UA54363C2 - AN ISOLATED DNA MOLECULE CODING MANS ERYTHROPOIETIN (VARIANTS), BIOLOGICALLY FUNCTIONABLE RING PLASMIDE OR VIRUS DNA-VECTOR, STRAIN OF eucaryote host- CELLS (VARIANTS), A METHOD FOR POLYPEPTIDE PREPARATION, A PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

New polypeptides are disclosed possessing in part or in full primary structural conformation and one or more biological properties of mammalian erythropoietin (ЕРО), which are characterised in the preferred forms by being the product of procaryotic or eucaryotic host expression of exogenous DNA sequence. Genomic DNA, cDNA and a constructed DNA sequences, coding partially or fully of the sequence of amino acid residues of ЕРО or for analogues thereof, introduced to self-replicating plasmids or viral vectors are used for transforming or transecting suitable procaryotic or eucaryotic host cells such as bacteria, yeast or vertebrate cells in the culture. Being isolated from culture media or cell lysates, or fragments products of DNA-sequences expression reveal, for example, immunological properties and in vitro and in vivo biologic activity of human or monkey erythropoietin. Chemically synthesized polypeptides having biological and immunological properties of EPO, improved methods for revealing the specific single chain polynucleotides in a heterologous cellular or viral sample, prepared from, for example, DNA, present in plasmid, or virus carrying cDNA or genomic DNA library are described.

Description

Description of the invention

This is a partial continuation of 9 applications for the US patent Mo561024, filed on December 13, 1983, which are in the process of simultaneous consideration; 582185, filed on February 21, 1984, and 655841, filed on September 28, 1984.

The present invention relates to the manipulation of genetic materials and, in particular, to recombinant techniques that make it possible to obtain polypeptides that possess a partially or completely primary structural conformation and/or one or more of the biological properties of naturally occurring zrithropostin.

A. Manipulation of genetic materials

Genetic material can be broadly defined as the chemical substances that program and control the production of components of cells and viruses and direct the answer! cells and viruses. The long-chain polymeric substance known as deoxyribonucleic acid (DNA) 79 contains the genetic material of all living cells and viruses, with the exception of some viruses programmed by ribonucleic acid (RNA). The structural links in DNA polymers are four different nucleotides, each of which consists of a purine (adenine or guanine) or a pyrimidine (thymine or cytosine) linked to a deoxyribose sugar to which a phosphate group is attached.

Addition of nucleotides in the form of a linear polymer occurs by fusing the 5'-phosphate of one nucleotide with the 3-hydroxyl group of the second nucleotide. Functional DNA is found in the form of stable double-stranded communities of single-stranded nucleotides (known as deoxyoligonucleotides), whose communities are formed by hydrogen bonding between purine and pyrimidine bases (i.e., "complementary" communities existing either between adenine (A) and thymine (T ), or between guanine (0) and cytosine (C)). By convention, a nucleotide is accepted! they are named after the names of their constituents, purine or pyrimidine-based, and complementary communities of nucleotides in double-stranded DNA (that is, A-T and 5-C). (3 is called "pair-based". Ribonucleic acid is a polynucleotide containing adenine, guanine, cytosine and uracil (/)), rather than thymine, connected to ribose and a phosphate group.

In short, the programming function of DNA is carried out mainly by means of a process in which specific sequences of DNA nucleotides (geniuses) are "transcribed" into a relatively unstable polymer of informational RNA (mRNA). In turn, mRNA serves as a matrix for the formation of structural, regulatory, and catalytic proteins from amino acids. This is the "transmission" process

MRNA includes the functioning of small strands of RNA (tRNA), which are transported and aligned with individual amino acids along the strands of mRNA, allowing the formation of nucleotides in the appropriate amino acid sequences. "Information" of mRNA, originating from DNA and creating the basis for 325 supply of tRNA and orientation of any of the twenty amino acids for the "expression" of a polyleptide, is in the form of triplet "codons" - consecutive grouped three nucleotide bases. In the known sense, the formation of a protein is the final form of "expression" of programmed genetic information provided by the nucleotide sequence of a gene. "Promoter" DNA sequences usually "precede" the gene in the DNA polymer and provide the C 50 site for the initiation of transcription into RNA. , binding proteins, which determine the frequency (or speed) of transcriptional initiation. Collectively called a "promoter/regulatory" or "control" DNA sequence, that is, such sequences that precede a selected gene (or a number of genes) in a functional DNA polymer contribute determining whether transcription and, ultimately, gene expression will occur. The DNA sequences that "accompany" the gene in the DNA polymer and provide a signal for the termination of transcription into "" mRNA are called "terminator" transcription sequences.

The focus of the microbiological technology of the last decade is the production of industrial and pharmaceutical substances with the use of microorganisms that either do not initially have genetically encoded information related to the desired product included in their DNA, or (in the case of mammalian cells in culture) do not express the chromosomal gene at a noticeable level in the usual way. Simply put, the gene that establishes the structure of the desired polypeptide product is either isolated from a "donor" microorganism, or chemically synthesized and then stably introduced into another microorganism, preferably a self-replicating unicellular microorganism, such as bacteria, yeast, or mammalian cells in culture. Existing machine

GF) equipment for the expression of genes in "transformed" or "transfected" microbial host cells works to create the desired product using xogenic DNA as a template for mRNA transcription, which is then translated into an uninterrupted sequence of amino acid residues. 60 This field of technology is widely represented in patent and literature publications related to "recombinant RNA" methodologies for the isolation, synthesis, purification and amplification of genetic materials used in the transformation of selected host microorganisms. US patent No. 4237224, issued by Sopep, her and I., for example, relates to the transformation of unicellular host microorganisms with "hybrid" viral or circular plasmid DNA, including the selection of xogenic DNA sequences. The methods of the Sopep patent, her AI, for the first time include the production of a transformation vector by means of enzymatically cleavable viral or circular plasmid DNA for o braiding of linear DNA strands. The selection of foreign ("xogenous" or "heterologous") strands of DNA, which generally include sequences encoding the desired product, is obtained in a linear form by using

Eastern enzymes. Linear viral or plasmid DNA is incubated with foreign DNA in the presence of cross-linking enzymes capable of carrying out the restoration process, and the "hybrid" vector! they are formed containing a selected segment of xenogeneic DNA, "twisted" into a viral or circular DNA plasmid.

Transformation of compatible unicellular microorganisms of the host with a hybrid vector ends with the formation of multiple copies of xogenic DNA in the population of host cells. In some 7/0 strains, the desired result is simply the amplification of foreign DNA, and the assembled, cultured "product" is DNA. More often, the goal of transformation is expression, carried out with the help of host cells from xenogeneic DNA in the form of large-scale synthesis of isolated quantities of industrially important protein or polypeptide fragments encoded by foreign DNA. See also, for example, US Patent No. 4,264,731 (issued Zpipe), 4,273,875 (issued Mapiz), 4,293,652 (issued Sopep) and 5B M European Patent Application Mo093619, published November 9, 1983.

The development of specific DNA sequences for splicing in a DNA vector is achieved by using a number of techniques that depend largely on the degree of "foreignness" of the "donor" in relation to the designed host, as well as on the size of the polypeptide expressed in the host.

Fearing oversimplification, it can be argued that there are three main alternative methods:

M) "separation" of the double-stranded DNA sequence from the genomic DNA of the donor; (2) chemical preparation

the DNA sequence that creates the code for the polypeptide of interest and (3) the synthesis of my double-stranded DNA sequence by means of enzymatic "reverse transcription" of mRNA isolated from donor cells. Indications are more methods that include education

DNA-complement" of mRNA, colloquially called "c" DNA" method.

Obtaining DNA sequences is often a vibor method when the complete sequence of amino acid residues of the desired polypeptide is known. The methods of obtaining DNA are described in i) the concurrently considered US patent application Mo483451, AGop, ei a!. (filed on April 15, 1983, and corresponding to PCT application 0583/00605, published on November 24, 1983 under the number U/083/04053), for example, create means to achieve such extremely desirable results as: provided by the presence of alternating codons, often occurring in the microorganism of the host selected for expression (for example, the provision of "preferred" codons of yeast or E, soy); avoiding the presence of ISE) untranslated "bintronic" sequences (usually found in genomic

mammalian DNA sequences and their mRNA matrices), which are easily processed by prokaryotic host cells; avoidance of unwanted "leader polypeptide" sequences, usually encoded by genomic DNA and cDNA sequences, however, often not easily cleaved from the polypeptide of interest with the help of bacterial or yeast host cells; provision of easy insertion of DNA into a suitable expression vector! in communities with the desired promoter/regulatory and terminator sequences; and the provision of easy construction of genes encoding a polypeptide fragment and analogues of the desired polypeptides.

When the complete sequence of amino acid residues of the desired polypeptide is not known, it is impossible to obtain DNA sequences directly, and isolation of DNA sequences encoding polypeptides using the cDNA method becomes a method of selection, despite the possibility;" disadvantages in the ease of assembly of expression vectors capable of providing the high levels of microbial expression indicated above. Among the standard methods for isolation of cDNA sequences is the preparation of plasmid-bearing cDNA "libraries" derived from the reverse transcription of mRNA, which is often found in donor cells, selected as those responsible for high-level gene expression (for example, cDNA libraries derived from pituitary cells, which are expressed in relatively large amounts of growth hormone products). Where a significant part of the amino acid sequence of the polypeptides is known, it is possible to use a tag, a probe sequence of a single-stranded DNA, duplicating a sequence presumably found in the "planned" cDNA

Me, in the methods of DNA/DNA hybridization, carried out on clonal copies of cDNA, which was denatured into this single-stranded form. (See, in the main, the disclosure and discussion of this field of technology, the presentation in US patent 4394443, issued in the name of U/eizetanp, her AI. and the latest indications of the use of long probes of oligonucleotide hybridization, the message in the works of Uailiyase, her AIY., Mis. . Asidz Kezv., 6, pp. 3543 - 3557 dv (1979), Keuev, ei ai., R.M.A.5. (0.5.A.), 79, pp. 3270 - 3274 (1982) and Uaue, et al., Mis. Asia Kev., 11, pp. 2325 - 2335 (1983). See also U.S. Patent No. 4,358,535, issued to Raikom, et al., relating to the method

F) DNA/DNA hybridization during diagnostics; publication of European patent applications Nos. Мо0070685 and Мо0070687 relating to light-emitting labels on single-stranded polynucleotide probes; work BRamiz, ey ai,, "A Mapia! yog Sepeiyis Epdipeegipu, Admapsed Vasiegiai Sepeiyisv", Soii Zrgipd Nagrog bo Garogayugu, So Zrgipd Nagrog, M.U. (1980) ag yr. 55 - 58 apa 174 - 176, concerning methods of hybridization of colonies and epidemic diseases; and Mem Epdiapa Mysieag (Vosiop, Mavzg5v.) brochure on "Sepe Zsgeep"

Nubgiaiganyop Tgapzieg Metbrgape taiiegiaizh, giving instructions for the transfer and hybridization of DNA and RNA,

MoMER-9721 catalog.

Among the most significant recent achievements in hybridization techniques for screening 65 recombinant clones is the use of labeled mixed oligonucleotide probes, each of which is potentially a complete complement of a specific DNA sequence in selective hybridization, which includes a heterogeneous mixture of single-stranded DNA and RNA. These methods are considered especially useful when generating cDNA clones obtained from sources that create extremely small amounts of mRNA sequences for the polypeptide of interest. In short, the use of strict hybridization conditions aimed at avoiding nonspecific binding may allow, for example, autoradiographic visualization of a specific cDNA clone at the end of planned hybridization

DNA is the only probe in the mixture that is its complete complement. See, mainly,

UUaMase, hey aI. , Cape Asidz Kev., 9, pp. 879 - 897 (1981); Btsdov, ei ai., R.M.A.5. (O.5.A. ), 78, pp. 6613 - 6617 (1981); Spoo, ei aiYi., Mayte, 299, pp. 178 - 180 (1982); Kygasnpi, ei aiI., R.M.A.5. (O.5.A. ), 79, pp. 585461 - 6464 (1982); OpKkyro, ey a. R.M.A.5. (0.5.A. ), 80, pp. 2196 - 2200 (1983); and Kotriinn, ey a).

R.M.A.5. (0.5.A.), 80, pp. 3218 - 3222 (1983). In general, the methods of mixed probes in the work of UMaiYiase, ei ai. (1981) more, more, more! by various developers until the moment when reliable results were reported in isolated cDNA clones using a 32-link mixed "pool" of oligonucleotide probes with a length of 16 bases (16-tag) of uniformly varying sequences

DNA together with a single 11-tag for performing two-position "positive" confirmation of presence

KDNK of interest. See the work of bipdeg-Zat, ei aI., R.M.A.5. (0.5.A.), 80, pp. 802 - 806 (1983).

The use of genomic DNA isolates is the least common of the three methods of developing specific sequences for use in recombinant techniques. This is especially true in the field of recombinant techniques aimed at ensuring the microbial expression of mammalian polypeptides, and is mainly due to the complexity of mammalian genomic DNA.

Thus, although there are reliable methods for the development of phage-carrying libraries of genomic DNA of humans and other species of mammals (see, for example, the work of I am/p, ei a!., Seyi, 15, pp. 1157 - 1174 (1978 ), referring to the methods for generating a human genomic library, commonly known as the "Mapia Library"; work Kat, ei a. R.M.A.85. (0.5.A.), 77, pp. 5172 - 5176 (1980) , dealing with the creation of a human genomic library based on the method of alternative restriction of DNA-nuclease fragmentation; the work of Viaitspeg, ei aiYi., Zsiepse, 196. pp. 161 - 169 (1977), describing the construction of a bovine i) genomic library), several relatively successful attempts have been made to use methods of hybridization to isolated genomic DNA in the absence of far-reaching prediction of amino acids or

DNA sequences. As one example, we can cite the work of Riddavs, ei ai., 9). My sister Arr. c zo Zepeisv, 1, pp. 3 - 18 (1981), where it is reported about the successful isolation of the gene encoding the alpha-subunit of the hypophyseal glycoprotein hormones of the human from the Mapias Library by using a "relatively long" probe, which includes a complete fragment of 621 pairs! Nitrogen based pre-separated (se

cDNA sequences for alpha subunits. As a second example, in the work Raz, ey ai., R.M. A. 5. (O.5.A. ), 80, pp. 1531 - 1535 (1983) reports on the isolation of human genomic clones for NO A-OK human with "

C5 is based on the use of a synthetic oligonucleotide with 175 pairs. And, finally, in the work Apaegsop, ei yu ai,, R.M.A.5. (0.5.A.), 80, pp. 6838 - 6842 (1983) reported the isolation of a genomic clone for bovine pancreatic trypsin inhibitor (BPTI) using a single probe having 86 base pairs in length and constructed according to the known amino acid sequence of BPTI .

The authors note the definition of modest prospects for the isolation of mRNA suitable for synthesis "

cDNA libraries, due to the obvious low levels of mRNA in the initially targeted sources of parotid 7-3) from glands and lung tissues. Then expressing hope for success in probing the genomic library using a mixture of labeled probes, they state: "For the most part, an oligodeoxynucleotide probe! ;" mixed sequences were used to isolate protein genes of unknown sequence from cDNA libraries. Such probes are usually a mixture of 8 - 32 oligonucleotides and have a length of 14 - 17 nucleotides, being typical representatives of every possible combination of codons for a small stretch (5 - b residues) of an amino acid sequence In harsh hybridization conditions, which put probes with improperly paired nitrogenous bases in a worse position, these mixtures are able to localize specific gene sequences in clonal libraries of low complexity. However, due to their small length and heterogeneity, 5p mixing of probes is often only the specificity necessary for probing such complexes

Me. sequences, like the genome of mammals. This makes such a method impractical for the selection of 4) protein genes of mammals when the corresponding mRNA is not available!" (Omitted links).

