable for the production of recombinant products. Basically, these techniques include isolating or synthesizing the desired gene sequence and expressing the sequence in either prokaryotic or eukaryotic cells using techniques generally available to one skilled in the art. Once the desired gene is isolated, purified and inserted into the transfer vector / i.e. cloned /, its availability in sufficient quantity is ensured. A vector with the gene cloned therein is transferred to a suitable microorganism or cell line, for example bacteria, yeast, mammalian cells such as COS-I / monkey kidney /, CHO / Chinese hamster ovary /, insect cell lines and the like, where the vector is replicated, when the microorganism or cell line proliferates, and from where the vector can be isolated using conventional means. This provides a continuously renewable source of the gene for further manipulations, modifications and transfers to other vectors or other loci within the same vector.

 Expression can often be obtained by transferring the cloned gene in the correct orientation and reading frame to a suitable site in the transfer vector so that translational reading from the prokaryotic or eukaryotic gene ends with the synthesis of a protein precursor containing the amino acid sequence encoded by the cloned gene linked to Met or amino - the terminal sequence of a prokaryotic or eukaryotic gene. In other cases, signals for initiating transcription and translation may be provided by a suitable genomic fragment of the cloned gene. Many specific protein cleavage techniques can be used to cleave the produced protein precursor at the desired point in order to release the desired amino acid sequence, which can then be purified by conventional methods. In some cases, a protein containing the desired amino acid sequence is produced without the need for specific cleavage techniques, and can also be released from cells into an extracellular growth medium.

Isolation of the genomic clone of human EPO
Human EPO was purified to homogeneity from the urine of patients suffering from aplastic anemia, as described below. Complete digestion of this purified EPO with trypsin gave fragments that were separated by reverse phase high performance liquid chromatography, recovered from the gradient fractions, and subjected to sequence microanalysis. The sequences of tryptic fragments are highlighted in tables 2 and 3 and are discussed in more detail below. Two of the amino acid sequences Val-Asn-Phe-Tyr-Ala-Trp-Lys and Val-Tyr-Ser-Asn-Phe-Leu-Arg were selected for the construction of oligonucleotide probes / to obtain a pool of oligonucleotides from 17 bases with 32-fold degeneracy and a pool of 18-membered members with 128-fold degeneracy from the first tryptic fragment, as well as two pools of 14-membered members with 48-fold degeneracy from the second tryptic fragment, respectively /. A 32-fold 17-membered pool was used to screen the human genomic DNA library in the Ch4 A / 22 / vector using a modification of the Woo and O'Malley / 47 / in situ amplification method to obtain screening filters.

 Arabic numbers in parentheses, (1) to (61), are used in the present description to refer to publications that are given in numerical order at the end of the description of the present invention.

 The phage hybridizing with the 17-member was packaged, distributed into small groups and tested with 14-member and 18-member pools. A phage hybridizing with a 17-member, an 18-member, and a 14-member was purified, and the fragments were subcloned into M13 vectors for sequencing using the dideoxy chain termination method described by Sanger and Coulson, / 23 / / 1977 /. The sequence region of one of the clones hybridizing with a 32-fold degenerate 17-membered is shown in Table 1. This DNA sequence contains nucleotides within the open reading frame that can accurately encode the tryptic fragment used to generate a pool of 17 membered oligonucleotides. In addition, DNA sequence analysis showed that the 17-membered hybridizing region was contained within an 87 bp exon coupled to a potential acceptor and splicing donor.

 Positive confirmation that these two clones (designated λ-HEPO1 and λ-HEPO2 /) are genomic EPO clones was obtained by sequencing additional exons containing another tryptic fragment encoding information.

Isolation of EPO cDNA Clones
Northern analysis / 56 / in relation to human fetal / age of 20 weeks / hepatic mRNA was performed using a single-stranded probe of 95 nucleotides obtained from clone M13 containing an 87 bp exon site. ; described in table 1. As shown in FIG. 1, a strong signal can be detected in mRNA of the fetal liver. The exact identification of this band as EPO mRNA was achieved using the same probe for screening the λ-cDNA of the fetal liver mRNA library / 25 /. Several hybridizing clones were obtained at a frequency of approximately 1 positive per 250,000 screened recombinants. The complete nucleotide and deduced amino acid sequences for these clones / λ-HEPOF L13 and λ-HEPOF L8 / are shown in Tables 5 and 6. EPO-coding information is contained within 594 nucleotides in the 5'-half of the cDNA, including the highly hydrophobic 27 amino acid leader and mature protein of 166 amino acids.

 Identification of the N-terminus of the mature protein was based on the N-terminal sequence of the protein excreted in the urine of people with aplastic anemia, as illustrated in Table 1 and as described in Goldwasser / 26 /, Sue and Sytkowski / 27 / and Yangawa / 21 /. It is currently unknown whether this N-terminus / Ala-Pro-Pro-Arg --- / represents the actual N-terminus found in EPO during circulation, or if some cleavage occurs in the kidney or urine.

 The amino acid sequences that are underlined in Tables 2 and 3 indicate those tryptic fragments or N-terminal region for which protein sequence information has been obtained. The deduced amino acid sequence exactly matches the tryptic fragments that were sequenced, confirming that the isolated gene encodes a human EPO.

The structure and sequence of the human EPO gene
Relative to the position of the DNA inserts of the four independent genomic human EPO clones are shown in FIG. 3. Hybridization analysis of these cloned DNAs using oligonucleotide probes and various probes obtained from two classes of EPO cDNA clones determined the position of the EPO gene within a region of approximately 3.3 kb shown by the shaded line in FIG. 3. A complete sequence analysis of this area / cm. Example 4 / and comparison with cDNA clones resulted in a map of the intron and exon structures of the EPO gene shown in FIG. 4. The EPO gene is divided into 5 exons. Part of exon I, complete exons II, III, and IV, as well as part of exon V contain protein-coding information. The remaining portions of exons I and V encode 5 ′ and 3 ′ untranslated sequences, respectively.

Unstable EPO expression in COS cells
In order to demonstrate that a biologically active EPO can be expressed in an in vitro cell culture system, expression studies in CO / 58 / cells were performed. The vector used for the intermediate steps, namely p91023 / B /, is described in Example 5. This vector contains the main late adenovirus promoter, the SV40 polyadenylation sequence, the SV40 replication initiator, the SV40 enhancer, and the adenovirus VA gene. Insert cDNA into λ-HEPOF L13 / cm. table 6 / was transferred to the vector p91023 / B / below the main late promoter of adenovirus. This new vector is identified as pPTF-L13.