Thus, in this field of technology, there is still a need for improved methods for rapid and efficient isolation of cDNA clones in those cases when little is known about the amino acid sequence and when "enriched" sources of mRNA tissue are not very accessible, so that they can be used in the construction of libraries of KKDNK. Such an improvement method! whites (F, whites are especially useful if they are used to isolate genomic clones of mammals, where sparse information is available regarding the amino acid sequences of the polypeptide encoded by the gene of interest. 60 V. Zritropostin as a polypeptide of interest

Zritropozz, the formation of red blood cells, is carried out continuously throughout a person's life to compensate for the destruction of cells. Zritropozz represents an extremely precisely regulated physiological mechanism that allows a sufficient number of red blood cells to work in the blood for proper oxygenation of the tissue, but not so much that! cells 65 Impeded blood circulation. The formation of red blood cells takes place in the bone marrow under the control of the hormone, erythropoietin.

Zrythropostin, an acid glycoprotein with a molecular weight of approximately 34,000 Daltons, can be found in three forms: so, p, and asialo. Form! o and D differ slightly in hydrocarbon components, however, they have the same potency, biological activity and molecular weight. The asialo form is an o or D-form with a cleaved terminal carbohydrate (sialic acid). Zrythropostin is present in very low concentrations in the plasma when the body is in a healthy state, in which the tissues receive sufficient oxygenation from the existing number of erythrocytes. A normal, low concentration is sufficient to stimulate replacement! red blood cells, which are normally lost in the process of aging. 70 The amount of zrithroposteine in the blood circulation increases in conditions of hypoxia, when oxygen transport by blood cells in the blood circulation decreases. Hypoxia can be caused by the loss of a large amount of blood during bleeding, the destruction of red blood cells due to radioactive irradiation, a decrease in oxygen consumption due to high altitudes or prolonged unconsciousness, as well as various forms of anemia. Under the influence of tissues exposed to hypoxic stress, 75 zritropostin begins to increase the formation of red blood cells by stimulating the conversion of primary precursor cells in the bone marrow into prothroblasts, which are subsequently brought to maturity, synthesize hemoglobin and are released into the circulation as red blood cells.

When the number of red blood cells in the circulation is greater than necessary for the normal oxygen demand of the tissues, erythropoietin in the circulation decreases.

See, in the main image, work! Tevia, ey aYi., EHR. Netaiyo!., 8v (Zirr. 8), 144 - 152 (1980); Topa, hey ay.,

U. Vioi. Spet., 256(24), 12666 - 12672 (1981); Soidjazzeg, U. Seyi. RPuvzioIi., 110 (Ref. 1), 133 - 135 (1982); Ripsp, Vioosa, 60(6), 1241 - 1246 (1982); ZuyumuvKki, ey aYi., Ehri. Netaiyo!., 8(Zirr. 8), 52 - 64 (1980); Matsdniop, App. Siip. ar. zZsi.,, 13 (5), 432 - 438 (1983); MUeivv, ey a), At. U. Mei. Kez., 44(10), 1832 - 1835 (1983); IHarrip, ei a, Ehr. Netaiyoi!., 11(7), 661 - 666 (1983); Vasim, ey a, App. M.M. Asai. su r; zZsi, 414, 66 - 72 (1983); Migrpu, ei ai.,, Asia. Naetaioiodis Oaropis, 46(7), 1380 - 1396 (1983);

Oezvzurgiv, ei aiI., Vgy 9. Naetatso!., 56, 295 - 306 (1984); and Ettapocei, ey aKi. , At. .). RPuvioi., 247 (1 RI o 2), E168-76 (1984).

Since erythropoietin is important in the process of formation of red blood cells, the hormone has a potential useful application both in diagnostics and in the treatment of blood disorders characterized by low or incomplete formation of red blood cells. See, mainly, work! Rappaipig-Oav, ey ay.,

Viosa, 63 (5), 1168-71 (1984) and Nadau, At. Great Dane, Ed. Netatso/i./Opsoi., 4, 191 - 196, (1982) , concerning zrithroposteine in possible therapies for sickle cell diseases, as well as Ezsprasy, ei ai. , 9. Siip. co

Ipmevi., 74(2), pp. 434 - 441, (1984), in which a therapeutic regimen for a uremic sheep is described based on the reaction of ip mimo to the infusion of plasma enriched with zritropostine, and a dosage of 10 | WITH

ERO/Kkg per day for 15 - 40 days as correction of anemia of the type associated with chronic renal failure. See also the work of Kgape, Nepgu Roga Nagr. Mea. ., 31(3), 177 - 181 (1983).

Recently, it was estimated that the presence of zritropoztin in large quantities will cure anemia in 1,600,000 people in the United States every hour. See, for example, Moghtizop, "Viorgosezvipd ip Zrase - ap Omegmiem", pp. « 557 - 571 ip Te Uuopa Vioiesn Kerogi 1984, Moiishte 2:O5A, (Opiipe Rybiisaiope, Mem MogKk, MM 1984). Recent studies have created the basis for predicting the effectiveness of zritropoztin therapy in a variety of diseases, disorders and hematological disorders: Medoumai, ei ai!., Asia. ts Naetaiyo!., 71, 211 - 213 (1984) (beta-thalassemia); MispipeKku, ey aYi. , in Rheaiaig., 105(1), 15 - 21 (1984) "" (cystic fibrosis); Soyevz, ei a). (pregnancy, menstrual disorders); Nada, ei ai., Asia. Readiaig. Zsapa., 72, 827 - 831 (1983) (early anemia of prematurity); Siaiz-MUaiKeg, es a! , Agsy. RIiuz. Mea. KenpNarbii. , 65, 370 - 374 (1984) (spinal cord injury); Yupp, ei ai., Eig. 9. Arri. RnuzioI., 52, 178 - 182 (1984) (space flight); MiMeg, ei ai!. , Vii. 9. Naeptaiio!), 52, 545 - 590 (1982) (acute blood loss); Odira, ei ai., 9. ar. Siip. Mead., t 103(4), 574 - 580 and 581 - 588 (1984); and Iirzspy", ei ai., Viosda, 63(3), 502 - 509 (1983) (aging); and

OO OaipiaK, ei aiYi., Sapseg, 51(6), 1101 - 1106 (1983) and Zspmagil, ei aiYi.,, Oyuiagupodoi., 109, 269 - 272 (1983) (various neoplastic states of the disease, accompanied by anomalous zritropozm).

Ф Previous attempts to obtain zritropoztin with a good yield of production from plasmas! blood or urine" evidence as relatively unsuccessful. Complicated and refined laboratory technique inevitably and, as a rule, leads to the collection of very small amounts of contaminated and unstable extracts containing zritropsztin.

US patent Mo3033753 describes a method of partial purification of zritropoztin from plasma! of sheep's blood, which ensures the production of small amounts of a general unadulterated extract containing zrithropostin. i) Initial attempts to excrete zritropoztin from urine led to unstable, biologically inactive preparations of the hormone. The US patent Mo3865801 describes a method of stabilizing the biological activity of a general substance containing zritropostine by recovering it from urine. The resulting preparation, containing 6o zritropostine, contains 9095 of the activity of zritropostin and is stable.

Another method of purifying human erythropoietin from the urine of patients suffering from aplastic anemia is described in MiuaKe, ei ai., 9U. Vioi Spet., My. 252; Mo15 (Aitsdivy 10, 1977), pp. 5558 - 5564. This seven-step technique includes ion-exchange chromatography, ethanol precipitation, gel filtration, adsorption chromatography, and produces pure erythropoietin with a potency of 70,400 units/mg of protein at 65 and a yield of 2,195.

In the US patent Mo4397840, known by TaKegaul, her AI., description! methods of obtaining a "zrythropostine product" from urine samples of a healthy person with weakly basic ionic changes, and it is claimed that the production of a product with a low molecular weight "does not have inhibitory effects against zrythropostine".

In the British patent application No. 2085887, Zyditoy, her AI., published in May 1982, a method for obtaining hybrid lymphoblastoid cells of a person is described and it is reported that the production levels are in the range from 3 to 420 units of erythropoietin per ml of cell suspension (distributed in cultures after reproduction by a mammalian host with a content of up to 107 cells per ml). At the highest levels of production, which it is claimed should be achieved!, the rate of production of erythropoietin 70 can be calculated to be between 40 and 4000 units/109 cells/48 hours in ip myo culture with subsequent transfer of cells from ip mimo propagation systems (See also the equivalent US patent

Mo4377513). They did it! There are many proposals in relation to the release of erythropoietin from tissue sources, including neoplastic cells, but the output results were extremely low. See, for example, work! OeiKtap, ei aiYi.,, Ehri. Netaiyo!., 11(7), 581 - 588 (1983); Tatroigip, ei ai., R.M.A.5. 75 (0.5.A.), 80, 6269 - 6273 (1983); KaivioKka, ei aY.,, SbSapp, 74, 534 - 541 (1983); Nadizhaga, ei ai., Viosa, 63 (4), 828 - 835 (1984); and Sporrip, eh! a/!., Viosad, 64 (2), 341 - 347 (1984).

Other bleaching technologies used to obtain purified zytroposin included immunological methods. A polyclonal, serum-derived antibody directed against zritropoztin is developed by injecting an animal, preferably a rat or a rabbit, with human zritropoztin. Injected human zritropoztin is recognized as a foreign antigenic substance by the animal's immune system and causes the production of antibodies against the antigen.

Different cells that respond to stimulation by means of an antigenic substance produce and release into the bloodstream antibodies that differ slightly from those produced by other reacting cells. Antibody activity remains in the serum of an animal when its blood is extracted. "M

Although unpurified serum or antibody preparation, purified as a serum fraction of immunoglobulin C, can be used in tests for the detection of complex formation with human erythropoietin, the substances experience the main inconvenience. That is, the serum antibody, which consists of all different antibodies produced by individual cells, is polyclonal in nature and forms a complex with the components in the general extracts, unlike a single "9 zritropoztin".

Of interest for the premises of the present invention are the latest achievements in the field and the development of whole cell cultures capable of producing a single type of antibody that is specifically immunologically active with a single antigenic determinant of the selected antigen. See with your own eyes the work of Spizpo!t, Nidy Thesppoiodu, Moi. 3, Moi, 57 - 63 (1983). Attempts were made to use cell fusion and hybridization techniques for the development of "monoclonal antibodies to xythroposin M" and to apply these antibodies to the isolated and quantitative determination of human xythroposin. As one example, we can cite the message that appeared in an annotated form in the work of I ee-Ntsapo,

Arzigashi Mo1463 oi Ray. Rhos., 41, 520 (1982), which mentions the successful development of liny cells "hybridoma! Mish-mishSh, bleaching monoclonal antibodies to human erythropoietin. As a second example, a detailed description of the preparation and use of a monoclonal, anti-zytroposin-c antibody appeared in the work of Mueizv, ei aiYi, R.M.A.5. (0.5.A.), 79, 5465 - 5469 (1982). See also the work of Zazaki, Viotei. Viospit. Asia., 42 (11/12), 5202 - 5206 (1983); Mapadamzha, ei ai., Viosd, 64(2), 357 - 364 "" (1984); Khapadaucha, ei ai., 9. Vioi. Spet., 259(5), 2707 - 2710 (1984); and US patent Mo4465624.

For the premises of the invention, reports on the immunological activity of synthetic peptides, which essentially duplicate the amino acid sequence 1 preserved in naturally occurring proteins, glycoproteins, and nucleoproteins, are also of interest. More specifically, a polypeptide! with a relatively low molecular weight indicates participation in immune reactions, similar in duration and duration to immune reactions of physiologically important proteins, (95) such as viral antigens, polypeptide hormones, and the like. Among the immune reactions of such 50 polypeptides is included and provoking the formation of specific antibodies in immunologically active animals. See, for example, work! I egpeg, ei ai., Seiyi, 23, 309 - 310 (1981); Kovv, ei aiYi., Maiige, 294, 654 - syu» 656 (1981); UUaMeg, ey aYi, R.M.A.5. (0.5.A.), 77, 5197 - 5200 (1980); Iegpeg, ei aYi., R.M.A.5. (0.5.A.), 78, 3403 - 3407 (1981); UUaneg, ey aiYi.,, R.M.A.5. (0.5.A.), 78, 4882 - 4886 (1981); Muopd, ei ai., R.M.A.5. (O.5.A.), 78, 7412 - 7416, (1981); Ogeep, ei ai.,, Seiyi, 28, 477 - 487 (1982); Midd, ei ai., R.M.A.5. (Sh.5.A.), 79, 5322 - 5326 (1982); Vagop, ei aii., Seiyi, 28, 395 - 404 (1982); Yugeeztap, ei ai., Maishge, 295, 158 - 160 (1982); and Iegpeg, Zsiepiyis Ategisap, 248, Mo2, 66 - 74 (1983). See also the work of Kaizeg, ei ai., about Zsiepse, 223, pp. 249 - 255 (1984), concerning the biological and immunological activity of synthetic peptides, which approximately possess the secondary structures of peptide hormones, however, they may not possess their primary structural by conformation The above-mentioned studies are, of course, related to the amino acid sequences of proteins other than zritropoztin, a substance for which no essential information about the amino acid sequence has been published. In the joint application for the US patent Mo463724, filed on February 4, 1983.). Edgie, published on August 22, 1984 as a European patent application Mo0116446, describes a hybrid cell line! mish-mish (А.Т.С.С. MonВ8209), which produces a highly specific monoclonal, anti-zytroposin antibody, which b5 is also specifically immunoinactive with a polypeptide containing the following sequence of amino acids:

MN»o-Aia-Rgo-Rgo-Aga-I esh-Pe-Suvz-Avzr-Zeg-Aga-Ma!-І ей-с1ИШ-Ago- Tug-I esh-І ets-с1Ш-Aia-І uv- UNO

The polypeptide sequence is the sequence that is attached to the first twenty amino acid residues of the mature human zytroposin, isolated in accordance with the method described in Miuake, et al., 9. Vioi. Spet., 252, 5558 - 5564 (1977), and on which an amino acid analysis was carried out using a gas-phase sequencer (Arriei Viozuvietv, Ips.) in accordance with the method of NemisK, M., ei ai., U. Vioi. Spet., 256, 7990 - 7997 (1981). See such a work by Zie, ei ai,, Rgos. May Asad All together. (O5A), 80, pp. 3651 - 3655 (1983), concerning the development of polyclonal antibodies against synthetic 26b-teg based on a different amino acid sequence, and work 7/0 ZUYUMuzKi, ей а|., у). And so on. Meipoadv, 69, pp. 181 - 186 (1984).

Although polyclonal or monoclonal antibodies, as described above, have very useful materials for use in immunoassays for the detection and quantitative determination of zritropoztin and can be very useful! in srodstvenno cleaning zritropoztina, it seems unlikely that it is material! for large-scale elimination, they can provide amounts of mammalian zritroposthin /5 MstochniKov, sufficient for further analysis, clinical examination and possible broad-based therapeutic use of the substance in the treatment of, for example, chronic kidney disease, in which tissue damage is unable to support the production of zritroposthin. Therefore, it is assumed that the best prospects for the full characterization of mammalian zritropoztin and the creation of large quantities of it for possible diagnosis and clinical application are the use of recombinant techniques for the implementation of large-scale microbial synthesis of the compound.

Despite the fact that significant efforts have been made in an attempt to whiten the DNA sequences encoding human zritropoztin or other mammalian species, none of them have been successful. This is mainly due to the lack of tissue sources, especially human tissue sources, enriched in mRNA so that! will allow the construction of a cDNA library, from which the DNA sequence encoding zritroposthin can be isolated using known methods. In addition, little i) is known about the continuous sequence of amino acid residues of erythropoietin, and therefore it is impossible to construct, for example, a long subnucleotide probe suitable for reliable use in DNA/DNA hybridization screening of cDNA and especially genomic DNA libraries. Illustratively, the sequence of twenty amino acids used for the generation of the above-mentioned monoclonal antibody produced by A.T.S.S. MoHN8209, does not allow the construction of a unique x, bO-basic oligonucleotide probe by the method described by Apdegzop, her, and others. It was found that the human gene for zytroposin can appear as "that single copy" in the human genome, and one way or another, the genetic material encoding human zytroposin is probably less than 0.0000590 - the total human genomic DNA that should be present in the genomic the library yu

To date, the most successful of known efforts in recombinant methods for creating DNA sequences suitable for use in microbial zxpression of isolated amounts of mammalian zytroposin have not reached their goal. As an example, we can cite the work of Ragreg, her AI.,, EHR. Netaiyo!., 11, Z(, pp. 14, Abrzigashi 101 (1983), which reports on the extraction of mRNA from the kidney tissues of baboons treated with phenylhydrazine, and the injected mRNA into the oocyte! Heporiv Yaemiv s doOvolno pla) with the temporary result of obtaining i.e., a mixture of "translational products", which had the manifested biological properties of zritropoztin. More recently, Ragreg, ei a!., Viood, 62, Mo5, Zurr. Mo1, Abrvzigashi, 392, ;" Ag Rade 122a (1983) reported on the translation of migo mRNA from the human kidney by frog oocytes. The resulting mixture of the translation product was estimated to contain about 220 units of the translation product having erythropoietin activity per microgram of injected mRNA. And although such levels of translation of xenogeneic mRNA from erythropoietin encoding erythropoietin were found to be completely low (compared to even the previously reported levels of translation of baboon mRNA into the desired product), it was considered that the result confirms the existence of the human kidney as an area of erythropoietin expression, allowing the construction of a cDNA library of an enriched human kidney, from which the desired gene can be isolated. (See also Ragbreg, Siyip. Kev., 31(4), 769A (1983). me) Since the filing of US patent applications MOMo 561024 and 582185, there has been a single 4) message about cloning and expression of what is recognized cDNA of human zytroposin in E. soya. In short, a number of cDNA clones were inserted into the plasmid! E. soybeans, and the product! fusion p-lactamase! hit marks! as immunoreactive with a monoclonal antibody to an unspecified "subtope" of human erythropoietin.

See Gee-Nyapo, Rgos. May Asad from (OA), 81, pp. 2708 - 2712 (1984).

For the first time, a new purification and isolation of a polypeptide product with a partially (i.f.) completely primary structural conformation (that is, a continuous sequence of amino acid residues) and one or more biological properties (for example, immunological properties and biological activity of mimo and migo) occurring in nature is proposed. zritropoztina, including its allelic variant!. These polypeptides also clearly differ in that they are the product of the expression of a prokaryotic or zukaryotic host (for example, bacterial, yeast, and mammalian cells in culture) of xogenic DNA sequences obtained by genomic or cDNA cloning, or by gene synthesis. The products of microbial expression in cells of vertebrates (for example, mammals and birds) can, in addition, differ in that they are free from associations with human proteins or other contaminants, b5 which can be associated with erythropoietin in its natural environment for mammalian cells or in extracellular fluids media such as blood plasma or urine. Product! typical yeast (for example, Zassagotusevs segemisidase) or prokaryotic (for example, E. soybean) host cells are free from association with favorite mammalian proteins. polypeptides can also include the amino acid residue of the initial methionine (at position -1).

The proposed new glycoprotein products include those that have a primary structural conformation that sufficiently duplicates the conformation of naturally occurring (for example, human) zritroposine so that! give the opportunity to possess one or more of the biological 7/0 properties of the latter, and those that have an average carbohydrate composition that differs from the composition of naturally occurring (for example, human) zritropostine.

Vertebrate cells (for example, СО5-1 and ХНО), proposed in the present invention, contain the first cells, always available, which can multiply continuously and rapidly and which during growth in culture are capable of producing more than 100 in their growth medium) (preferably, more 500) and most preferably 75, more than 10,000 - 5,000))) of erythropoietin per 106 cells in 48 hours, as determined by radioimmunoassay.

Also offered are synthetic polypeptides that completely or partially duplicate the continuous sequence of amino acid residues of zritropoztin, which is explained here for the first time!. These sequences, by sharing the primary, secondary or tertiary structural and conformational characteristics with naturally occurring erythropoietin, can possess biological activity and/or immunological properties together with the naturally occurring product so that they can be used as biologically active or immunological substitutes for erythropoietin in therapeutic and immunological applications. processes. Accordingly, monoclonal and polyclonal antibodies are offered, produced by standard methods, which are immunoreactive with such polypeptides and, preferably, also immunoreactive! with naturally occurring zrithroposteine.

The present invention illustrates the cloning of the DNA sequence of the species' beginnings of monkeys! и и) human, as well as polypeptide sequences, conveniently deduced from there and representing, respectively, the primary structural conformation of the species beginnings of monkeys and humans.

A new, biologically functional vector is also offered! viral and circular plasmid DNA, including DNA sequences of the invention, and microbial (for example, bacterial, yeast and mammalian cells) organisms! the host, which is stably transformed or transfected with the following vectors. Accordingly, a new method is proposed! production of useful polypeptides, including the cultivation of such transformable or transfected microbial hosts under conditions that facilitate large-scale expression of xenogeneic, vector-carrying DNA sequences and

Isolation of the desired polypeptides from the nutrient medium), cell lysates or fractions of cells (membranes.

Isolation and purification of microbially expressed polypeptides proposed by the present invention can be carried out using traditional methods, including, for example, separation by preparative chromatography and immunological separation of preparations containing monoclonal and/or polyclonal antibodies. - c Explained the sequence of amino acid residues of zritropostin, the present invention offers complete and/or partial construction of DNA sequences encoding zritropostin and "" containing such favorable characteristics as the introduction of codons "preferred" for expression by selected non-mammalian hosts, provision of sites for cleavage by means of restriction endonuclease enzymes and provision of additional initial, final or intermediate DNA sequences, which contribute to the construction of easily expressed vectors.

Accordingly, the construction (and development by means of specific mutagenesis of cDNA and genomic DNA) of DNA sequences encoding microbial expression of polypeptide analogs or zritroposine derivatives that differ from naturally occurring forms in terms of the identity and location of one or more amino acid residues (that . or the middle part of the polypeptide); and which possess some or all properties of forms found in nature.

The proposed new DNA sequences include all sequences suitable for the expression in prokaryotic or zukaryotic host cells of polypeptide products that have, at least, part of the primary structural conformation and one or more of the biological properties of erythropoietin, which include: (a) DNA sequences shown in Tables M and

MI, or their complementary threads; (B) DNA sequences that hybridize (in the conditions of hybridization illustrated here, or more stringent conditions) to DNA sequences defined in (a), or their fragments; and (c) DNA sequences, which are due to the degeneration of the genetic code of duties! hybridize to the DNA sequences defined in (a) and (b) above.

Especially coverage in part (b) of the genomic DNA sequence, which encodes forms of allelic variants of monkey and human zytroposin and/or encodes zytroposin of other mammalian species. 65 Especially coverage! in part (c) the construction of DNA sequences, co-iodizing EPO, EPO-fragment and

EPO analogs whose DNA sequences may include codons that promote the translation of informational RNA in invertebrate hosts.

The class of polypeptides that are coded in parts is covered by the proposed invention

DNA complement to the upper part of the genomic DNA sequence shown in Table MI, i.e. "complementary inversion of proteins", as described in Tgatopiape, et al., Mysiwis Asiaz Kezeagsi, 12, pp. 5048 - 5059 (1984).

Also covered by the invention are pharmaceutical compositions containing an effective amount of the polypeptide products of the present invention together with suitable diluents, stimulants and/or carriers, allowing the creation of zritroposine therapy, especially in the treatment of anemic 7/0 disease states and, above all, such anemic states that lead to chronic renal failure.

The proposed polypeptide product can be "labeled" by covalent association with a detectable marker substance (for example, radiolabeled 129I) to provide reagents suitable for detecting and quantifying erythropoietin in solid tissue and liquid samples, such as 75 Blood or urine. DNA products can also be labeled with detectable markers (such as radiolabels and non-isotopic labels such as biotin) and used in DNA hybridization processes to localize the position of the erythropoietin gene and/or the position of any related gene family in the chromosomal map of humans and monkeys! and other species of mammals. They can also be used to identify a disordered erythropoietin gene at the DNA level and be used as genetic markers for

Identification of adjacent genes and their disruption.

As described in detail below, the present invention, in addition, provides a significant improvement in methods of detection of specific single-stranded polynucleotides of unknown sequence in a heterogeneous cellular or viral sample, including multiple single-stranded polynucleotides, in which: c (a) a mixture of single-stranded polynucleotide probes with uniformly varying based sequences, and each of the specified probes is potentially specific i) complementary to the base sequence, which is presumably unique for the detected polynucleotide, (B) fix the sample on a dense substrate, and (c) process the substrate, which has a fixed sample on it, to weaken the further binding of polynucleotides to it, with the exception of the hybridization path to polynucleotides in the specified i-th sample, with (4) the processed substrate, which has a fixed sample on it, temporarily brings the sword into contact with the specified mixture of their probes under conditions conducive to hybridization only between fully complementary polynucleotides, and that (e) specific polynucleotide is detected by monitoring for the presence of a hybridization reaction between it and a fully complementary probe within the specified mixture of labeled probes, as evidenced by the presence of the highest density of labeled material on the substrate in the locus of a specific polynucleotide in comparison with the background density of the labeled material resulting from the non-specific binding of the 70 ZzonDdOovB label to the substrate. - with these techniques, they are especially effective! in situations that dictate the use of 64, 128, 256, 512, 1024 or more mixed polynucleotide probes, having a length of 17 to 20 based on DNA/DNA, or RNA/RNA, "" or DNA/RNA hybridizations.

As described below, the instruction is more than an improvement of the method! clearly allowed to identify cDNA clones encoding monkey zrithropostin within the library obtained from mRNA of kidney cells from anemic monkeys. More specifically, a mixture of 128 uniformly varying 20-tag probes, based on amino acid sequence information derived from alternating fractions of human erythropoietin, was used in colony hybridization techniques to identify seven "positive" zytropoietin cDNA clones within a total of 200,000 colonies. Even more, the practice of applying the improved methods of the present invention allowed to quickly isolate three positive b clones from the screening limits of 1,500,000 sterile spots that make up the human genomic library. c» This was completed using the above mixture of 128 20-teg probes together with a second set of 128 17-teg probes based on amino acid analysis of a distinct continuous sequence of human erythropoietin.

The instructions above are illustrative only! constitute the first known example of the use of multiple mixed oligonucleotide probes in DNA/DNA hybridization processes aimed at the isolation of genomic clones of mammals, and the first known example of the use of a mixture of more than 32 oligonucleotide probes in purified cDNA clones.

Numerous aspects and advantages of the invention will become apparent to specialists in this field upon consideration of the following detailed description of the invention, which provides an example of the practical application of the invention in its preferred embodiments.

In accordance with the present invention, white allocations and characterizations! DNA sequences encoding a partially or completely polypeptide sequence of the human and monkey zritropoztina species (hereinafter "ERO"). In addition, monkey and human DNA became the subject of zukaryotic and 65 prokaryotic zxpression, which creates a large number of polypeptides that exhibit biological (for example, immunological) properties of EPO found in nature, as well as biological activities of mimo and migo EPO.

Monkey DNA was first isolated from a cDNA library constructed from mRNA derived from the kidney tissue of a monkey in a chemically induced anemic state and whose serum, as determined by immunology, contained higher levels of EPO compared to the serum of normal monkeys. Isolation of the required cDNA clones containing EPO-encoding DNA was carried out by using DNA/DNA colony hybridization using a pool of 128 mixed, radiolabeled, 20-tag oligonucleotide probes, and included rapid screening of 200,000 colonies. The calculation of oligonucleotide probes of proteins is based on the information of the amino acid sequence, represented by 7/0 enzymatic fragmentation and determination of the sequence of small samples! human ERO.

White human DNA was isolated from the human genomic DNA library. Isolation of clones containing

EPO-encoding DNA was carried out by hybridization of DNA/DNA plaques with the use of more than 128 mixed 20-tag oligonucleotide probes, as well as a second pool of 128 radiolabeled 17-tag probes, the sequences of which were based on amino acid sequence information obtained from a different enzymatic fragment of EPO human

Positive colonies and white plaques are checked with the help of dideoxy-programming of the clonal DNA sequence using a set of 16 sequences within the pool of 20-tag probes, and selected white clones are subjected to nucleotide sequence analysis with the final deduction of the primary structural conformation of EPO polypeptides encoded by it. The deduced polypeptide sequence showed a high degree of homology to each other and 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 inserted into the "shuttle" DNA vector, which was amplified in E. soya and used for transfection of mammalian cells in culture. The cultural growth of the transfected host cells was expressed in preparations floating on the surface of the culture medium containing up to 3,000 tu EPO per ml i) culture liquid.

Try the following! presentations with the aim of illustrating the invention and especially directions for the method, previously implemented for the identification of EPO encoding monkey cDNA clones and human genomic clones, methods resulting from this identification, as well as sequences, development of expression systems and immunological confirmation zkspressii ERO in such systems. X,

More specifically, Example 1 is aimed at determining the amino acid sequence (ie

Human EPO fragments and the construction of mixtures of radiolabeled probes based on the results of sequence determination. Example 2 is directed, mainly, to the method), inclusion in - c5 identification of positive cDNA-clones of monkeys, and thus ensures obtaining information related to the treatment of animals and preliminary analysis of radioimmunoassay (KIA) of animal sera. Example C is aimed at obtaining a cDNA library, screening hybridization of colonies and checking positive clones, determining the DNA sequence of a positive cDNA clone, as well as generating information on the primary structural conformation (amino acid sequence) of monkey EPO polypeptides. Example 4 is directed to the method of inclusion in the identification of positive human genomic clones, and thus provides information related to the source of the genomic library, methods of hybridization of plaques and verification of positive clones. Example 5;" aimed at determining the DNA sequence of a positive genomic clone and generating information on the amino acid sequence of human EPO polypeptides, including comparison with information on the sequence of monkey EPO. The example would be directed to the method! construction of a vector including EPO-encoding DNA derived from a positive cDNA clone of monkeys, use of the vector for transfection of CO5-1 cells and culture growth of transfected cells. Example 7 is directed to the method of constructing a vector including EPO-encoding DNA, originating from a positive human genomic clone, using the vector to transfect CO5-1 cells and

Cultural growth of transfected cells. Example 8 is directed to the method of immunoassay,

Me. carried out on the supernatant liquids of the media obtained from the cultural growth of transfected cells in 4) in accordance with Examples b and 7. Example 9 is aimed at the biological activity of ip mygo and ip mimo microbially exposed ERO of Examples 7.

Example 10 is directed to the development of mammalian host zxpression systems for cDNA EPO of monkey species of two M human genomic DNA, including Chinese hamster ovary cells (HNO") and to the immunological and biological activity of the products of such zxpression systems, as well as the characterization of such products. Example

F) 11 is aimed at obtaining genes that control human ERO and analogues of ERO, genius! which include a number of preferred codons for expression in E. soy and yeast host cells, as well as on systems! such an expression. Example 12 refers to the immunological and biological activity profiles of the expression products of the systems shown in Example 11.