Twenty-four hours after transfection of this construct into strain M6 of COS-1 / Horowitz et al., J. Mol. Appl. Genet. 2: 147-149 / 1983 / the cells were washed, transferred to serum-free medium, after which the cells were harvested after 48 hours. Then, using the quantitative radioimmunoassay for EPO / 55 /, the level of EPO release into the culture supernatant was investigated. In a separate experiment, a vector containing EPO cDNA from λ-HEPOF L13 was transformed into COS-1 cells, and the media were collected as described above. EPO in the media was then quantified by any of the two in vitro biological assays, namely, ³ H-thymidine and CFU-E / 12, 29 /, and any of the two in vivo assays, namely the “hypoxic mouse” and "starving rat" / 30, 31 / / cm. table 9, example 17 /. These results indicate that biologically active EPO is produced in COS-1 cells. Using Western blotting using an anti-EPO polyclonal antibody, erythropoietin produced by COS cells showed mobility on SDS-polyacrylamide gels identical to that of native EPO obtained from human urine (Example 8). Thus, glycosylation of a COS-1-produced EPO can be similar to what occurs in the case of native EPO.

 Various vectors containing other promoters can also be used in COS cells or in cells of other mammals or eukaryotes. Examples of these other promoters useful in the practice of the present invention include early and late SV40 promoters, a mouse metallothionein gene promoter, a promoter found in long terminal repeats of bird or mammalian retroviruses, a polyhedral baculovirus gene promoter, and others. Examples of other types of cells, as well as mammalian cells, such as CHO / Chinese hamster ovary /, C127 / monkey epithelium /, ZTZ / mouse fibroblast /, CU-1 / African green monkey kidney / and insect cells, such as cells from Spodoptera frugiperda and Drosophila melanogaster. These additional promoters and / or cell types may provide the ability to regulate the timing or level of EPO expression, the production of specific EPO type cells, and the buildup of large quantities of EPO-producing cells in less expensive, more easily controlled conditions.

 An expression system that retains the advantages of expression in mammals, however, takes less time to produce a cell line with a high expression level, is formed by insect cell lines and the DNA virus that is reproduced in this cell line. The virus is a nuclear polyhedrosis virus. It has a genome in the form of double-stranded circular DNA with a size of 128 kb. The nucleocapsid is rod-shaped and packaged in two forms, namely, the non-occluded form (membrane budding virus) and the occluded form (packed in a protein crystal in the infected cell nucleus). These viruses can be routinely propagated in vitro in insect cell culture and applied to all conventional animal virology methods. Cell culture media are usually nutrient saline and 10% fetal calf serum.

 In vitro viral growth is triggered when an unoccupied virus (HOB) enters the cell and advances to the nucleus, where it replicates. Replication is nuclear. During the initial phase / 8-18 hours of post-infection / viral application, nucleocapsids collect in the nucleus and subsequently pass through the plasma membrane as NEW, spreading the infection through the cell culture. In addition, some nucleocapsids subsequently (another 18 hours of post-infection) remain in the nucleus and become clogged in a protein matrix known as polyhedral intracellular inclusion (PVV). This form is not contagious in cell culture. The matrix consists of a protein known as polyhedrin, molecular weight 33 cd. Each PVV is approximately 1 mm in diameter, and up to 100 PVV can be in the core. It is clear that later, in the infection cycle, a large amount of polyhedrin is produced, it makes up to 25% of the total amount of cellular protein.

 Since PVV does not play a role in the in vitro replication cycle, the polyhedrin gene can be removed from the viral chromosome without any effect on in vitro viability. When using the virus as an expression vector, the applicant replaced the region encoding the polyhedrin gene with the foreign DNA to be expressed, placing it under the control of the polyhedrin promoter. This led to a viral phenotype that does not form PVV.

 This system has been used by several researchers, the most famous of which are Pennock et al. And Smith et al. Pennock et al. / Cregory D. Pennock, Cnarles Shoemaker and Lois K. Miller, Molecular and Cell Biology 3:84, p. 399- 406 / reported highly efficient expression of the bacterial protein β-galactosidase when it is placed under the control of the polyhedrin promoter.

 Another expression vector based on the nuclear polyhedrosis virus is presented by Smith et al. / Gale E. Smith Max D. Summers and M.J. Fraser, Molecular and Cell Biology, May 16, 1983, pp. 2156-2165). The authors demonstrated the effectiveness of their vector by expressing human β-interferon. The synthesized product, as expected, was detected glycosylated and isolated from insect cells. Example 14 describes modifications of a plasmid containing the polyhedron gene of the nuclear polyhedrosis virus Antographa Californica / AcNPY /, which allow the easy insertion of the EPO gene into the plasmid so that it can be under transcriptional control of the polyhedrin promoter. The resulting DNA is transfected with intact chromosome DNA from wild-type AcNPY to insect cells. The event of genetic recombination leads to the replacement of the polyhedrin region of the AcNPY gene with deoxyribonucleic acid from the plasmid. The resulting recombinant virus can be identified among viral offspring by the presence of EPO gene DNA sequences. This recombinant virus during reinfection of insect cells, as expected, produces EPO.

 Examples of EPO expression in CHO, C127 and ZTZ, as well as in insect cells are presented in examples 10 and 11 / CHO /, 13 / C127 and ZTZ / and 14 / insect cells /.

Recombinant EPO obtained in CHO cells, as in Example 11, was purified by conventional column chromatography methods. The relative amounts of sugars present in the glycoprotein were analyzed by two independent methods: (I) Reinhold, Methods in Enzymol. 50: 244-249 / Methanolysis / and (II) Takemoto, H. et al. Anal. Biochem. 145: 245, 1985 pyridyl amination, together with the independent determination of sialic acid. The results obtained by each of these methods were in excellent agreement. Thus, several determinations were made, which gave average values, where N-acetylglucosamine for comparative purposes is given as a value of 1:
Sugar - Relative molar level
N-Acetylglucosamine - 1
Hexoses: - 1.4
Galactose - 0.9
Mannose - 0.5
N-Acetylneuraminic Acid - 1
Fucose - 0.2
N-Acetylgalactosamine - 0.1
It is noteworthy that significant levels of fucose and N-acetylgalactosamine were reproducibly observed when using both independent sugar analysis methods. The presence of N-acetylgalactosamine indicates the presence of O-linked protein glycosylation. The presence of O-linked glycosylation was further noted by the analysis of the glycoprotein in SDS-PAGE after its digestion by various combinations of glycosidic enzymes. In particular, following the enzymatic cleavage of the entire N-linked carbohydrate of a glycoprotein using peptide-endo F N-glycosidase, the molecular weight of the protein as determined by SDS-PAGE analysis was further reduced after sequential digestion with neuraminidase.

 The in vitro biological activity of purified recombinant EPO was tested by C. Krystal, Exp. Hematol., 11: 649, 1983 / bioassay for spleen cell proliferation / with protein determinations calculated based on amino acid composition data. After repeated determinations, the specific in vitro activity of purified recombinant EPO was found to be more than 200,000 units / mg of protein. The average value was in the range of 275000-30000 units / mg protein. In addition, a value higher than 300,000 was also observed. In vivo activity ratios / analysis of polycythemic mice, Kazal and Ersleu, Am. Clinical Lab., Sci., Volume B, p. 91/1975 // for in vitro activity tested for recombinant material were in the range of 0.7 - 1.3.