EXAMPLE 1

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

Human EPO is isolated from urine and subjected to tryptic cleavage, which leads to the development and secretion of 17 separate fragments in amounts approximately estimated at 100-150 picomoles. 65 Fragments are arbitrarily assigned numbers and analyzed for amino acid sequence by microanalysis using a gas-phase sequencer (Arriei Viozuzvietv) to obtain information about the sequence given in Table 1 below, where the application is an alphabetic code and "X" denotes a residue that is not uniquely determined.

Table I

Fragment y The result of the analysis of the sequence tav А-Р-Р-В tav v-k-sh-k te d-I.-6-A-9-K t13 M-1-E-v8 t16 d-y-5-6 -1-K tiv s-E-K t21 k--K-K 125 u-14-1-E-A-K t2va 1-1-s0-0-5-K t26v Sh-u-t-6- E-A-S-B 127 T-I-T-A-0-T-5-K t28 E-A-I-5-R-R.-0-A-A-M-D-D-R -I-I tzo E-d-E-X-1-T-1-6-X-A-E-N-X-5-1-

M-E-X-1I-T-U-R 131 U-y-5-M-E-(-8 tz3 5-0 -T-1-4-4-K t35 M-M-k-X- A-N-K t38 b-0-A-1 -I -U-X-5-5-0-8-K-

E-R-I-0-(-N-U-0-K

V. Calculation and construction of mixtures of oligonucleotide probes

Amino acid sequences, shown in Table I, are considered in the context of the degeneration of the genetic code with the aim of clarifying whether it is possible to apply mixed probe techniques to methods with

DNA/DNA hybridization on cDNA and/or genomic DNA libraries. This analysis shows that within the MoT35 fragment there is a series of 7 amino acid residues (MaI-Azp-RNe-Tug-AIa-Tgr-I uv), which can be unequivocally characterized as being coded by one of 128 possible DNA sequences, covering 20 steam is based. Therefore, the first set of 123 20-teg oligonucleotides is synthesized using standard phosphoamidity methods, see, for example, the work of Veysade, et al., Tegygapedop s zo I ebegv, 22, 1859 - 1862 (1981) on a solid support, in accordance with sequence given below in Table II. ise)

Table ІІ с

Remainder - Uai - d8p Re t 818 Games Ace h;E

З" sa ttb AAb AT SA 0 ASSO TT 0-5 t A A A y Io) s s

Additional analysis shows that within the MoT38 fragment there is a series of b amino acid residues (Sip-Pgo-Tgtr-SISH-Pgo-yesh), on the basis of which it is possible to obtain a pool of 128 mixed "oligonucleotide 17-tag probes, as shown in Table ІІ below

Table ІІІ - - Remainder - bip Rtgo iv biv rto ev ' y? z' stu ssA Ass stt ss A 5 s b

Гос с 175 Oligonucleotide probe is inserted into the 5-end of gamma-Z2R-ATP, 7500 - 8000 Si/mmol (ISM), using polynucleotide kinase T., (MEM). p EXAMPLE 2 p A. Methods of processing monkeys and KIA-analysis

Females of Supotoiidiz monkeys Masasa Gazistsagiaz (2.5 - 3 kg, 1.5 - 2 years) are treated by subcutaneous injection of a solution of phenylhydrazine hydrochloride (pH 7.0) in a dosage of 12.5 mg/kg for 1, 3 and 5 days. The hematocrit is controlled before each injection. On the 7th day, or as soon as the hematocrit level drops below 2595 from the initial level, serum and kidneys are collected after the introduction of ketamine hydrochloride in a dosage of 25 mg/kg. Material collection! immediately freeze in liquid nitrogen and store at a temperature of -707C.

V. KIA for ERO

A radioimmunological study is used for the quantitative determination of EPO in samples in (F.) in accordance with the following methods: d. zritroposine, labeled 125), and the test tubes are incubated at a temperature of 02 C for at least another 15 hours. Each test tube contains 500 μl of incubation mixture containing 5 μl of diluted immune serum, 10,000 srt of 125I-zrythropostin, 5 μl of trasylol and 0 - 250 μl of EPO standard , or an unknown sample, as well as РВ5 containing 0.195 В5BA to fill the remaining volume.

The serum used is the blood of a rabbit immunized with 1905-nI/mm 65 pure human urinary erythropoietin in the second sample. The final dilution of the antiserum is adjusted so that! the content of 129I-erythropoietin bound to the antibody did not exceed 10 - 2095 of the total input amounts. In general, this corresponds to the final dilution of the antiserum from 1 : 50,000 to 1 : 100,000. 125|-zitropostine bound to the antibody is precipitated by adding 150 μl of biarp A. After a 40-minute incubation of the sample! centrifuge and the sediments in the test tube are washed twice with 0.75 ml of 10MM solution

Tiv-NSII, having a pH of 8.2, containing 0.15 M Mass, 2 µM EOTA and 0.05905 Ttyop X-100. The amount of residual residues is determined with the help of a gamma-quantum counter to determine the percentage of 125I-zitropositin binding.

Quantities bound by pre-immune sera are read from all final values for correction of non-specific precipitation. The zytroposin content of unknown samples is determined by comparison with a standard curve.

The above method is applied to the monkey serum obtained above in part A, as well as to the processed monkey serum. Normal serum levels have been determined to be approximately 36 tU/ml, whereas serum levels from treated monkeys range from 1000 to 1700 tU/ml.

EXAMPLE C

A. Construction of the cKDNK-library of monkeys

Reporter DNA is isolated from the kidneys of normal and anemic monkeys by means of the technique using guanidinium thiocyanate described in the work by SpPig/p, ei ai!., Viospetivigh, 18, p. 5294 (1979), and poly (A)" mRNA is purified by omitting two ! through a chromatographic column with oligo(at)-cellulose, as described on pages 197 - 198 in the work of Mapiaiiv, ei aiYi., "Moiesshag Siopipd, A I aroghaiogu Mapia!" (Soyd Zrgipod5

Narbog I arogayogu, Soia brhipoz, Nagrog, M.U., 1982). The cDNA library is built in accordance with the modification of the basic methods described in the work of OKauata, ey a. , My Apa Sey VioiI., 2, pp. 161 - 170 (1982).

The key points of the currently preferred methods are the following: (1) pOS8 is used as a sole vector, Pv is cut and then provided with a tail 60 - 80 cm long based on the oligo aT; (2) Nipsia cleavage is used to cleave the AT-tail oligo from one end of the vector; (3) the synthesis of the first strand and the joining of the tail with the help of oligo db are carried out in accordance with the published methodology; (4) cleavage of VatNi is used for cleavage of the d4O-tail oligo from one end of the vector; and (5) the replacement of the RNA strand with DNA is carried out in the presence of two binding units (SATSTAAAASSOCTSSSSSSSSS and ASOSTSTTTTA) at a three-fold molar excess relative to the oligo-tailed vector.

V. Methods of hybridization of colonies for screening cDNA libraries of monkeys

Transformed E. soyii are spread with a density of 9,000 colonies on 10 x 10 cm nutrient cups containing 10 kg/ml of amlicillin. Sepebsgeep filter! (Mem/Epdiapa Misieag Sayaiod MoMER-972) is pre-moistened on a VNI-SAM cup (37g/l brain/heart bactoextract, 2g/l Sazatipo acids and 15g/l (o agar containing 50O0mcg/ml chloramphenicol) and used for raising colonies from the dish. Colonies are grown in the same medium for 12 hours or more to amplify the number of plasmid copies. Amplified colonies (sides of colonies) are processed serially by placing a filter over two pieces of ZMM Whatman saturated with the following solutions: (1) BOMM glucose - 25mM Ttgiv-NSII (pH 8.0) - 1MM EOTA (pH 8.0) for 5 minutes, (2) O0.5M Mason for 10 minutes, and « (3) 1.0M Ttgiv-NSII (pH 7.5) for three minutes.

Filter! then dry in air in a vacuum for two hours at a temperature of 807C. t s Then the filter! they are subjected to cleavage with Proteinase K through treatment with a solution containing 5 Ωkg/ml of protease enzyme in Buffer K (0.1 M Ttiz-HCI (pH 8.0) - 0.15 M Masi - 10 mM EOTA (pH 8.2) - 0.296 5O5I. » Specifically, 5 ml of the solution is added to each filter and the cleavage is continued at a temperature of 557C for 30 minutes, after which the solution is removed.

The filters are then treated with 4 ml of pre-hybridization buffer (5 x Z5PE - 0.595 505 - 100 μg/ml 1 Z E. soybean DNA - 5 x VER). Preliminary hybridization treatment is carried out at a temperature of 557C, usually for 4 hours or more, after which the solution is removed.

The hybridization process is carried out as follows. Add 3 ml of hybridization buffer (5 x 55 PE - 0.595 505 - 100 μg/ml of yeast tRNA) to each filter containing 0.025 picomoles of each of the 128 b 50 sequences of the probes presented in Table I (the complete mixture is designated as the EMP mixture), and filter! they are kept at a temperature of 48"C for 20 hours. This temperature is 2"C lower than the lowest of the "C" temperature values! dissociation (Ta), determined for any of the probes.

At the end of the hybridization filter! washed three times for 10 minutes on a rolling pin with a solution of 6 x 55 - 0.196 805 at room temperature and washed from two to three times with a solution of b x 35C - 195 505 at the temperature of hybridization (487).

Gee! Autoradiography of the filters revealed seven positive clones among the screened 200,000 colonies.

Analysis of the initial sequence of one of the imaginary cDNA clones of monkeys! (designated as clone 83) which is carried out to check the results by modifying the method described by UmMaiIase, ei ai!., Sepe, 16, pp. 21 - 26 (1981). Briefly, the DNA plasmid from cDNA clone 83 of monkeys is linearized by Esoki cleavage and 60 is denatured by heating in a boiling water bath. Nucleotide sequence is determined by the dideoxy method, described by Zapdeg, ei ai!., R.M.A.5. (0.5.A.), 74, pp. 5463 - 5467 (1977).

A set of ERM-mixture probes consisting of 16 sequences is used as a seed for sequence reactions.

C. Determination of cDNA sequences (EPO) of monkeys! 65 Analysis of nucleotide sequences of clone 83 is carried out using the methods described

Mezzipo, Meijipodz ip Epgutoiodu, 101, pp. 20 - 78 (1983). In the IM table of representations! the result! pre-restriction card analysis for EsokKiI/Nip! cloned fragment of clone 83 with approximately 1600 base pairs. Approximate location of the recognition sites of restriction endonuclease enzymes expressed by the number 3 and the E. soybean site at the 5 end of the fragment. Determination of the nucleotide sequence is carried out by finding the sequence of separate limiting fragments, with the aim of pairing overlapping fragments. For example, overlapping sequence information, represented by the analysis of nucleotides in the restriction fragment, is designated C113 (ZaiyzZA at "111/5ta! at 324), and determination of the sequence in the opposite order is designated C7Z (Alishi at "424/B8EII at 203).

Table iiU site of recognition of the restriction enzyme Approximate location (i)

YesoVI 1

BvizA 11

BtvI 180

VZEE1I 205

Zkai 32A

312 regions of the world

Vish yes

RVE A26 dish 430 nwai 1 d101 546

RBC 601

RI vod si 191 605 di 782 o 2191 788

K881 792

RE 807 year 81 (ze)

A1chI 927 nsoi zab |se)

Zvizd 1014 with actions 1072 virgins 1115 " lychees 1225

VZ 1301 IS o) Yazai 1343 I 1384 NipotII 1449 li 1850 "

Multiplex 1585 C 70 Sequence determination of approximately 1,342 base pairs (within the region from the 3s region upstream to the EsokI site and the Nipayi site) and analysis of all possible reading systems contribute to the development of the DNA and amino acid sequence information presented in Table M. In the table, the imaginary initial amino acid residue of the amino terminus of the matured EPO (as verified by the correlation of the previously specified analysis of the sequence of twenty amino terminus residues) is indicated by the number y1. The presence of 15 methionine-determining ATS codon (marked -27), "directed against the flow" of the final alanine residue of the initial amine, as the first residue designated for the amino acid sequence of the mature protein, is an indicator of the likelihood that EPO was originally expressed in the cytoplasm in with the precursor form, which includes a "leading" region of 27 amino acid residues, which is inserted before the mature EPO enters the bloodstream. Areas of potential glycosylation within (22) 50 polypeptide designations! asterisks The calculated molecular weight of the translation region is 21117 c» daltons, and the molecular weight of the 165 polypeptide residues that make up the mature EPO of monkeys is 18236 daltons.

F) name) 60 b5

Table U

Translation of monkey ERO cDNA

Base

САТСССОоССССССССТТебАСАСССОСССТОТоССТосАСбСССССССССССССССОСССССОО

СОСТСДАСТССССССАСТССДССССТССССССТОСТОСССССССССОССАСТССССОССОСТ

-27 -20

Mei biu Mai Ni bi Suz Rto A1v TIr

SSASSSSSSSSSSSSSSSSASATO obo Sto SAC o baAA tot sstT boss tob -10

Sech Ttr Se Ses Se beg bez Mai 5eg Ge Rto Ge biu Me Rgo sto tob stt sto Sto tst sto st ts sto sst sto o sto ssA -15:1 10

Mai bho biu dia Rgto Rgo Dgu se 112 Suz dzr bet Agu Ma1i Se stS ssSo bos ss sSsA SSA SOS sStTS ATS TST SAC AbsS ssA st hundred 20 f

SI Ago Tut cheshe bid ATa Sub bio A1a bi Azp ma) Tvg Mee

САС АСС TАС STS o TTo bAb bSS dAb САС bSS bAS AdT sSTS ACC At zo z 40 biu Suz Zeg bi 5eg Suz beg ieu Ab5p bi dzp I1e TnI Uai Rgo

BOS TST TSS o baAA AS TOoS dos TtTo DAT CASS DAT ATS dsS ST ssA

Table U (continued) 50 azr taI Suz Maii Avp Rie Tug Aia Tgtr uz Ago Mee Si Mai Siu bAS ASS o ddd Sstt AdS ttS TAT SSS tob AAb ASb ATb bAb TS bob vo 70 bip bip dia Mai bi mai Tgr biu biu es dia Sebe 5eg bi

SAS Sas bsST STA bdd btsS tob SAS SoS sto os sto ST TSA SadA vo - dia mai Se dto biu bip dia Mai Se dia Azp 5eg 5eg bip Rto sst sto sto sbo bos sSAb oss sto tto osS dAS TStT tTosS sAb SS s 90 100 rRne bis Rgo (eu bip ce niv Mei Ad5r uz dia Ii 5eg b3)u (He o

TTS bAb SSS Hundred SAB Hundred SAS ATO SAT AddA SSS dtS AST obS art 110

Atgo beg I1e TNg Tag Ge Geo Dt Aiia sem siu Aiv bip bi dia

SOS ABS ATS ASS AST Sto stt Sat bso Sto bbA SSS sSAb saAA boss 120 150 1126 5eg Me Rgo Azr dia dia beg dia Aia Rgo (ve Ag Tntg I1e o zo ATS TS Sto SSA bAT person ssSs tTSo OST OST SSA ST SAD des ts 180 (Se)

Tit A1a Azr Tng Rye Suz Suz Me RMe Atu Uvi Tug 5eg Azp Rne

AST ST BAS dStT TTS ToS Ada StTS TTS sСsA sSTS TAS ToS o ddt o ttTe

Table U (continued) about "I 150 1v6a and mes Agu Siu sub Ge Sub Se Tut Trtg biu 619 A1a Su AgO Ago 325 ste ee SbA Al Sto dAs sto TAS dSS bob bab bos ToS Abb AbA IF) 165 biu Ar Ag OR

SOS BAS DSA TSA SSABSTOSSTOSAVSTOBSSASASATSASSASSASTOSSTSASSAASA

СТОССТОССАССССССТСССТСАССАССТССССААССССАССАССАВССОСТСССССТССТССТСССТ

СОССАБСССТСССАТОСАСАССАССАСТОССАБССАТССАСАСТСАСССССССАССАССАССАСССАССАССАССАССАССАССАССАССАССАССАССАССАСС with

ASSADOSATTSASASASSASSSTTTAADASTSASSASSSDSASASAAATOSASASOSAADDASASST

; - САССТСАСССОССАСССТОСАДАААТТСАТОСАССАСАССССТТТОБАСССААДТТТАССTo

TTTTTSSASSTASSATSSASSOSASSASSATSATSTSSASAADSTTASSTSSSADOSTSTSASTT

СТСААССССТСАСОСССАСТССССССТСССТССССААСССССССТСАССССАСААААТАТТТ sl TTOSADTSSTOSSASSASSDDAAADTTASSSASSASTTTTTOSASSTTSSSASSSTATTTSASA

СТСТОСТОСССААССАССССССТАССССТОСАССТОССАТОССАСТСАСААСССТСАДСАС с» ДОСАТОобОоСоСстТеСсСсСТсСтТобттТСстТоСстТОбосСтТосСААССТТ ніаЗтІ (95) Полипептидная последовательность в Таблице М может бьїіть без труда подвергнута анализу на присутствие б 50 Чрезвьчайно гидрофильньх областей и/или вторичньх конформационньїх характеристик, являющихся показателем возможно очень иммуногенньїх областей, например, при помощи способов, предложенньйх Норр, еї

Fe a, R.M.A.5. (О0.5.А. ), 78, pp. 3824 - 3828 (1981), and Kuye, ey a). , 9. Mine. Viol., 157, pp. 105 - 132 (1982), and/or Spoi, ei ai., Biospet., 13, pp. 222 - 245 (1974) and Aidmapsez ip Epgutoiodu, 47, pp. 45 - 47 (1978) ). Computerization of the analysis in accordance with the method of Knorr, ei aiI., is possible thanks to the program designated PEP Keygepse bBesiop 6.7, presented by |IpieiPdepeiisv, Ips., 124 MOpimegeiu Amepie, Raio o A, Saiyogpia.