 It is interesting to compare the above characteristic of a glycoprotein with the characteristic of a recombinant CHO-produced EPO previously described in International Patent Application WO 85/02810 / published June 20, 1985 /. A similar comparative analysis shows that the values for fucose and N-acetylgalactosamine are 0, while the ratio of hexose: N-acetylgalactosamine is 15.09: 1. The absence of N-acetylgalactosamine indicates the absence of O-linked glycosylation in the previously described glycoprotein. In contrast to this material, the recombinant CHO-produced EPO of the present invention described above contains significant and reproducibly detectable amounts of both fucose and N-acetylgalactosamine, contains less than 1/10 of the relative amount of hexoses, and is characterized by the presence of O-linked glycosylation. Moreover, the high specific activity of the above recombinant CHO-based EPO can be directly associated with glycosylation features.

 Biologically active EPO obtained by eukaryotic expression of the cloned EPO genes of the present invention can be used for in vivo treatment of mammals by doctors and / or veterinarians. The amount of active component, of course, will depend on the severity of the regimen, the chosen route of administration and the specific activity of the active EPO, and ultimately will be determined by the attending physician or veterinarian. Such an amount of active EPO as determined by the attending physician is referred to herein as an “effective amount of EPO for treatment”.

 For example, in the treatment of sheep-induced hypoproliferative anemia associated with chronic renal failure, an effective daily amount of EPO was found to be 10 units / kg for 15-40 days. See Eschbach et al., J. Clin. Invest. 73: 434/1984 /.

 Active EPO may be administered by any route appropriate to the conditions used. Injection of EPO into the bloodstream of a mammal is preferred. It will become apparent to a person skilled in the art that the preferred route of administration varies with the mode selected.

 While active EPO can be administered as a pure or substantially pure compound, it is preferable to have it in the form of a pharmaceutical composition or preparation.

 The compositions of the present invention, both for veterinary and medical use, contain an active EPO protein, as described above, together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic agents. The carrier (s) may be "acceptable" in the sense of being compatible with other components of the composition and not harmful to its recipient. It is desirable that the composition does not include an oxidizing agent and other substances with which, as you know, peptides are incompatible. The compositions may be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of binding the active component to a carrier that includes one or more accessory components. Basically, formulations are prepared by uniformly and thoroughly mixing the active component with liquid carriers or finely divided solid carriers, or both, after which, if necessary, the product is formed.

 Compositions suitable for parenteral administration include sterile aqueous solutions of the active ingredient with solutions, preferably isotonic with the blood of the recipient. Such compositions can be prepared by dissolving the solid active component in water to obtain an aqueous solution, and sterilization of the specified solution can be achieved in disposable or multi-dose containers, for example, sealed ampoules or tubes.

 EPO / cDNA, as used here, includes a mature EPO / cDNA gene, which is preceded by the ATG codon, and EPO / cDNA encoding allelic variants of the EPO protein. One allele is illustrated in Tables 2 and 3. The EPO protein includes the 1-methionine derivative of the EPO protein / Met-EPO / and allelic variants of the EPO protein. The mature EPO protein is shown by the sequence in Table 2 and begins with the sequence Ala, Pro, Pro. Arg ..., the beginning of which is indicated by the number "1". Met-EPO should start with the sequence Met, Ala, Pro.Pro. Arg ....

 The following examples are given to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It is clear that in the proposed methods, modifications can be made within the essence of the present invention. All temperatures are expressed in degrees Celsius and are not adjusted. The mark for micron or micro, for example microliter, micromole, etc., is "micron", for example, microliter, micron, etc.

Examples
Example 1. Isolation of the genomic clone of EPO
EPO is purified from the urine of patients suffering from aplastic anemia, mainly as described previously / Miyake et al., J. Biol. Chem., 252: 5558/1977 //, except that the phenolic treatment is omitted and replaced by heat treatment at a temperature of 80 ^o for 5 minutes to inactivate neuraminidase. The final purification step is fractionation on a C-4 Vydak column of high performance liquid chromatography / Separation Group / using a 0 - 95% acetonitrile gradient with 0.1% trifluoroacetic acid / TFA / for 100 minutes. The position of the EPO in the gradient is determined by gel electrophoresis and analysis of the 11-terminal sequence / 21, 26, 27 / of the main peaks. EPO elutes at 53% acetonitrile and approximately 40% of the protein is detected subjected to reverse phase high performance liquid chromatography. The fractions containing EPO were evaporated to 100 μl, adjusted to pH 7.0 with ammonium bicarbonate, digested with 2% TRPC-treated trypsin / Worthington / for 18 hours at 37 ^° . The tryptic digest is then subjected to reverse phase HPLC as described above. Optical density is recorded at 280 and 214 nm. Well-separated peaks are evaporated to almost dryness and directly analyzed for the N-terminal amino acid sequence / 59 / using the Model 480A Applied Biosystems Gas Phase Sequencer. The obtained sequences are shown in tables 2 and 3. As described here above, two of these tryptic fragments are selected for the synthesis of oligonucleotide probes. From the sequence Val-Asn-Phe-Tyr-Ala-Trp-Lys / amino acids 46-52 in Tables 2 and 3), a 17-member with 32-fold degeneracy is obtained:
AAA
TTCCANGGGTAGAAGTT
and 18-member with 128-fold degeneracy
AAA
CCANGCGTAGAAGTTNAC
From the sequence Val-Tyr-Ser-Asn-Phe-Leu-Arg / amino acids 144 - 150 in tables 2 and 3 /, two pools of 14-mers are obtained, with 32-fold degeneracy each
TTGN TT TTGN TT
TACACTAACTTCCT and TACACTAACTTCTT
which differ in the first position of the leucine codon. Oligonucleotides are labeled at the 5'-end of ³² P using polynucleotide kinase (New England Biolacs) and gamma ³² P-ATP / New England Nuclear /. The specific activity of oligonucleotides varies between 1000 and 3000 inch ³ / mmol of oligonucleotide. The human genomic DNA library in the bacteriophage lambda / Lawn et al., 22 / is screened using a modification of the in situ amplification technique originally described by Woo et al. / 47 / / 1978 /. About 3.5 x 10 ⁵ phages are plated at a density of 6000 per 150 mm Petri dish / NZCYM medium / and incubated at 37 ^° until plaques become visible, but small (approximately 0.5 mm). After cooling at 4 ^{° C.} for 1 hour, duplicate replicas of the resulting patterns were transferred onto nylon membranes (New England Nuclear) and incubated overnight at 37 ^{° C.} in plates with fresh NZCYM media. Then the filters are denatured and neutralized by flotation of each for 10 minutes on a thin film of 0.5 N NaOH - 1 M NaCl and 0.5 M Tris / pH 8 / - 1 M NaCl, respectively. Following vacuum drying at 80 ^{° C.} for 2 hours, the filters are washed in 5 x SSC, 0.5% SDS for 1 hour, and cell debris on the surface of the filters is removed by careful scraping with a dry cloth. This scraping reduces the background binding of the probe to the filters. The filters are then washed with water and pre-hybridized for 4 to 8 hours at 48 ^° in a 3M solution of tetramethylammonium chloride, 10 mM NaPO ₄ / pH 6.8 /, 5 x Denhardt's, 0.5% SDS and 10 mM EDTA, 17 membered labeled with ³² P, then added at a concentration of 0.1 pmol / ml and hybridization is carried out at a temperature of 48 ^o for 72 hours. Following hybridization, the filters are washed in 2 x SSC / 0.3 M NaCl - 0.03 M sodium nitrate, pH 7 / at room temperature and then for 1 hour in 3M TMACl - 10 mm NaPO ₄ / pH 6.8 / at room temperature and 5 to 15 minutes at hybridization temperature. Approximately 120 strong duplicate signals are detected after 2-day autoradiography with an amplifying screen. Positive signals are selected, grouped into pools of 8, reseeded and again screened in triplicate using 1/2 pool of 14-membered on each of the two filters and 127-membered on the third filter. The conditions and the 17-member for sowing and hybridization are as described above, except that the hybridization for the 14-mer is carried out at a temperature of 37 ^o . Following autoradiography, a probe of 17 nucleotides was removed from the filter in 50% formamide for 20 minutes at room temperature, and the filter was re-hybridized at 52 ^° with an 18-membered probe. Two independent phages hybridize with all three probes. The DNA of one of these phages (designated λ-HEPO1 here) is completely digested with Sau3A and subcloned into M13 for DNA sequence analysis using the dideoxy chain termination method proposed by Sanger and Consol / 23 / / 1977 /. The nucleotide sequence and the deduced amino acid sequence of an open reading frame encoding a tryptic fragment of EPO / underlined region / are described here. Intron sequences are given in lowercase letters, exon sequences / 87 nucleotides / are given in capital letters. Sequences that correspond to acceptor sites / a / and donor / d / splicing are underlined. /Cm. table 4 /.