EXAMPLE 4 ko A. Human genome library

The genomic library of the human fetal liver, carrying the SNI4A phage, obtained in accordance with the 60 methods of Gamp, ei., Seyi, 18, pp. 533 - 543 (1979), is preserved for use in plaque hybridization studies.

V. Methods of plaque hybridization for screening the human genome library

Phage particles are lysed and DNA is fixed on filters (50,000 plaques per filter) in accordance with the methods described by MUso, Meipoaz Ip Epgutoiodu, 68, pp. 389 - 395 (1979), with the exception of 65 that SepebZsgeep Riv filter was used! (Mem/Epdiapa Misieag Sagaiod MoMER-976) and MAMAM cups (mass, 5g; Massi»-6NlioO, 2g; M2A-Amin A, 10g; yeast extract, 5g; acid! sazatipo, 2g; maltose, 2g, and agar , 15 g per liter).

Air-dry the filter! burn at a temperature of 80"C for 1 hour and then cleave with proteinase K, as described in Example 3, part B. Preliminary hybridization is carried out with

Using 1 M Mass - 195 5O5-buffer at a temperature of 55"C for 4 hours or more, after which the buffer is removed. Hybridization and post-hybridization washes are carried out as described in Example 3, part B. They are used as a mixture of 128 20-tag probes, designations ERU, as well as a mixture of 128 17-teg probes of the Table

And (denoted as ERO mixture). Hybridization is carried out at a temperature of 487C using a mixture of ERM probes. Hybridization using a mixture of EPO probes is carried out at a temperature of 46 °C, i.e., 4 °C below the smallest multiple of T for the members of the mixture. Removal of hybridization probes for re-hybridization is performed by boiling in 1 x 355C - 0.196 5O5 for two minutes. Autoradiography of filters reveals three positive clones (active with both mixtures of probes) among the screened 1,500,000 phage plaques. Verification of positive clones for EPO coding is achieved by determining the DNA sequence and electron micrographic visualization of the heteroduplex 7/5 formation with the monkey cDNA of Example 3. This technique also gives proof of the presence of multiple introns in the genomic DNA sequence.

EXAMPLE 5

Analysis of the nucleotide sequences of positive clones (designation! 7PEI) is carried out, and the results obtained to date are presented! in Table MI.

MI table

DASSTTSTSSOSTTSSASSASSSASSSTATTTTSSOSADSTSASSAAASSSASSSSATSTSTToASTSTESOSSSA

АСАСССОбАТОССССССАССССАОСТССССООАСССССССТTTSСАСАСАСССССТССССАСТСССС

AAbSSSTOSSSAASSOSSSTOSASTSSSSSTOSSSSSPVASSSSAVSSSSSSOSASSABSSSSSATSASSSSASSASOS

АСОТСТАСАССАССССОСТСАСОССССОСсССАССССТСАдСссСАСОССТОССТОССССССССОССОССССССОСССССССССОСССОСССОСССОСТ

STOSSSSSSSSSTOBSSSSSSSSSTSASSSSASSASSSSSTSTSSSSSSSSSSSSSSSSSSSSSSASASATO (8)

SASATADASASSSSSSVASSSSSSSSSSASSASSSSHSHASASTOSSToSOSSASSSSSSOSSSoSTSSSSSTOSSbSsTe

СОССССАСССОСТСТСССТОССbСАССССССАСССССОССАССОССОСССКОССОССОССОССОССОССОССОСССОССОСССОССОСССОССОССССОССОСССОСССОССССОСССОССССОСССОСССОСССОСССОСССОССССОСССОСССОСССОСССОСССОСССОСССОСССОСССОСССОСССОСССОССОССОССО

STTOSASSASSSOSSSTSTSSTSTASSSSSSSTOOOSTOBSSSSTOSASSSSSSSSSTTTSSSSOSSSATSASSXX

-27 -24 I«o)

Meb biu MUa1i niv

СССоСТСАССОБСОССССССААСТСОТСАСССАССССССССААСССАЕССАЕ АТе bob hundred САС s

СТОАСТАССССССССТСОССОССОССвООСССОССОСССТТtТсstoTTtsАСССССssATtTTASSOSSSSСССССССССОСССТ

UI table (continued) "g

ATTSSSSAAAASSSTSSSTOSSTTSADOSASSOSSSSASTTTSTSAASSASSSSobAAASSoSssASSoSooToso

ССАСССТССАССТОССОСОСОСАСТСССССБАСТСТСТСССАТСССАДАДАССТОСССТОТСССССССА

САСТТТососттососАССАСстtttsvosttstostotosasttetetesttotsaAstetstoo (1.5.1

TTSSASASSSASASSATSAATAAASSSASSASSSASSASSSTOASTOSTTSSATOSTTSSSASASSAAAOSASSAS

STOBSSSASASASSSTOSSSATSDABSDASSTOTSSTTSSASSASSSASSSTTTSToSSSsSSsssbostedeTST « -23 -20 skosstesstatstottstao bla tt set SOS to ST tob TT TS st TS ST 8 s ce bet tsvo Rgo (vu 91u tsets Rgo Mvi Te su dIa Rgo Rgo dgd sech ce Su» sto tso sto sst sto StoTS sSsA S StTS boss SSS SSA SSA SOS ST ATS Test i 10 20 t i - Azr beg Ago Uai cem Si Agu Tut ce se Si Aia iu5 bi Aia bi dep I12

SDS ABS SoA stTS STO SAS DSS TAS st tTTo bAb SSS AAS SAS SSS SAS DAT ATS ti dse STSASASSSSSTTSSSSSASSSATTSSSASAASTSASOSTSASOSSTTTSSASSAAASTOSTOSTOSSSATS 1 SSSOADSSTOSSASTTSSTTTOSOSTOSASTTOSSASTASASASTOSSSSSSSATASAATAASTS

MI table (continued)

Cheka"

TOSTSSSSSSADASSATASSTSAAASTASSSAASSASSADASSASSASSASSASATSSTASSSSTToSOSSASbO 7 zo o tat biu Suz Aia Sich

ССАБАСССТTSАБСССАСССТСАСТССССССССТСТСТСТТТСАС АССс СОС tot ссСТ SAD (o) . 40 ni Suz bet Ge dop bi Azp 112 Tig Ma1i Rgo Azr Tpg (in Mai two Rpie Tug

Sas tToS des TT ADdT SAS DAT ATS AST stos SSA SAS ASS AAA STT AAT o TTS TAT "yu" 50 55

A1v Tgr Guz dk9 Mei 0619 bSS tob DAS ASb ATS SAC STSASTTSSTTTTTTTTTTTTTTTTSSTTTTTTTTOSASAATSTSATT

TOSSAOSSSTOATTTTOSSATODDASSSASAAATSATSSOSSSADASSTAAAATOSASSASSASSASASSATSASSST

25 СССТОССОСАСАСОСТСАССТСТАААТССССАСССТСАСАТОСССССАСАТОССААATToСsTTsАСССССТ (F) ССАСТЕССАССАССАССАССАВСАСТАСТСДСАССССССАСТСТСТАСАДАСАТТТАААААААТТАТССАС ke STSAASTOSTSСАТСССТОСТАССССССАСАСАСАТТТОСАДСССТОСАСССССССАССАССССТСАСССАССАА

ТТТОАСССТОСАСТСАССТСТОАТСАСАССАСТОСАСТССАСССТСАСТСАСАСАСТСАСССССТСТСТСА

60 b5

UI table (continued)

ADADASAADASADAADASADADATAATSASSSOSTOTATOSAATASATTSATTTATTSATTSASTSASTSAST

САСТСАТСАТСАТСАТСАТСААСААСТСТАТТОСАТАССТИСТОТТТОССАССТTeSsToSTtoo

СОСТОСТСАССОССАССАВОВАССССТСАСАСТСОСТСАССТСОАСТССССАСАСТССАСССССТОТАС

56 at 7

Uuai biu biu bip A1ia Uai biu uai Tgr bib b1u ieu Aia Ge Se 5et bi div

STS bob САС САС СС o bTA SAA StsS tob SAb boss sto ss sto Sto TSb bAA ost vo

Mai ces Ago biu bip dia Sam Se Uai dop Zeg Zeg bip Rgo Tgr 619 RgO iieo sts sto sob bos Sas bss sto tt sts o dAS o tst TssS SAS ssb tob SAS ses sto 100 710 bip ce Niz wai Azr Guz A1a Uai beg biu yech Ago eh yeu Trg Tig Ge yiv

САС STO SAT STO SAT ddd SS sto dst sbS Stt SOS dos StTS AS AST ste ST 110 115

Atu Aia Gen biu dia bip sob ssStT Sto SA ССС САb СТСАСТАССДССССАСАСТТSTOSTТОСССТTTSTSTAАСАДАОСССА

СААСССТСТТССТААССАСТАССАССААСТОТСССТАТССТСТСССТTTСТСТССАСТОСАСССАССССТСТ 116 120

Guz bio A1ia I16 5eg Rgo Rtgo Azr Dia DIia betg Div div sttttstosTtToosAS AAS SAA OSS ATS TSS SST SSA SAT bsb bss tsA st ost

MI table (continued) 130 150

Rgo Mem Agu Tyg YII)1e Tnt dia dzr Tyg Re go Guz Se Rpe dgo Mai Tug eh

SSA STS SSd DSA dDTS dST SST SAS AST TT SOS ADA STS TTS SoA bts TAS tes 150 160 dzp RYe (vy Ago b1iu Muz ce tsuz (Se tug tpg biu bim A1v Suz dgud TI biu

DAT tTTS Hundred sob SA AU Hundred ADS STS tTdS ASA bob bab OSS tTbS AS ASA bob 166 dr dgo ОР

САС DSA AND ССАССТОТОТССАССТОООСАТССАССАССТСССССССАААСТОСТСТССССАСА

СССТСССССССССТССТСААСССССССССАСОССОСТССАСССССССССССССОСССССКАЦСОССССССКИЦ

ДОТОССАСААТСАSATSTСАСССОССАСАСSDASTSTССАСАСАССАДSTСТСАСАТСТААССАСТСТСАС with

ASBOSSADSTTSAASSSSSSSaSASSASSLASSATTSASASASSASSST TT AAASTSASSSSASASASSSATOS

TOSODASASSSSTOSDASSTSSASTSSOSSSASSSTOSADAATTTTSATOSSASSASSASSSTTTOSASSOSSATTTAS about

STOTTTTSOSASSTASSSATSASSSASSASSATOASSTOSASAASTTASSTOSSAASSTTSASTTSTSSASS

ТСТСАСССССАССОСАСТОССТСТСТОССССАААСССССССТТОАСАСССССССТСТСССААССАССААСАСС

AHSATHOSOOSTOSSSTSTOOSTSTSATOSOSTSSADSTTTTSTSTATTSTSAASSSTATTSASASASTSAA

ASASVATATSAS Fo

In table MI, the initial continuous DNA sequence denotes the upper strand of 620 s based on the fact that, apparently, it is a non-translated sequence immediately preceding the translated part of the human EPO gene. More specifically, the sequence contains - from the C-END of the gene, which leads to the translated DNA region encoding the first four amino acids! (ie -27 to -24) of the leader sequence ("pre-sequence"). Four pairs based in the sequence preceding the last one encode the beginning of the leader, however, they have not yet been determined unambiguously and therefore designated "X". Then follows an intron of approximately 639 base pairs (of which 439 base pairs have a certain sequence, and the remaining 200 base pairs are designated "1.5.7, " immediately preceding the codon for glutamine, which is designated as residue -23 translated from the c polypeptide. - the sequence encodes amino acid residues through the alanine residue (designated as residue 1-1 of the amino acid sequence of the mature human EPO) to the codon specifying threonine at position 26, followed by the second intron consisting of 256 bases, as specifically indicated. The zxon sequence following this intron for amino acid residues 27 - 55 is continued by the third intron, containing 612 base pairs. c The next zxon encodes residues 56 - 115 of human EPO, then there is a fourth nitron of 134 base pairs, which is continued by the zxon encoding residues 116 - 166 and the "stop" codon (TA). Finally, in the Table MI is identified as a sequence of 568 pairs based on the fact that it constitutes 2) untranslated region 3 of the human EPO gene, two pairs based on which ("X") have not yet been uniquely determined by the sequence.

Me, Table MI serves to identify the primary structural conformation (amino acid 4) sequence) of a matured EPO human, which includes 166 established amino acid residues (with a calculated molecular weight of 18399). The Table also shows the DNA sequence encoding the leader sequence of 27 residues together with the DNA sequence 5 and 3, which may be important for the promoter/operator functions carried out by the gene operon. Areas for potential glycosylation of the human mature EPO polypeptide are marked with asterisks in the Table. It is necessary (Ф, to note that the specific amino acid sequence in Table MI is probably the sequence of allelic forms of human erythropoietin found in nature. The basis for this position was found in the results of continuous attempts to determine the sequences of human erythropoietin in the urine of Mzolyatov, which made it possible to have information about that a significant amount of erythropoietin molecules have methionine at residue 126 as opposed to serine, as shown in the Table.

Table MI, below, illustrates the degree of polypeptide sequence homology between human and monkey EPO. In the upper continuous line of the Tables, letter designations of applications! for 65 represents the deduced translated sequences of human EPO, starting from residue -27, and the lower continuous line shows the deduced polypeptide sequence of monkey EPO,

starting from the number -27, denoting the remainder. Asterisks are used to highlight sequence homology. It should be noted that the deduced sequences of EPO of humans and monkeys reveal an "additional" lysine (K) residue in the proposed 116 (for humans). The reference to Table MI means that this residue is located at the edge of the putative mRNA junction in the genomic sequence. The presence of a lysine residue in a human polypeptide sequence is verified by determining the sequence of a human cDNA clone obtained from mRNA isolated from CO5-1 cells, which was transformed with human genomic DNA in Example 7, below.

UTI table

Comparison of human and monkey ERO polypeptides -20 -10 «1 10 20 zo Go) 79 Человек MOMNESRAMI MI. O.I RIBIRMI.SARRVIISOZAME EM S EAKEAEMIT TOSAYENSZIMEMITMROTK жк ЕКижкикижжж жкжЖжжжже ЖКЖККыжKKкккжКККЕжKKКжккий Кк ка и века кавивых

Monkey MOUNESRAMIMI TSI.5I.M5IRISI RURSARRESISOZAMI EM SEAKEDEMUTMOSZE5SZIMEMITMUROTK 50 6o 70 o 90 100 110

Человек UMEHAMKAMEUSODFAMEMMOST ATGI. ZEAMI KSODI I. UMZ5ORMERIOI NMOKAUBSIAZSTTT A SAKE жк КИККЕККЖКЖЖЧКЖЖЖЭЖЖ ЖЖЖЖЕКЖЖ ЖжЖЖЖЕж ЖЖ жижеж кжавыжжеж Kk

Monkey MUMEUHAMKAMEMSODAMEMUMOVI A ZEAMI ASOAMIAMZ5ORRERI OYI NMOKAI5SI BIT VA SAO-ye 120 130 180 150 160 s

Человек AtТ5РРОДАБААРИНТИТАОТЕНКИАМУ5МРИ АСК КИ UTSEADSATSON жЖжж ККЕКЖЖЕКЖЕКЕКЖЕКА ЖЖЖЖЖКЖЖжЖежЕЖККЖжКжкжKK ЖЖ о

Monkey DIBI RODAZADRI YATITAOTESKI EVMU5MEI VOK KI UT SEAsSAASsOoV

EXAMPLE 6

In the initial attempts of microbiological synthesis of isolated amounts of zytroposin polypeptide material, encoded by monkey cDNA, obtained according to the method of example 3, the selected co-expression system is a system containing mammalian host cells (for example, cells СО5-1, А.Т.С.С. , MoSkKI -1650). The indication of the cell is transfection by means of a "shuttle" and a vector that has the ability to spontaneously replicate in the host E. soya (thanks to the presence of

DNA derivatives from rVK322 plasmids) and mammalian hosts (thanks to the presence of DNA derivatives from 5M40 viruses). WITH

More specifically, the expression vector is built according to the following methods. Plasmid clone 83, 10) obtained in example 3, is amplified in E. soy bacteria and when EsokiI and Nipai are cleaved, approximately 1.4 kb. Monkey DNA, which hydrogenates zrithropostin. From plasmids! pRVK322 separately isolate 4.0 t.p.n. of the Nipaip/zaiI fragment. About 30 base pairs of the "linker" fragment of Esoki/ZaiY are obtained from DNA containing repetitions of MiZtr10 KE DNA (R and G. laboratories). The indicated "linker" includes in sequence the EsoKi-sticky end, followed by the recognition sites of 55iY, Ztai, Vatni, and Khvai, and the sticky end of Zai. - c Indications of more than three fragments are stitched together to obtain approximately 5.4 t.p.n. intermediate plasmid ("REKZ"), and the EPO-DNA of which borders on one side when using "banks of useful restriction sites"» of recognition of probe nucleases. After that, the REKZ plasmid is cleaved by NipampySh and Ba with the production of erythropoietin DNA and the transformation of Esoki into the "linker" ZaiI (MIZtr10). The indicated above DNA fragment with a length of 1.4 kb is cross-linked with the VatNi/Za site! approximately 4.0 t.p.n. plasmid rVK322 and the second 1 "linker" of the M1Ztr10 Nipa!Pp/Watni KE fragment, which also has approximately 30 base pairs. Fragment 1» of the M13 "linker" is a NipaP-sticky end, followed by the recognition sites of Rzyi, Zai!, Hbai and the sticky end of VatNi. The cross-linking product again becomes a useful intermediate plasmid ("RVK-ERO"), (95) containing ERO-DNA, which is bounded on both sides by "stacked rows" of the restriction site. b 50 The vector selected for the expression of EPO-DNA in CO5-1 cells ("PrOBMI 1") is constructed in advance for the possibility of selection and autoduplication in E. soya. The indicated characteristics are achieved due to the initiation of this replication and the sequence of DNA genes resistant to the action of ampicillin, which are present in the region carrying out the contraction of nucleotides 2448 to 4362 of the rVK322 plasmid. The specified sequence is structurally modified by introducing a "linker" that ensures recognition

It should be done immediately after adding nucleotide 2448 to the indicated vector. Along with the selection of vectors for expressing EPO-DNA, other useful properties include the ability to self-replicate in CO5-1 cells and the presence of a sequence acting as a viral promoter in mammalian cells. Name) These characteristics are achieved by starting the sequence of DNA replication and the sequence of the "late" DNA gene, which is a viral enhancer, which is present in a chain containing 342 base pairs, reducing the number of nucleotides from 5171 to 270 of the 5M40 genome. A unique restriction site (Watni) is formed on the vector and is immediately adjacent to the viral promoter due to the use of a commercially available "linker" sequence (SoParogaiime Kezeagsp).

In addition, a sequence of 237 base pairs (derived from the nucleotide number from 2553 to 2770 of the ZM40 virus) containing the polyadenylation signal of the "late gene" of the viral meNK 65 (commonly known as the transcription terminator) is introduced into the vector. The specified fragment is placed in the vector in the appropriate orientation relative to the viral promoter of the "late gene" through a unique site

cotton wool In the specified vector, the second mammalian gene is also represented, which is in a position that is not material relative to the possible transcription of the gene, which is placed on the specified unique site between the sequences of the viral promoter and terminator. (The instruction contained a mammalian gene

It has approximately 2,500 pairs of basic dihydrofolate reductase! (ONEK)-muscle minigene, isolated from plasmid pMoO-1, (zazzeg, ei a. R.M.A.5. (0.5.A.), 79, pp. 6522 - 6526, (1982)) WE again the majority active components of the plasmid! ROZMI 1 consists of 2448 - 4362 nucleotide plasmids! rVK322 along with nucleotides 5171 - 270 (342 base pairs) and 2553 - 2770 (237 base pairs) of viral DNA containing the 5M40 virus. 70 In accordance with the methods described earlier, for example, Mapiaiyov, et al., etc., the erythropoietin-encoding DNA is isolated from the RVC-ERO plasmid as a Watni fragment and inserted into the ROZMI 1 plasmid cut by Watni. To confirm the introduction of the EPO gene in the correct orientation in two of the obtained cloning vectors (duplicated vectors! H and I) an analysis is carried out using a restriction enzyme (see Fig. 2, illustrating the plasmid ROSMI 1-MKE. Vectors with an incorrect /5 EPO orientation -genes are left as regulators of negative values in transfection experiments designed to determine the levels of EPO expression in hosts, which are transformed using vectors with the correct orientation of EPO-DNA.

Vector H, I, E, X and b are combined with a DNA carrier (DNA of liver and spleen of muscle). To implement the transfection, double-bomm plates are used, using the method! calcium phosphate microprecipitation. Dullikatny bOhm plates are also transfected with a DNA carrier as an "imitation" of controlling the transformation of negative values. After 5 days, the entire culture medium is tested for the presence of polypeptides, which have the immunological properties of zritropoztin of natural origin.

EXAMPLE 7 p

A. System of initial EPO expression using СО5-1 cells

Selected at the initial stage of the microbiological synthesis of secretable amounts of i) human erythropoietin polypeptide material encoded by the EPO clone of human genomic DNA, the system also includes expression in mammalian host cells (for example, cells

So5-1, A.T.S.S. MoSKI-1650). The human ERO gene is initially subcloned into the "Shuttle" vector, which has the ability to autonomously replicate in both E. soy bacteria hosts! (thanks

DNA derivative from plasmids! rVK322) and into the cell of mammalian CO5-1 (thanks to the presence of the ISE virus)

ZM-40 virus-derived DNA). The shuttle vector containing the EPO gene is then transfected into cells with

Co5-1. EPO-polypeptide material is formed in transfected cells and secreted into the cell culture medium. -

More specifically, the feature expression vector is built according to the following techniques. DNA isolated from the LLE clone! sheep, containing the genomic ERO gene, are cleaved by Watni and Nipai restriction endonucleases and isolate 5.6 kb. DNA fragment, as found, containing complete

ERO gene. The specified DNA fragment is mixed and ligated with the bacterial plasmid risv8 (VeyPezaa

Kezeagsi and Arogayogiev, Ips.), which is cleaved in a similar way, creating an intermediate plasmid « 40. "pOS8-Nie", thus forming a suitable source of the restriction fragment. Vibrant vectors for EPO-DNA expression in CO5-1 cells are constructed in advance. Plasmid remM4zEi, which also contains DNA sequences, enables selection and autoduplication in EE. soybeans The specified "" characteristics are achieved by the onset of replication and the sequence of the DNA gene resistant to ampicillin, which is present in the region spanning nucleotides from 2448 to 4362 of the bacterial plasmid rVK322. The structure of the specified sequence is changed by introducing a linker, providing the following Nipai recognition site! adjacent to nucleotide 2448. Plasmid romM45Ei is also capable of autoduplication in cells CO5-1.

He) nucleotides from 5171 to 270). The specified fragment is modified with the introduction of a linker that provides a 5p EsoKI recognition site adjacent to nucleotide 270 and a linker providing a site

The ZAI recognition site adjacent to nucleotide 5171. This vector also contains a 1061-seq fragment based on the ZM40 virus (number of nucleotides from 1711 to 2772 with an additional linker that provides a ZAI recognition site adjacent to the next nucleotide 2772). Within the specified fragment, there is a unique sequence of recognition of Vatna. Summarizing the above, the remMAZEI plasmid contains unique Watni and Nipai recognition sites, making it possible to introduce the human EPO gene. Specifying the sequence makes it possible to implement replication and iF) selective selection in E. soy and replication in cells СО5-1. For the introduction of the ERO gene into the pOM45Eii plasmid, the pOS8-NiE plasmid was cleaved by Vatni and Nipai by restriction endonuclease and 5.6 kb were isolated. EPO, coding DNA fragment. Plasmid raem4ZzEi is also cleaved by Watni and Nipai, and the main part of the fragment, containing 2513 base pairs, is isolated (while preserving all necessary functions). The specified fragments are mixed and stitched, forming the final vector "RZMONYERO" (see Fig. 3). The Ztot vector is bred in E. soya, and the DNA vector is isolated. To confirm the introduction of the EPO gene, a restriction enzyme analysis is carried out.

Plasmid DNA ReManyEro is used for the expression of signs of ERO polypeptide material in 65 K cells CO5-1. Explaining in detail, RZUMONIERO plasmid DNA is combined with a DNA carrier and transfected into triplicate bOM plates of CO5-1 cells. As a DNA control, the carrier alone is transfected in CO5-1 cells. The cell culture medium is selected after 5 and 7 days and tested for the presence of polypeptides that have immunological properties characteristic of naturally occurring human EPO.

B. The second system of expression of EPO signs in CO5-1 cells.

Another system of expression is used to obtain improved products of human erythropoietin-polypeptide material, coded with the help of a clone of human genomic DNA, forming

EPO in cells СО5-1 (А.Т.С.С. MOSTI -1650).

In the previous system, the signs of EPO are expressed in CO5-1 cells with the use of its own 7/0 promoter, which is present within the restriction fragment of Watni relative to

NipaiSh, having a length of 5.6 t.pl. In the following, the constructed EPO gene alternates with obtaining the expression of signs using the late promoter of the 5M40 virus.

More specifically, the restriction fragment of human genomic EPO, representing the cloned 5.6 kb. Cotton wool is modified according to the following technique. As described above, the pOS8-NiE plasmid is cleaved by Watni and the restriction endonuclease Vz5iEII. The restriction endonuclease VVIEII cleaves within 5.6 kb. The EPO gene is located in a position that represents 44 base pairs of 5 to the initiating ATO, encoding the precursor peptide and approximately 680 base pairs of 3 to the Nipai restriction site. Then a fragment containing approximately 4900 base pairs is isolated.

A DNA fragment with a synthetic linker, containing ZaiYi and Vz8eiEiY sticky ends and the internal region of the 2o recognition of Cotton, is subjected to synthesis and purification. The indicated two fragments are mixed and cross-linked with the rVKZ322 plasmid, which is cut with the help of Za and VatNi to obtain an intermediate plasmid!

RVEKONE. The human EPO genomic gene can be isolated from it in the form of a Watni cleavage fragment with 4,900 base pairs, carrying the complete structure of the gene with a single ATO of 44 base pairs and 3 bases relative to the site

Cotton wool, adjacent to the region of the end of amino group coding. with

The resulting fragment is isolated and introduced as a Watni fragment into the plasmid ROZMI 1 of the cleaved Watni zxpression vector (see example 6). The resulting plasmid rom 90NyERO, as shown in i) fig. 4, is used for the expression of features of the EPO polypeptide material from cells CO5-1, as described in examples b and 7A.

EXAMPLE 8 so z The culture medium grown from 6 transfected cultures of CO5-1 cells of example 6 is analyzed using a radioimmunological analysis according to the method described in example 2, part B. Each x sample is analyzed at the values of an aliquot sample! 250, 125, 50 and 25 μl. Supernatant layers from the growth of cells, "simulated" transfected or transfected with the help of vectors with incorrect orientation of EPO genes, had negative values for immunoreactivity. For each sample of two supernatant liquid layers obtained from the growth of SbOo5-1 cells, transfected with vectors (H and I) with the correct orientation of EPO DNA, 95 inhibition of 729I-EPO bound to the antibody ranged from 72 to 8895, all the values of which are placed in the upper position of the standard curve. Therefore, it is impossible to accurately calculate the true concentration of zritropoztin in the floating layers of the culture. According to preliminary calculations, it was 300 tI/ml, however, it exceeded the values of the largest aliquot sample (250 µl). - c A sample of the culture fluid obtained according to example b, and 5- and 7-day culture fluid, described in example 7A, were subjected to radioimmunological analysis to compare the activity of "" recombinant monkey and human ERO materials relative to the naturally occurring human ERO, which meets the standard The results are presented graphically in Fig. 1. In short, the presentation of the results unexpectedly showed that the recombinant monkey EPO is significantly competitive for the human anti-EO antibody, although it was not possible to completely inhibit the binding under the test conditions. The maximum 90 values of inhibition for the recombinant in human erythropoietin, however, it almost approaches the standard indicators. The parallel nature of the dose-effect curve suggests the immunological identity of the sequences (prototypes) in general. Before performing calculations of monkey EPO concentrations in culture fluids, they should be carried out! repeat It was estimated at the indicated high values of dilution and it was found that they fluctuated in the interval from 2.91 to 3.12 O/ml. Quantitative levels of human ERO production are set respectively at 392 tSH/ml for a sample with 5-day growth and 567 tSH/ml for a sample with 7-day growth. Calculation of levels of ERO production of monkeys in the example of 7V systems! expressions were similar or better.

EXAMPLE 9

F, Culture fluids, obtained according to examples b and 7, were subjected to the analysis of ip mygo to determine the activity of EPO in accordance with the method of Soifmazzeg, ei aI., Epdoshypoiodu, 97, 2, pp. 315 - 323 (1975). Numerous monkey EPO values for the examined culture liquids ranged from 3.2 to 4.3 O/ml. Culture fluids of human EPO also showed activity in the obtained calculations during the analysis of ip mygo and, in addition, the indicated activity can be neutralized under the action of the "anti-EPO" antibody. Recombinant monkey ERO culture liquid! according to example 6, it was also subjected to quantitative determination of biological activity according to the general method of Soyez, et al., Mayige, 191, pp. 1065 - 1067 (1961), and Nattopa, et al., App. M.U. Aside. zZsi., 149, pp. 516 - 527 (1968). Indication of 65 levels of activity ranged from 0.94 to 1.24 U/ml.