 Example 2. Northern analysis of mRNA of the human fetal liver.

5 μg of human fetal liver mRNA / obtained from a 20-week fetal liver / and adult liver mRNA are electrophoresed on a 0.8% agarose formaldehyde gel and transferred onto nitrocellulose using the method of Derman et al., Cell, 23: 731/1981 /. The single-stranded probe is then obtained from the M13 matrix containing the insert shown in table 1. The primer is a 20-member originating from the same tryptic fragment as the original 17-membered mer probe. The probe is prepared as previously described by Anderson et al., PNAS / 50 / / 1984 /, except that following SmaI / which produces the desired fragment of 95 nucleotides in length containing 74 nucleotides of the coding sequence / the small fragment is purified from the M13 matrix by chromatography on a column with Sepharose C14B in 0.1 N NaOH — 0.2 M NaCl. The filter is hybridized to approximately 5 x 10 ⁶ pulses per minute of this probe for 12 hours at 68 ^° , washed in 2 x SSC at 68 ^°, and exposed to a reinforcing screen for 6 days. A single-stranded marker mRNA of 1200 nucleotides (indicated by arrow) was accelerated in an adjacent lane / Fig. 1/.

Example 3. Fetal liver cDNA
A probe identical to that described in Example 2 was prepared and used to screen a fetal liver cDNA library obtained in the vector λ-Ch21A / Toole et al., Nature / 25 / / 1984 //, using standard screening techniques / Berten Davis, Science, / 54 // 1978 //. Three independent positive clones / are designated here λ-HEPOF L6 (1350 base pairs), λ-HEPOF L8 (700 bp) and λ-HEPOF L13 (1400 bp) were isolated by screening 1 × 10 ⁶ plaques. The full insert of λ-HEPOF-L13 and λ-HEPOF L6 was sequenced after subcloning in M13. / Tables 7 and 5, respectively. In λ-HEPOF L8 only parts of the insert were sequenced, the rest are assumed to be identical to the other two clones. / Table 7 /. 5 'and 3'-untranslated sequences are represented by lowercase letters. The coding region is in capital letters.

 With respect to tables 2 and 3, it should be mentioned that the deduced amino acid sequence shown below the nucleotide sequence is numbered starting from the number 1 for the first amino acid of the mature protein. The putative leader peptide is indicated at the top of the amino acid designations. The cysteine residues in the mature protein are additionally indicated by SH, and potential N-linked glycosylation sites are indicated by an asterisk. Amino acids that are underlined mark residues identified by sequencing of the N-terminus of the protein or sequencing of tryptic EPO fragments, which are described in Example 1. Partial underlining indicates residues in the amino acid sequence of tryptic fragments that could not be uniquely identified. The cDNA clones λ-HEPOF L6, λ-HEPOF L8 and HEPOF L13 are deposited and are available from the American Type Culture Collection, Rockville, Maryland, with accession numbers ATCC 40156, ATCC 40152 and ATCC 40153, respectively.

Example 4. Genomic structure of the EPO gene
The relative sizes and positions of the four independent genomic λ-HEPO clones 1, 2, 3, and 6 / from the HaeIII / AluI library are illustrated by overlapping lines in FIG. 3. The thickened line indicates the position of the EPO gene. The scale (in t.p.o.) and the positions of known restriction endonuclease cleavage sites are shown. The region containing the EPO gene is completely sequenced for both strands using directed exonuclease III-genated series of deletions in this region. A schematic representation of five exons encoding EPO mRNA is shown in FIG. 4. The exact 5'-boundary of exon 1 is currently unknown. Protein-coding regions of exons are darkened. The complete nucleotide sequence of exons is shown in Table 4. The known limits of each exon are outlined by continuous vertical stripes. Genomic clones / λ-HEPO1, λ-HEPO2, λ-HEPO3, and λ-HEPO6 are deposited and are available from the American Type Culture Collection, Rockville, Maryland, with accession numbers ATCC 40154, ATCC 40155, ATCC 40150 and ATCC 40151, respectively.

Example 5. Construction of the vector p91023 / B /
The transformation vector was pAdD26SVpA / 3 /, described by Kaufman et al., Mol. Cell Biol., 2: 1304/1982 /. The construction of this vector is shown in FIG. 5A. In short, this plasmid contains the mouse dihydrofolate reductase cDNA gene / DFHP /, which is under the transcriptional control of the main late promoter of the adenovirus 2 / Ad2 /. The 5'-splicing site is indicated in adenoviral DNA, and the 3'-splicing site derived from the immunoglobulin gene is present between the main late promoter of adenovirus 2 and the DFHP coding sequence. The SV40 early polyadenylation site is present downstream of the DFHP coding sequence. The site of prokaryotic origin pAdS26-SVpA / 3 / comes from pSVOd / Mellon et al., Cell, 27: 279/1981 // and does not contain pBP322 sequences known for inhibiting replication in mammalian cells / Lusky et al., Nature 293: 79/1981 //.

 pAdD26pA / 3 / is converted to plasmid pCVSVL2, as shown in FIG. 5A. pAdD26VSpA / 3 / is converted to plasmid pAdD26SVpA / 3 / / by deletion of one of the two pstI sites in plasmid pAdD26SVpA / 3 /. This is accomplished by partial digestion of PstI, using enzyme deficiency in such a way that a subpopulation of linearized plasmids is obtained, in which only one PstI site is cleaved, then they are treated with Klenov enzyme, ligated to restore the ring and screened for deletion of the PstI site located at 3'- the position of the polyadenylation sequence from SV40.