EXAMPLE 10

In the previous examples, recombinant human or monkey EPO material is obtained from vectors used to transfect CO5-1 cells. The indication of the vector is carried out by replication in CO5-1 cells due to the presence of the T-antigen of the 5ZM40 virus inside the cell of the origin

Duplication of ZM40 on specified vectors. Although the result is a vector! they reproduce the useful qualities of EPO in CO5-1 cells, their variability is only temporary (within 7-14 days) due to possible losses of the vector. In addition, only an insignificant 956 reproduced CO5-1 becomes productively transfected with the indicated vectors. A real example describes the system! expression of traits using Chinese hamster ovary (CHO) UONEK cells and a selectable marker, ONEK. For a discussion of related expression systems, see US Patent No. 4399216 and European Patent Application No. 117058, 117059, and 117060, published August 29, 1984).

In the cells of SNO ONEK (Bykh-VI) SNO Kt, Opatsyb, ei aiYi.,, Rgos. May Asai. All together. (О0О.5.А.), Moi. 77, 4461 (1980), the enzyme dihydrofolate reductase is missing! (ONEK) due to mutations occurring in structural genes, and, therefore, the presence of glycine, hypoxanthine and thymidine in the culture medium is necessary. Plasmid! rOBMI-MKE (Example 6) or rOBMI-NOERO (Example 7B) are transfected together with a DNA carrier into ONEK:" CHO cells, which are grown in a medium containing hypoxanthine, thymidine, and glycine in BOM culture plates. Plasmid RMONiERO (Example 7A) mixed with the pMo2 plasmid, which contains the muscle gene based on dihydrofolate reductase!, cloned into the vector of the bacterial plasmid! rVK322 (according to Savgzeg, et al., above).

A mixture of plasmids and carrier DNA is transfected into ONEK" CHO cells. (Cells that take up one plasmid will usually also take up the second plasmid). After 3 days, the resulting cells are dispersed by trypsinization on several 100 mm culture plates in a medium that lacks hypoxanthine and thymidine. Only those cells that are transformed into a stable state by means of the ONEK gene show survival in this environment of the EPO gene. Colonies of living cells become visible after 7-21 days.

Indication of a colony of transformants after dispersion by trypsinization can continuously reproduce in an environment with hypoxanthine and thymidine deficiency, creating a new strain! cells about (for example, SNO rOBMI-MKERO, SNO reManieERO, SNO rOBMI -9NyERO).

Culture fluids obtained from the above-mentioned cell strains are subjected to radioimmunological analysis for the presence of recombinant monkey or human EPO. The medium for CHO strains roOzMI-MKERO contains EPO with the same immunological properties as EPO obtained from CO5-1 cells transfected with the plasmid rOzMI-MKERO. A sample of the culture fluid "with a 65-hour incubation" contains 0.60 U/ml monkey EPO.

Culture fluids obtained from CHO r5MONIERO and CHO rorami-YaniERO contain recombinant human EPO, which has the same immunological properties as EPO, obtained from CO5-1 cells transfected with the plasmid reMaNiERO or rOBMI-9NiERO. A sample of culture fluid with a 3-day extract obtained from SHO p5MOoNyERO contains 2.99 /ml human ERO M, and a sample with a 5.5-day extract obtained from SHO rOBMI -YAONIE RO contains 18.2 U/ml ERO of a person, as shown by the results of radioimmunological analysis.

The quality of EPO, obtained by means of the above-mentioned cell strains, can be increased due to "amplification of genes, creating a new strain!" cells with greater efficiency. Dihydrofolate reductase enzyme! (ONEK), which is a product encoded by the ONEK gene, can be inhibited by methotrexate (MTX). In particular, cells proliferating in hypoxanthine and thymidine deficient media are inhibited or killed by methotrexate. Under appropriate conditions (for example, at a minimum concentration of MTX), it is possible to obtain cells that are stable and capable of growing in

MTX. It was found that these cells become resistant to MTX due to the strengthening of a number of their ONEK genes, resulting in an increase in the production of the ONEK enzyme. Living cells can, in turn, be treated with increased concentrations of methotrexate, as a result of which the number of UONEK genes increases in cell strains. "A transient gene (for example, EPO), transferred to an expression vector together with the ONEK gene or transformed with it, as is often found, increases the number of reproduced ones. 20 increasing its concentrations (ONM, ZONM and LOOnNM). Samples, c" representing the bb-time culture medium at each stage of amplification, according to radioimmunological analysis, contain 0.60, 2.45 and 6.10 U/ml of zrithropostin, respectively. Strain CHO roOBMI-d9NiERO cells are treated with methotrexate (MIH) with a serial increase in its concentrations: ZONM, 5OnNM, 10OnNM, 20OnNM, 1mM and 5mM. A sample representing a 3-day supernatant

Gee! liquid, after treatment with methotrexate (MIH) with a concentration of 00nM contains, according to radioimmunological analysis, 3089 and 129 IU/ml of human erythropoietin. Samples representing a 48-hour culture medium treated with methotrexate with a concentration of TO0OHM and 1 μM contain, according to radioimmunological analysis, 46b and 1352 OM/ml of human osritroposthin, respectively 60 (the average number obtained as a result of 3-time testing of samples). According to the specified method, 1 x 109 cells are sown in a Petri dish containing 5 ml of medium. After 24 hours, the medium is removed, replacing it with bBml of fresh serum-free medium (OMEM with a high glucose content, to which 0.1 nM of non-essential amino acids and I-glutamine are additionally added. Zritropostine is allowed to accumulate for 48 hours in the medium , which does not contain serum. Then the medium is collected for testing by means of a radioimmunoassay, and the cells are trypsinized and then counted. The average values of the samples as a result of the radioimmunoassay are about 467 and 1352 IU/ml of erythropoietin for cells grown when treated with methotrexate with a concentration of TO0NM and μm , respectively, provide the actual yield of zritropoztin of the order of 2335 and 6750 u per cup.

The number of cells per cup is 1.94 x 106 and 3.12 x 109 cells, respectively. Thus, the rate of effective production in the specified cultivation conditions has values of 1264 and 2167 010. 5 cells in 48 hours.

Description of cell cultures! represents a genetically heterogeneous population. In order to isolate genetically heterogeneous clones with the highest production capacity, 70 M standard selection methods are used (see section A, part 2 "Roipiv Yu Sopvideg ip (pe SVagasiegigayop oi

Seyi Hipez Ovey (about Rhodis Vioiodiev), June 1, 1984, Department of Scientific Testing of Biopreparations, Center for Medicines and Biopreparations, US Food and Drug Administration.

The above-described productivity of CHO cell lines producing zritropoztin can be improved by using appropriate methods of cell cultivation. Reproduction of mammalian cells 75 Animals in the culture medium generally requires the presence of serum in the growth medium. The method of obtaining erythropoietin from CHO cells in a serum-free medium greatly facilitates the rectification of erythropoietin from the culture medium. The method described below makes it possible to economically obtain zritropoztin in large quantities in a serum-free environment, sufficient for production.

Cells of lyminy SNO rOBMI-YA9NyERO, grown in standard conditions of cell cultivation, for 2nd seeding use vials with revivable cell culture. Cells are propagated in the form of a suspension cell line in flasks containing a cultured cell culture in a medium consisting of a 50:50 mixture of high-glucose UOMEM and E12 Nat, to which 595 of fetal bovine serum (BS5), I-glutamine is additionally added , penicillin and streptomycin, 0.005nM of non-essential amino acids and methotrexate with the appropriate concentration. Cell cultures in the form of a suspension provide an opportunity for rapid reproduction to large volumes of zritropoztin, emerging from CHO cells. CHO cells, grown in suspension, are used for seeding in rotating vessels! and 9) at the initial seeding density of 1.5 x 10" viable cells per 850 cm? of the indicated vessel containing 200 ml of medium. During the 3-day period, the indicated cells are given the opportunity to grow until the formation of a monolayer of a relatively densely adjacent line of cells. Used in this phase of growth H) cell medium is similar to the medium used for cell cultivation in suspension. By the end of the 3-day cell growth period, the serum-containing medium is separated and replaced with 100 ml of serum-free medium. The serum-free medium contains a 50:50 mixture of OMEM with a high content of glucose and E12 Nat, in Geo) which was additionally introduced with 0.05 mM of non-essential amino acids and I-glutamine. The rotating vessel with the prepared medium is placed in the incubator for 2 hours and then it is removed again, replacing it with 100 ml of fresh, serum-free medium !. At 1 - With temporary incubation in a serum-free medium, a decrease in M) concentration of contamination of proteins with whey occurs. Then rotating these vessels are again placed in an incubator for 7 days, during which accumulation of zritropoztin occurs in a serum-free culture medium.

By the end of the 7-day phase of zritropoztin production, the old medium is removed, replacing it with a fresh medium that does not contain serum, which is necessary for the second cycle of production. As an example of the practical implementation of the specified system! of production, a sample that has been in a serum-free environment for 7 days is presented, which, according to radioimmunological analysis, contains 3,892 and 409 h» Sh/ml of zritropoztin. Taking into account this calculation of the cell density of the order of 0.9 - 1.8 x 109 cells per cm, each rotating vessel with a volume of 850 cm 2 contains from 0.75 to 1.5 x 108 cells, and based on this, thus, the rate of production of zritropoztin within 7 days in 100 ml of culture! reaches values in the range from 750 to 1470 Ш0/1059 cells in 48 hours. i. The supernatant liquid from the cell line ХО розми-МКЕРО amplified in methotrexate with "" concentration of 1 ОНМ, is subjected to radioimmunological of the analysis for testing the activity of erythropoietin ip migo and ip mimo. The sample, prepared in a concentrated medium during radioimmunological analysis, contains 41.2 1.44 IU/ml; 41.2 - 0.064 U/ml MkK-zrythropoietin, as determined by the analysis data on

Ge») 20 biological activity of ip mio, and 42.5 u- 5 O/ml according to the analysis of determination of biological activity T/l mimo. With a certain sequence of amino acid residues of polypeptide products, the presence of zritropoztin products is detected, and the dominant type includes the residue of the "leading" sequence, adjacent to the supposed termination of amino groups with alanine. At present, it is not clear whether this is the result of improper processing of the polypeptide core in 25 CHO cells, or whether it reflects a difference in the structure of the terminal amino groups of monkey erythropoietin

HF) relative to human erythropoietin. kyu The supernatant liquid prepared from the cell line ХНО зМИ-9НиЕРО is subjected to three tests. According to radioimmunological analysis, a 5.5-day sample contains about 18.2 CHO/ml of recombinant human zytroposin in the indicated environment; in the analysis of ip miigo 15.8 - 4.6 O/ml and in the analysis of 60 ip mimo 16.8 and 3.0 O/ml.

The supernatant liquid, prepared from the cell line ХО роБзМИ-ЯОНиЕРО with gradual amplification of methotrexate with a concentration of 100nM, is subjected to three tests. According to radioimmunological analysis, the 3rd-day sample contains 3089 and 129 CPM/ml of recombinant human zrythropostin; when the tested sample was 2589, it was 71.5 O/ml and when the tested sample was 2040, it was 160 Х/ml. The sequence of amino acids shows the termination of amino groups corresponding to that presented in table MI.

The cellular conditioned medium prepared from CHO cells, which is transfected with the pROSMI-MKE plasmid in methotrexate with a concentration of 1OnM, is combined, and the methotrexate is dialyzed in

The course of several days, as a result of which a culture is formed with the activity of zritropoztin on the order of 221 and 5.1 U/ml (zritropoztin-cellular conditioned medium) (ERO-SCM). To determine the action

ERO-SSM on the level of hematocrit in the Vair/C line of normal muscle and pass the following experiment.

Cell conditioned medium prepared from untransfected CHO cells (CCM) and

ЕРО-ССМ, regulated using phosphorus-buffered solution (РВ5). Cell-conditioned 7/0 medium is used on the control group of animals (Z mice)), and for experimental groups (2 mice per group) 2 dosage levels of ERO-SCM are used: 4 units per injection and 44 units per injection. During the 5-week course, 7 mice are given intraperitoneal injections three times a week. After the 8th injection, the average hematocrit values in the control group were found to be 50.495, for the group with 4 units of ERO-SCM they were 55.1905 and with 44 units of ERO-SCM - 67.9905.

The products of the expression of mammalian cells can be easily extracted in a fairly purified form from the culture medium using OLC (C/) with an ozonol gradient, preferably at a pH value of -7.

A preliminary test was carried out to determine the properties of recombinant glycoprotein products obtained from the conditioned medium of the expression of CO5-1 cells and CHO of the erythropoietin gene from human urine, using both UV-Viegpro (staining) and ZO5-RACE analysis.

Indications of research show that the erythropoietin material produced by CHO cells has a slightly higher molecular weight than the products of the expression of CO5-1 cells, which, in turn, is slightly larger than the human urine extract of the pool source. All products are heterogeneous to some extent. Neuraminidase enzyme treatment! to remove sialic acids! leads to the fact that, as a result, recombinant products of СО5-1 and ХО cells have approximately the same molecular weight, which, however, both one and the other, is greater than the molecular weight of the resulting urinary extract of i) a person containing asialog groups. Processing with the enzyme E-zndoglucosidase! (EC 3.2.1) of the recombinant product of CHO cells and the product of urine extract (for the complete removal of carbohydrates from both products) results in the production of almost homogeneous products, which, in principle, have the same (in zo) molecular weight.

Purified erythropoietin obtained from human urine and recombinant erythropoietin produced from cells of CHO, ix, erythropoietin according to the present invention are subjected to analysis to determine the carbohydrate composition according to / (In the method described by ideep, ei aiY., Meiodz ip Eplutoiodu, 83 (Rai 0, 139 - 191 (1982), which is modified by using the hydrolysis method described by Mezzeg, ei ai., Apa)i. Viospet., 142, 58 - 67 "(1984). Determined experimentally the indicators of the carbohydrate composition for the urinary isolate in the form of molar ratios in the indicated products) have the following values: hexose, 1.73;

M-acetylglucosamine, 1; M-acetylneuraminic acid, 0.93; fucose, 0; and M-acetylgalactosamine, 0.

Corresponding values for recombinant products (derived from cell line ХО роЗМИ -Я-УНОЕ РО

3-day culture medium), reinforced with methotrexate at a concentration of 1ООНМ) has the following "indicators: hexose, 15.09; M-acetylglucosamine, 1; M-acetylneuraminic acid, 0.998; fucose, 0; and with M-acetylgalactosamine, 0.

Getting results! coincide with the results of the described several analyzes of UUeviegp Bioi and 505-RACE. ;" Glycoprotein products obtained according to the present invention, thus, are achievable products, the primary structural conformation of which is sufficiently

Reproduce the conformation of zritropoztins of natural origin to give them one or more biological properties, and the quantitative composition of carbohydrates, which differ from the composition of zritropoztins of natural origin. t- EXAMPLE 11 oo The proposed example relates to the entire cycle of production of erythropoietins through the assembly of two structural genes encoding the sequence of human erythropoietin based on nucleotides,

Me. presented in table MI and containing, respectively, "preferred" codons for the expression of 4) characters in E. soybean and yeast cells (5. segemiviae).

In the specified example, the construction of genes encoding analogues of zrithropostin is additionally described.