The tripartite adenovirus leader and virus associated genes (VA genes) are inserted into plasmid pAdD26SVpA / 3 / (d) as shown in FIG. 5A. First, pAdD26SVpA / 3 / / d / cleave PvuII to create a linear molecule open inside the 3'-part of the three elements that make up the tripartite leader. Then pJAW43 / Zatu et al., Cell 16: 851/1979 // digest XhoI, process a Klenov fragment, digest PvuII, and a 140 bp fragment containing the second part of the triple leader is isolated by acrylamide gel electrophoresis / 6 / in Tris-borate buffer / Maniatis et al., above /. Fragment 140 bp then crosslinked with the PvuII-primary plasmid pAdD26SVpA / 3 / / d /. The crosslinking product was used to transform E. coli to give tetracycline resistance, and the colonies were screened using the Grunstein-Hoghess method using a probe labeled with ³² P hybridizing with a 140 bp fragment. DNA is obtained from positively hybridizing colonies to test whether the reconstructed PvuII site is the 5 ′ or 3 ′ end of a 140 bp inserted DNA specific for the second and third late adenovirus leaders. The correct orientation of the PvuII site is on the 5'-side of the 140 bp insert. This plasmid is designated tTPL in FIG. 5A.

 The SV40 virus AvaIID fragment containing the SV40 enhancer sequence is obtained by digesting the SV40 AVAII DNA, blunting the ends with a Klenov fragment PolI, stitching the XhoI linkers with the resulting fragments, digesting XhoI to open the XhoI site and isolating the fourth largest fragment / D / by gel electrophoresis . This fragment is then ligated with pTPL digested with XhoI to obtain the plasmid pCYSYL2-TPL. The orientation of the SV40 fragment D in pCYSYL2-TPL is such that the late promoter of the SV40 virus is in the same orientation as the main late promoter of the adenovirus.

 To introduce adenovirus-associated genes (VA genes) into pCYSYL, 2-TPL, the plasmid pBP322 with the HindIII B fragment of type 2 adenovirus is first constructed. The type 2 adenovirus DNA is digested with HindIII and the B fragment is isolated by gel electrophoresis. This fragment is inserted into the plasmid pBR322, which is previously digested with HindIII. After transformation of E. coli with ampicillin resistance, the recombinants are screened for the presence of the HindIIIB insert, the fragment, and the orientation of the insert is determined by restriction digestion. pBR322-AdHindIIIB contains the HindIIIB fragment of type 2 adenovirus in the orientation shown in FIG. 5B.

 As shown in FIG. 5B, VA genes are readily obtained from the plasmid pBR322-AdHindIIIB by digestion of HpaI, addition of EcoRI linkers and digestion of EcoRI followed by reduction of the 1.4 kb fragment. A fragment having sticky EcoRI ends is then sutured to the EcoRI site of a PTL previously digested EcoRI. After transformation of E. coli HB101 and selection for tetracycline resistance, the colonies are screened by filter hybridization with DNA specific for the VA genes. DNA is obtained from positively hybridizing clones and characterized by its digestion with restriction endonucleases. The resulting plasmid is designated p91023.

 As shown in FIG. 5C, two EcoRI sites in plasmid p91P23 are removed by complete digestion of p91023 EcoRI to give two DNA fragments, one about 7 kb in size and the other about 1.3 kb in size. The last fragment contains VA genes. The ends of both fragments are filled using a Klenov fragment PolI and two fragments are then ligated. Plasmid p91023 / A /, containing VA genes and similar to p91023, but lacking two EcoRI sites, was identified by screening Grunstein-Hogness fragments of the VA gene, as well as by traditional restriction analysis.

 One PstI site in plasmid p91023 (A) is deleted and replaced with an EcoRI site. p91023 (A) completely cleaved with PstI and treated with a Klenov fragment PolI to generate sticky ends. With the blunt PstI site of plasmid p91023 (A), EcoRI linkers are crosslinked. The linear plasmid p91023 (A) with EcoRI linkers attached to the PstI site is separated from the excess linkers and fully digested with EcoRI, and then crosslinked. Plasmid p91023 / B / shown in FIG. 5C, after reduction, was identified as having a structure similar to plasmid p91023 / A / but containing the EcoRI site instead of the previous PstI site. Plasmid p91023 / B / is deposited and is available from the American Type Culture Collection, Rockville, Maryland under accession number ATCC 39754.

 Example 6

cDNA clones (λ-EPOF L6 and / λ-EPOF L13, Example 3) were inserted into plasmid p91023 / B / to obtain pPTFL6 and pPTFL13, respectively. 8 μg of each of the purified DNA is then used to transfect 5 · 10 ⁶ COS cells using the DEAE-dextran method / below /. After 12 hours, the cells were washed and treated with Chloroquin (0.1 mM) for 2 hours, washed again and incubated for 24 hours in 10 ml of medium containing 10% fetal calf serum. The medium is then changed to 4 ml of serum free medium and collected after 48 hours.

 The preparation of immunologically active EPO is quantified by radioimmunoassay as described by Sherwood and Goldwasser / 55 /. Antibody delivered by Dr. Judith Sherwood. The iodinated isotope indicator is obtained from the homogeneous EPO described in Example 1. The sensitivity of the sample is approximately 1 ng / ml. The results are shown below in table 8.

 Example 7

 EPO cDNA / λ-HEPOF L13 / is inserted into the plasmid p91023 / B /, the COS-1 cells are transformed and harvested as described above / Example 6 /, except that the chloroquine treatment is omitted.

The in vitro EPO biological activity is measured using either a colony-forming sample with mouse fetal liver cells as a source of CFV-E, or a ³ H-thymidine uptake assay using spleen cells from mice injected with phenylhydrazine. The sensitivity of these assays is approximately 25 mU / ml. The in vivo biological activity of EPO is measured using either the hypoxic mouse method or the starving rat method. The sensitivity of these assays is approximately 100 mU / ml. No activity was detected in any of these simulated conditioned environments. The results of the study of EPO expressed by the HEPOF L13 clone are shown in Table 9 below, where the reported activities are expressed in units / ml using a commercial, quantitative EPO / Toyobo, Inc./ as standard.

Example 8. Analysis in SDS-polyacrylamide gel EPO from COS cells
180 ng of EPO released into the environment of COS cells transfected with EPO cDNA / λ-HEPOF L13 / in a vector 91023 (B / / above) is subjected to electrophoresis on a 10% Laemllin SDSNAA gel and transferred onto nitrocellulose paper / Towbin et al. ., Proc. Natl. Acad Sci. USA 76: 4350/1979 /. The filter is probed with an anti-EPO antibody as described in table. 8, washed and re-probed ¹²⁵ I-staf. The A-protein filter is autoradiographed for two days. Native homogeneous EPO is described in example 1, it is also subjected to electrophoresis either before / lane B / or after iodination / lane C / / see FIG. 6 /. Used markers include ³⁵ S-methionine-labeled serum albumin / 68000 d / and egg albumin / 45000 d /.

Example 9. Construction of RKI-4
From plasmid PSV2 DHFP / Subramani et al., Mol. Cell. Biol. 1: 854-864 / 1981 // isolate a BamHI-PvuII fragment containing the early SV40 promoter adjacent to the mouse dihydrofolate reductase gene / DHFR /, SV40 enhancer, small tron antigen intron, SV40 polyadenylation sequence / fragment A /. The remaining fragments are obtained from the vector p91023 / A / above / as follows: p91023 / A / is digested with PstI in a single PstI site near the adenovirus promoter to linearize the plasmid and either crosslinked with PstI-EcoRI synthetic converters and recycled / creating PstI-EcoRI-PstI sites in the original PstI site; 91023 / B '//, or is treated with a large fragment of DNA polymerase I to destroy the PstI sites and crosslinked with a synthetic EcoRI linker and recycled / creating an EcoRI site in the original PstI site; 91023 / B /. Each of the two obtained plasmids 91023 / B / and 91023 / B '/ was digested with Xba and ECORI to obtain two fragments / F and G /. By combining fragment G from plasmid p91023 / B / and fragment F from plasmid p91023 / B '/, as well as fragment G from plasmid p91023 / B / and fragment F from plasmid p91023 / B' /, two new plasmids are created that either contain a site EcoRI-PstI, or the PstI-EcoRI site, in place of the original PstI site. The plasmid with the PstI-EcoRI site, where the PstI site is closest to the main late promoter of the adenovirus, is named p91025 / C /.

 Vector p91023 (C) is digested with XhoI until ready, and the resulting linearized DNA with sticky ends is blunted by the reaction of filling the ends with a large fragment of E. coli DNA polymerase I. A 340 bp HindIII-EcoRI fragment containing the SV40 enhancer, prepared as follows, was sutured to this DNA.

 A HindIII-PvuII fragment from the SV40 virus that contains the SV40 replication source as well as an enhancer is inserted into the plasmid Clac / Little et al., Mol. Biol. Med., 1: 473-488 / 1983 //. The Clac vector is prepared by digesting Clac BamHI DNA, filling the sticky end with a large fragment of DNA polymerase I, and digesting HindIII DNA. The resulting plasmid / CSVHPlac / regenerated the BamHI site by crosslinking at the blunt ends of PvuII. The EcoRI-HindIII fragment is obtained from cSVHPlac, sutured to the EcoRI-HindIII PSVOd fragment / Mellon et al., Above / which contains the plasmid replication source, and the resulting plasmid pSVHPOd is selected. A 340 bp EcoRI-HindIII fragment is then obtained. Origin / SV40 enhancer plasmids PSVHPOd are blunted at both ends with a large fragment of DNA polymerase I and crosslinked with the XhoI-digested, blunt-ended vector p91023 / C / described above. The resulting plasmid / p91023 (C) / Xho / blunt ends plus EcoRI / HindIII / blunt ends SV40 origin plus enhancer /, in which the orientation of the HindIII-EcoRI fragment is such that the BamHI site inside the fragment is adjacent to the VA gene, called pES105. Plasmid pES105 is digested with BamHI and PvuII, as well as separately with PvuII, and the BamHI-PvuII fragment containing the main adenovirus promoter / fragment B / and the PvuII fragment containing the plasmid tetracycline resistance gene / as well as other sequences / fragment C / are isolated . Fragments A, B and C are crosslinked and the resulting plasmid shown in FIG. 7, isolate and denote PKI-4. Plasmid PKI-4 is deposited with the American Type Culture Collection Rockville, Maryland, where it is available under accession number ATCC 39940.

Example 10. The expression of EPO in CHO cells - Method I
DNA / 20 μg / from the plasmid pPTFL13 described above / Example 6 / was cleaved with the restriction endonuclease ClaI to linearize the plasmid and crosslinked with ClaI-digested DNA from the plasmid pAdD265SVp / A / I / 2 μg /, which contains the intact dihydrofolate reductase gene driven by the main late promoter of adenovirus / Kaufman and Sharp, Mol. and Cell Biol., 2: 1304-1319 / 1982 //. This cross-linked DNA is used to transfect DHFR-negative cells CHO / DUKX-BII, Chasin LA and Urlaub G. / 1980 / PNAS 77: 4216-4220 /, and after two days of growth, cells that included at least one DHFR gene are selected for alpha-nucleotide-free medium filled with 10% dialyzed fetal bovine serum. After growing for two weeks on selective media, the colonies are removed from the original plates, grouped into groups of 10-100 colonies per pool, re-plated and grown to confluence on alpha-free nucleotide media. The supernatant of the medium for growing pools before methotrexate selection is tested for EPO by radioimmunoassay / RIA /. Pools that show the production of EPO are grown in the presence of methotrexate / 0.02 μM /, and then subcloned and re-examined EPO Cla 44.02-7, a subclone from EPO Cla 44.02 pool, releases 460 ng / ml EPO into a medium containing 0, 02 μM MTX / table 10 /. EPO Cla 44.02-7 is a cell line selected for EPO production and deposited with the American Type Culture Collection under accession number ATCC CRL 8695. This clone is subjected to phased selection at increasing MTX concentrations, resulting in cells that show even higher levels EPO. For pools that are negative for RIA, methotrexate-resistant colonies obtained from equivalent cultures growing in the presence of methotrexate / 0.02 μM / are again tested in EPO pools by RIA. Those cultures that are not positive are subcloned and cultured at even higher concentrations of methotrexate.

 Stepwise methotrexate selection (MTX) is achieved by repeated cell culture cycles in the presence of increasing concentrations of methotrexate and selection for surviving microorganisms. At each cycle, EPO is measured in the culture supernatant by RIA and in vitro biological activity. The levels of methotrexate used in each step amplification are 0.02 μM, 0.1 μM and 0.5 μM. As shown in table 10, after the first selection cycle at 0.02 μM MTX significant levels of EPO are released into the culture media.

Example 11. The expression of EPO in CHO cells - Method II
DNA from clone / λ-HEPOF L13 was digested with EcoRI, and a small RI fragment containing the EPO gene was subcloned into the EcoRI site of the RKI-4 / cm plasmid. Example 10 /. This DNA / RKFL13 / is then used to transfect DHFR-negative CHO cells directly / without digestion /, and selection and amplification are carried out as described in example 10 above.

 RKFL13 DNA was also introduced into CHO cells by fusion of protoplasts and microinjections. Plasmid RKFL13 has been deposited and is available from the American Type Culture Collection, Rockville, Maryland, accession number ATCC 39989.

 A preferred clone of a single colony is deposited and is available from the American Type Culture Collection Rockville, Maryland, accession number ATCC CPI 8695.

Example 12. Expression of a genomic clone of EPO in COS-1 cells
The vector used for expression of the genomic clone of EPO is pSVOd / Mellon et al., Supra. PSVOD DNA is completely digested with HindIII and blunted with a large fragment of DNA polymerase I. The genomic EPO clone, λ-HEPO3, is completely digested with EcoRI and HindIII, and the 4.0 kb fragment containing the EPO gene is isolated and blunted as described above. The nucleotide sequence of this fragment from the HindIII site to the region above the polyadenylation signal is shown in FIG. 4 and Table 4. The EPO gene fragment is inserted into the pSVOd plasmid fragment, and correctly constructed recombinants in both orientations are isolated and checked. Plasmid CZ2-1 has the EPO gene in the "a" orientation / i.e. with the 5'-end of the EPO closest to the beginning of SV40 /, the plasmid CZ1-3 is in the opposite orientation / orientation "b" /.

 Plasmids CZ1-3 and CZ2-1 transfect CO-1 cells as described in Example 7, and the media are collected and analyzed for immunologically reactive EPO. Approximately 31 ng / ml EPO was detected in supernatants of cultures with CZ2-1 and 16-31 ng / ml with CZ1-3.

 Genomic clones HEPO1, HEPO2 and HEPO6 are inserted into COS cells for expression in a similar manner.

 Example 13. Expression in C127 and ZTZ cells.

Construction of pBPYEPO
A plasmid containing the cDNA sequence of EPO under the transcriptional control of the mouse metallothionein promoter associated with the complete DNA of bovine papillomavirus is obtained as follows.

pEPO49F
Plasmid SP6 / 5 was purchased from Promega Biotec. This plasmid was digested until EcoRI ready, and a 1340 bp EcoRI fragment. from λ-HEPOF L13 was inserted using DNA ligase. The resulting plasmid, in which the 5'-end of the EPO gene is closest to the SP6 promoter / as determined by the digestion of BglI and HindIII /, is designated pEPO49F. In this orientation, the BamHI site in the PSP6 / 5 polylinker is directly adjacent to the 5 ′ end of the EPO gene.

pMMTneo BPV
Plasmid pdBPV-MMTneo / 342-12 / / Law et al., Mol. Cell Biol., 3: 2110-2115 / 1983 //, shown in FIG. 8 completely, digest BamHI to obtain two fragments — a large fragment of ~ 8 kb in length containing the BPV genome and a smaller fragment of ~ 6.5 kb in length containing the origin of PMI 2 replication and the resistance gene to ampicillin, a metallothionein promoter, a neomycin resistance gene, and SV40 polyadenylation signal. The digested DNA is recycled with a DNA ligase, and plasmids that contain only a 6.8 kb fragment are identified by digestion with restriction endonucleases EcoRI and BamHI. One such plasmid is designated pMMTneo BPV.

pEPO15a
pMMTneo BPV fully digested BglII. pEPO49f was completely digested with BamHI and BglII, and a fragment of approximately 700 bp containing EPO cDNA was obtained by gel isolation of BglII-digested pMMTneo BPV and a 700 bp BamHI / BglII EPO fragment. sutured, and the resulting plasmids containing EPO cDNA were identified by colony hybridization using the oligonucleotide d / GGTCATCTGTCCCCTGTCC / probe that is specific for the EPO gene. Of the plasmids that are positive at the entrance of the hybridization assay, one / pEPO15a / that has EPO cDNA in orientation, such that the 5 ′ end of EPO cDNA is adjacent to the metallothionein promoter, is identified by digestion of EcoRI and KpnI.

pBPV-EPO
Plasmid pEPO15A is fully digested with BamHI for linearization. Plasmid pdBPV-MMTneo / 342-12 / was also digested fully with BamHI to obtain two fragments 6.5 and 8 kb in length. A fragment of 8 kb in size, which contains the whole genome of the bovine papilloma virus, is isolated in a gel. pEPO15a / BamHI and a 8 kb BamHI fragment are crosslinked to each other into a plasmid / pBPV-EPO / which contains a BPV fragment, identified by colony hybridization using the d / P-CCACACCCGGTACACA-OH / oligonucleotide probe, which is specific to the BPV genome. HindIII pBPV-EPO DNA digestion indicates that the direction of transcription of the BPV genome is the same as the direction of transcription of the metallothionein promoter / as in pdBPV-MMTneo / 342-12 /, see FIG. nine/. Plasmid pdBPV-MMTneo / 342-12 / is available from the American Type Culture Collection, Rockville, Maryland, accession number ATCC 37224.

Expression
The following methods are used to express EPO.

Method I
PBPV-EPO DNA was prepared and approximately 25 μg was used to transfect 1 x 10 ⁶ C127 / Lowy cells and others, J. of Virol., 26: 291-298 / 1978 / CHO using standard calcium phosphate / Grahm precipitation techniques and al., Virology, 52: 456-467 / 1973 //. Five hours after transfection, the medium is removed, the cells are treated with glycerol, washed and fresh λ-medium containing 10% fetal bovine serum is added. After forty-eight hours, the cells are trypsinized, diluted 1:10 with DME medium containing 500 μg / ml G418 / Southern et al., Mol. Appl. Genet., 1: 327-341 / 1982 /, and grown for two to three weeks. G418-resistant colonies are then isolated individually and grown in the presence of G418. The cells are then washed, fresh media containing 10% fetal bovine serum are added, which are harvested after 24 hours. Conditioned media tested by radioimmunoassay and in vitro biological assay are EPO positive.

Method II
C127 and 3T3 cells transfect 25 μg of pBPV-EPO and 2 μg of pSV2neo / Southern et al., Above / as described in method I. This represents an approximately 10-fold molar excess of pBPV-EPO. Following transfection, a procedure similar to that described in method I is used.

Method III
C127 cells transfected with 30 μg pBPV-EPO as described in method I. Following transfection and dilution (1: 10), fresh media were changed every three days. After about 2 weeks, colonies of BPV-transformed cells appear. Separate foci are collected in 1 cm wells of a microtiter plate, grown to a sublayer monolayer, and analyzed for EPO activity or antigenicity in conditioned media.

Example 14. Expression in insect cells
Construction pIYEY EPOF L13
The PIYEY plasmid vector has been deposited and is available from the American Type Culture Collection, Rockville, Maryland under accession number ATCC 39991. The vector is modified as follows:
pIYEYNI
pIYEY was digested with EcoRI to linearize the plasmid, blunted using a large fragment of DNA polymerase I, and a single NoI linker was inserted.

GGCGGCCGCC
CCGCCGGCGG
stitching with a blunt finish. The resulting plasmid is designated pIYEYNI.

pIYEYSI
pIYEY digest SmaI until linearization of the plasmid and insert a single SfiI linker
GGGCCCCAGGGGCCC
CCCGGGGTCCCCGGG
stitching with a blunt finish. The resulting plasmid is designated pIYEYSI.

сшиванием с тупым концом. Полилинкер вставляют в обеих ориентациях. Плазмиду, в которой полилинкер ориентирован таким образом, что сайт BglII внутри полилинкера является близлежащим к полэдриновому генному промотору, называют pIYEYSIBgKp. Плазмиду, в которой сайт KpnI внутри полилинкера является близлежащим к плэдриновому генному промотору, называют pIYEYSIKpBg. Число пар оснований, которые были потеряны между первоначальным KpnI-сайтом в pIYEYSI и полиэдриновым промотором, не определено. pIYEYSIBgKp депонирована и доступна из American Type Culture Collection, Rockville, Maryland, под входящим номером ATCC 39988.pIYEYSlBgKp
pIYEYSI digest KpnI prior to linearizing the plasmid and from about 0 to about 100 bp. cleaved from each end by digestion with a double-stranded endonuclease Bal31. All non-blunt ends obtained are blunted using a large fragment of DNA polymerase I, and a polylenker is inserted

stitching with a blunt finish. The polylinker is inserted in both orientations. A plasmid in which the polylinker is oriented such that the BglII site within the polylinker is adjacent to the polyhedrin gene promoter is called pIYEYSIBgKp. A plasmid in which the KpnI site within the polylinker is adjacent to the pladrin gene promoter is called pIYEYSIKpBg. The number of base pairs that were lost between the original KpnI site in pIYEYSI and the polyhedrin promoter has not been determined. pIYEYSIBgKp is deposited and is available from the American Type Culture Collection, Rockville, Maryland, with accession number ATCC 39988.

pIEYSIBgKpNl
pIYEYNI completely digested KpnI and PstI to produce two fragments. A larger fragment that contains the plasmid start of replication and the 3'-end of the polyhedrin gene is obtained by gel isolation / fragment A /. pIYEYSIBgKp is digested until ready for PstI and Kpn to produce two fragments, and a smaller fragment that contains the polyhedrin gene promoter and polylinker is obtained by gel isolation / fragment B /. Fragments A and B are then combined with a DNA ligase to form a new plasmid pIYEYSIBgKpNI, which contains a partially deleted polyhedrin gene, into which the polylinker is inserted, and also contains a NotI site / replacing the destroyed EcoRI site / and an SfiI site that covers the flank of the polyhedrin gene plot.

 pIYEPO pIYEYSI BGKpNI is digested until EcoRI is ready to linearize the plasmid, and an 1340 bp EcoRI fragment from λ-HEPOF L13 is inserted. Plasmids containing the EPO gene in an orientation in which the 5 ′ end of the EPO gene is adjacent to the polyhedrin promoter and the 3 ′ end of the polyhedrin gene is identified by digestion of BglII. One of these plasmids with the orientation described above is designated as pIYEPO.

EPO expression in insect cells
Large amounts of pIYEPO plasmid are obtained by transformation of the E. coli JMIOI-tgl strain. Plasmid DNA was isolated by the clarified lysate technique (Maniatis and Fritsch, Gold Spring Harbor Manual /) and further purified by CSCI centrifugation. The DNA of the wild-type Antographa californica polyhedrosis virus strain L-1 / AcNPY / is obtained by phenol extraction of viral particles and subsequent purification.

These two DNAs are then transfected into Spodoptera Frudiperda IPI B-SF-21 cells / Vaughu et al., In vitro, Volume B, pp. 213-217 / 1977 /, using the calcium phosphate transfection technique / Potter and Miller, 1977 /. A wild-type AcNPY DNA clamp and 10 μg pIYEPO are used for each dish of transfected cells. Cups are incubated at a temperature of 27 ^o C for 5 days. Then the supernatant was collected and the expression of EPO in the supernatant was confirmed by radioimmunoassay and an in vitro biological sample.

Example 15. Purification of EPO
The conditioned media of COS / 121 / cells with EPO concentrations of up to 200 μg / liter are concentrated to 600 ml using ultrafiltration membranes with a molecular weight of 10,000, such as Millipore Pellican, equipped with 5 square meters. ft /0.465 sq.m / membrane. Assays are performed using RIA as described in Example 6. After ultrafiltration, the concentrate is diafiltered against 4 ml of 10 mM sodium phosphate buffer at pH 7. Concentrated and diafiltered conditioned media contain 2.5 μg EPO in 380 mg of total protein. The EPO solution was further concentrated to 186 ml, and the precipitated proteins were separated by centrifugation at 110000g for 30 minutes.

The supernatant that contains EPO / 2.0 mg / is adjusted to a pH of 5.5 with 50% acetic acid, is allowed to mix at 4 ^° C. for 30 minutes, and the precipitate is recovered by centrifugation at 13,000 x g for 30 minutes.

Chromatography on carboxymethylsepharose
The supernatant after centrifugation / 20 ml /, containing 200 μg EPO / 24 mg total protein /, is placed on a column packed with CM-Sepharose / 20 ml /, equilibrated with 10 mM sodium acetate, pH 5.5, washed with 40 ml of the same buffer solution . EPO bound to CM-Sepharose was eluted with a 100 ml NaU / 0-1 / gradient in a 10 mM sodium phosphate solution, pH 5.5. The fractions containing EPO / total amount of 50 μg in 2 mg of total protein / are grouped and concentrated to 2 ml using an Amicon YM10 ultrafiltration membrane.

Reverse phase HPLC
Concentrated fractions with CM-Sepharose containing EPO were further purified by reverse phase HPLC using a Vydac C-4 column. EPO is fed to a column balanced in 10% solvent B / Solvent A is 0.1% CF ₃ CO ₂ H in water; solvent B is 0.1% CF ₃ CO ₂ H in CF ₃ CN /, with a low flow rate of 1 ml / min. The column is washed with 10% B for 10 minutes, and the EPO is eluted with a linear gradient of B / 10-70% // for 60 minutes. Fractions containing EPO are combined into groups of ~ 40 μg EPO in 120 μg of total protein and lyophilized. Lyophilized EPO is re-diluted in a 0.1 M Tris-HCl solution at pH 7.5 containing 0.15 M NaCl and re-chromatographed by reverse phase HPLC. Fractions containing EPO are grouped and analyzed by SDS-polyacrylamide / 10% / gel / Laemlli, UK, Nature / electrophoresis. The combined fractions of EPO contain 15.5 μg of EPO in 25 μg of total protein.

 The invention has been described in detail, including preferred embodiments thereof. The specialist, however, will understand that when considering the description of the invention and the drawings, improvement options may arise within the scope and essence of the present invention defined in the attached claims.

List of references
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Claims (6)

 1. A method of producing human erythropoietin by culturing in a suitable medium mammalian cells transformed with a DNA sequence encoding human erythropoietin and separating the obtained erythropoietin from cells and from the medium, characterized in that the mammalian cells are transformed with a recombinant vector (plasmid) comprising a DNA sequence encoding human erythropoietin selected from the group consisting of DNA shown in table 3, or its fragments, including at least the sequence t ATG encoding initial Met (position - 27), by AGA, coding terminal Arg (position 166); DNA presented in table. 4, or fragments thereof, including at least a sequence from the ATG encoding the initial Met (position 617), by the AGA encoding the terminal Arg (position 2761).  2. The method according to p. 1, characterized in that the culture medium contains fetal serum.  3. The method according to claim 1 or 2, characterized in that the host mammalian cells are COS, CHO, C127 or 3T3 cells.  4. The method according to claim 3, characterized in that the mammalian cells are 3T3 cells.  5. The method according to claim 3, characterized in that the mammalian cells are Chinese hamster ovary (CHO) cells.  6. The method according to claim 1, characterized in that the said DNA sequence is included in the vector, containing, in addition, DNA of bovine papillomavirus.

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