In short, the protocol used, in general, representations! in the above described Ap, ei ai. (M/o ov 83/04053). Instructions of the genius of construction! for the initial assembly of the constituent oligonucleotides in a complex duplex, which, in turn, assembles! into three discrete parts. Indication of part of the construction! for (F, amplification and when removed from the amplification system, they can be assembled sequentially or through the ligation of a fragment with a complex structure into a suitable vector of expression.

The MIP - KHIM tables show the scheme and assembly of the resulting gene encoding the translation product of human Zrytropostina, which lacks any leading or preceding sequence, but contains the original methionine residue at the position -1. Moreover, the gene introduced into the main part of the E. soybean cell gives preference to codons and, thus, the specified construction is called the "ESERO" gene. b5

MIA table

Oligonucleotide part 1 ESERO 1. DAATTSTASADASSATSASSSTAATADAATA 2. СSATATTTTTTATTASSSTSATOSTTTSTAS z. АТобСТОСССССОТsStToАТСССАС 4. СТССАСТСССАСАСАССССССССАССАС 70 5. ТССАСАСТСТССААССТСТАССТСТСТС 6. СТТССАССАССТААССТТССАСАСААСТ 7. САДССТАСААССТСААААСАСТ c. STOSTSATSTTTTSASSTTSTTTTAS 9. ASSASTOSTTTSTOSTSAAAASTSTTTS 10. SADASAAAASTST TSASSASAASSA 11. TTTSAAAAAAASATASSSSTASS 12. SATSSSSSTASSSTAATSTTTTTSSTT

Table IX

Part 1 ESERO

Hbai

EsSoVI 1 3 s 285 AATTSTAS AAASSATSAS SSTAATADAA TADTObSTSS bSSsssTSTo

САТС ТТТССТАСТС ССАТТАТТТТ АТТАСОСАЕС ССССССАСАС Go) 2 4 2

АТСТСССАСИ ССАСАСТТСТ ССААССТТАС STOSTOVAАС СТААСААСС

TASASSSTS SAABA SSTTSSAATS SASSASSTTTO SATTTSTTSS so 6 7 9 and so tTeAAAAasAtTs DSSASTOSTT STOSTOAASA STSTTSITTO AASSAYDASA

ASTTTTTSTAS SVSSAA SASSASTTTST SASAAS ttesttttet 8 10 so

KrpI Watni ttasostASs HUD z

AATSSSAToS SSTAS 12 I c) - p. and 1 s" (95)

Ge») s» ko 60 65

Table X

Oligonucleotide part 2 ESERO

And and. ДАТТСССТАССАСАССААССТ 2. СТТААССТТОТСТОоСТАСССС 70 z. TAASTTSTASSSTTSSADASSTAT 4. TTSSATASSTTTSSAASSSSTASAD 25. СSAASTTSSTSAASADSSSASTTTSAAST 6. SSLDASTTSSAASTOSTTSTTSASSAdS 7. TTVBSASSSTSTOSSASTOSTSASSsSSO 8. СССТСОСТСАССАССАСССССТ 9. ABOSTOTASSTOSSTOBSSSСССССА 10. СССАСТОССТССССАСССТССТССАССА 1. ТТСССАСССТСАССАСТТТАССА o 15. СОСДАССОСТОСАСТОСАТОТТОАС 14. ССТИТТСААСАТоСАССТОСАССОС so 7 15, ДАДССАСТСТОБССТСАСАТСТО ish 16. САТССАСАСТСАСВСССАСАСТАСТ to her

And that's all

YAKE: piskeme oiemeh peseem posle zlteetsyne zmosnettoh etrteeko Tetetenee Testenosi them 5 in 7 i Veres vie STBdesdeeh Esh stseseAvee

I will die ME t 16 1 g" (95)

FO 50 be» o name) 60 b5

Iablitsa Khiya

Part 3 ESERO and. САТССАСАТСТСТСАСТАСТОТоС 2. АСОСАССАСАСТАСТСАСАСАТСТо 170 5. ТОССТОСТСТОССТОоСАСАСАДАСАСС 4. САТАСССТСТТТСТОТОСАСССАСАДОС 5. СТАТСТСТССОССССАТОСТОСАТСТ 6. САССАСАТССАССАТССООСССАСА 7. ССТОСАССОСТОССТАССАТСАСТо 8, ДТСАССАСТОАТОСТАСОСАССаСсТ о 7 в, СТСАТАССТТССССАААСТСТТТСС 10. АТАСАССАДАСАСТТТОСССААССТ 11. ТСТАТАСТСТААСТТССТСССТОСТА се 25 12. САСТТТАССАСОСАССААДСТТАСАСТ о 15. ДАСТСАААСТСТАТАСТОСССАдоС 18. СССАТОСТТССССАСТАТАСАСТТТ о (Се) 15. ATOSSSTASTOSTSASSSSSTAATAS so 16. TSBASTATTASSSSTSASSASTAS «t

HITI table

Part 3 ESERO Fr

BA tssAVAtststo

STSTASASAS « to ASTASTSTOS Kesstestst estesass AAASASSETA teistssess ne s TSATVASASe VASA sesasotote tttstes sASSses g» y y I t T At t

SStASSASS nn voAsosAt wat sya k-reY TeSAAss 5 E y AAASTSTT TSSESTATAS tstaisttss tesetost ASTEAAST i» STT TAS ASSKEATDT MATTEANo AESSASSAI Kit (95) 15 zaii

TATASTSSSS AASSSDTSSSSS TASTSSTSAS SSSTAATAS o 50 AtTATSDASSbS teseg let ATSASSASSTo SSSATTATS dost 1 16 syu" (F. ko bo b5

KhIU table

Ten ESERO 4 and 9 Khba1 meed1a

BECOMES AAASSATSAS SSTAATAAAAA TAAToSSTosS sssssststs

TTTObTASTS SSATTATTTT ATTASSSASS СОбССССАС

ATSTOSSAST SSASASTTST SSAAASSTTAS STSSTSSAAS STAAASADSS

ТАСАССТОА ССТСТСДАСА ССТСССАATS САССАССТТС САТТСТТСС

TbAAAAATS ASSASTOSTTT STOSTSAASA STSTTSTTTS AASSAAAAASA

ASTTTTTSTAS TSSTSASSAD SASSASTTTST SASAASAAAAS TTOSTTTTST

ТТАССТАСС АСАСАССААС STTAASTTTST АСССТТССАА АССТССАА

DATOSSATSS TSTSTOSTTS SAATTSDASA TOSSADSSTT TOSATASSTT

79

SAASSASTTS TTSSTSAAST TSAAASSSSTS SSASSASSSTO ASSASTSSST

СССТСТСТС ССТОССССССССТССТССТССТ АААСТСССТST САбССОТобо

СССАСАТСАС ССАССССТССЭ СТСАССАССА TTTСАССАСА СТССССАСССС

20

ТТОбСбАССТ ССАССТАСАА СТСТТССТС АТАСАССССА STSTASASAS

ASTASTSTOS ToSSTOSTST bOSTSSASAS ADAASASOSTA TSTSToSSSS

TSATSASSASS ASSSASSASSA SSSASSSTSTS TTTSTOSSAT ASASASSSSS s 25 bbАТОСТССА TSTOSTOSАС СССТОССТАС САТСАСТОСТ SATASSTTСС

SSASSASSST ASASSASSTO SSASSASSATS STATSASSASSA STATSSAASS

SSADASTTT TSSTETATAS TSTAASTTSS TSSSTSSTAA ASTSAAASTS

ССТТТСААА АССАСАТАС АСАТСААСС АСССАССАТ ТСАСТТТСАС

5aiy (ze) 30 LATASTOSSS AASSATSSSSE TASTOSTSSA COMPOSE

ATATSASSSSS TTSSTASSSS ATSASSASSTo SSSATTATSA SST Ge)

In more detail, Table MI illustrates the use of oligonucleotides for the generation of Part 1 of the so gene

ESERO, encoding the amino terminal residues of a human polypeptide. Oligonucleotides are assembled into a duplex (1 and 2, C and 4, etc.), which are then cross-linked to obtain Part 1 of ESERO, as in Table IX. It should be noted that the assembled part includes the corresponding sticky ends of the final EsoKiI and Vatni, and "along the flow" of the sticky end of EsoK! The recognition site of the restriction enzyme of Khva is directed, and the recognition site of Krpi is directed "upstream" of the sticky end of Watna. Part 1 can be easily amplified using the M13 phage vector used to check the sequence of this part.

Some difficulties unexpectedly arise when extracting a part as a Khrai/Krpi fragment from DNA KE, "du generated in E. soybean, probably due to methylation of the base recognition site of Krpi within the limits of the host. Therefore, the phage DNA is isolated and reproduced in a double-stranded form of migo by stretching with a primer, and then the required double-stranded fragment is isolated. :z» Parts 2 and 3 of the ESERO gene (Tables; XI and XIII) are constructed in a similar way from oligonucleotides of Tables X and XIII, respectively. Each part is amplified on the M13 vector used to check the sequence, and isolated from phage DNA. As can be seen from Table XI, Part 2 of ESERO is built with the sticky ends of Esoki and Vatni and can be separated as a fragment of Krpi/Vaoi. In the same way, part of ESERO is obtained with sticky ends of VatNi and Za and can be isolated from KE DNA as a fragment of VoaiPzaiY. t. The three parts obtained in this way can be easily assembled into a continuous DNA sequence with (Table XIM), which coordinates the polypeptide of the complete human EPO with the methionine codon of the amino terminus (ATO) for the translational initiation of E. soya. It should be noted that "against the flow" of the original ATO (o) a series of base pairs is directed, sufficiently duplicating the sequence of the ribosome binding site! c" of the highly expressed OMR-Y gene of E. soybean.

Any suitable expression vector can be used to transfer ESERO. The specific vector selected for the expression of the ESERO gene as a "temperature-sensitive" plasmid pSEM536 is a derivative of the plasmid pSEMA14 (ATCS 40076), as described in the concurrently considered US patent application Mob36727, filed in August 1984, Spapez ER. Moitis. More specifically, RSEM536

GF) split Hbvai and Nipan; a large fragment is isolated and used in a double-sided spliced with the 7 ESERO gene. Parts 1 (Khvai/KrpiI), 2 (Krpi/Vai) and Z (VopP/Za!I) are pre-assembled in the correct order in

M13 and isolate the EPO gene from there as a single fragment of Hvai/Nipa!i. This fragment includes part of the polylinker from the M13 trophage, which tightens the sections of the and Nipa. Control over expression in the resulting expression plasmid, ro5Zb, is carried out with the help of the lambda P | promoter, which itself can be controlled by the C-vb57 repressor gene (such as the one obtained in the K12ANIigr thread of E. soybean).

The constructed ESERO gene can be modified in different ways to encode zritropostine analogues, such as (Azp2, dev-Rho? according to PeUNERO and (Niv LPNERO, as described below. v5 A. |(Azp2, dev-Rho? according to PeYapERO

Plasmid 536, carrying the ESERO gene constructed in CHM Tables as an insertion of Hvai in Nipai, is cleaved by Nipai and

Come on The latter's probe nuclease cleaves the ESERO gene in a unique recognition site that has 6 base pairs, tightening the last base of the codon encoding Azr C with the second base of the codon Agu U.

The sequence of the Hvrai/XpoY linker, which has the following sequence, is constructed:

Khwai 1 2 Shshch 78 9

Mek dia Azp Suz Ar , KhPOI 5'-STAb ATS SST DAT TS bAS-5 , z'-tAs sSbA TTA ACC sto AbST-5

Linker HvaiYi/Hnoi! and a fragment of the sequence of the ESERO gene Xpo//NipaiII is inserted into a large fragment 70 obtained as a result of the cleavage of Khvai and Nipai plasmid RSEM526 - a derivative plasmid! RSEM414

TA, T.S.S. 40076) - as described in the simultaneously pending US patent application No. 636727, filed on August 6, 1984 by Spagpez R. Moghtiz, for the generation of a plasmid carrying a DNA sequence encoding the expression of E. soybean forms of Me! the desired analogue.

V. (Iniz PERO

Plasmid 536 is cleaved by Nipaii and Xpoi, as in part A above. Linker Khrai/Khpo! built with the following sequence: hvaiYi h1 2 Z a 5 6 7 8 9 Khpoi 77 Mef div Rgo Rgo Agd sechu I1e NIi5 d5ro, 5'-STAb ATO bST SSb SsSA SsT Sto ATS SAT bAS-X z zZ'-TAS SbA bos bot bSA SAS TAS STA ST ASST-5

The linker and fragment of the ESERO Xpo//Nipa sequence are then inserted into PCEM526 to generate a plasmid-carrying DNA sequence encoding the expression of E. soya forms! Me! the desired analogue.

The construction of the manufactured gene (ZSERO"), which includes the yeast preferred codon, occurs as described in the following Tables XM - XXI. Just as in the case of the ESERO gene, the complete construction contains the formation of three sets of oligonucleotides (Table XM, XMII and XIX ), which is formed in the duplex and assembled in parts (Tables XMI, XM and XX). It should be noted that the synthesis of SC is partially facilitated by the use of some suboptimal codons in both ZSERO and ESERO Ge) constructions, i.e., oligonucleotides 7 - 12 of the part 71 of both genes were identical), as were the identities of oligonucleotides 1 - b part 2 in each gene.

Table ХМ пн со 3о Oligonucleotide ЗСЕРО part 1 (Се) со 1. ADTTSADSSTTSSATAAAASASST her for 2. STOSASSSTTTTTATTSSAASSTTTS yu z. ССАССААСАТТСАТСТСТСАСТС й, ТСТСБАСТСАСАСАТСААТСТТо « ес 70 5. САСАСТТТТССАДАСАТАСТТСТ То в) "» 6. СТТССААДСААСТАТСТТТССААААС п 7. СААССТАДАСАДОСТСААААСАТС 1 8. СТОСТСАТСТТТТСАССТТСТТ ТАС т 9. АССАСТОСТТСТОСТОААСАСТСТТС (95) 10. САДАСАДСАСТОТ ТСАССАСААССА (22) сю» 11. ТТТСДАССААДАСАТТАСССТАССС 12. САТССССТАСССТААТОТТТТССТТ

F) name) 60 b5

Table KUT

Part 1 BSERO

EsoiI nipacPI 1

VATISA DOSTTOSATA st. TSVAASSSTAT z

EN Mrmekme MET FV EMAK and 710 in t,

SSAAASATAS TTSTTOBAAS STDAASAASS TSAAADASATS VSSASTOSTT setttetAte Merma sdtttettee ASTTTTTSTAS KEETESsx

From her KrpI VatnI

SsToStodASA STSTTSITTO AASSADAASA TTASSSSTASS S 7/5 САССАСТТСТ I TtbSttttot AАТОССАТО SStАС

Table XVIII

Oligonucleotide BSERO parts 2 20 and. ААДТТСССТАССАСАССААССТ 2. СТТААСТТоСТSTСТОСТАСССС with. TAASTTSTASSSTTSSADASSTAT c 25 o a. ТТССАТССТТССАДСССТАСАА 5. ССААСТТССТСААСАДССАСТСДАСТ зо 6. ССАДАСТСААСТОСТСТТСАССАДС о (Se) 7. ТТОБСАDoСТTTСОСССТСТТАСТСТО сo 8. ССТТСАСАТААСААСОССАААССТTo " 35 9. ADOSTSTTTTTTASASSSTSAdSSST u 10. ААСАДСТСТСАСССТСТААС "СССОСТСАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТАСТААААААААСАДААСТААСАДААСАДААСА program category's section is 11. ТОСТТСССАСТСАСАААСТТАДСС - с и 15. ААССАТСССААТТОСАСССТССАТ a 14. СТТТАСССАССТОСАААТТОСАА sl 45 15. ДАДОСССТСТСТСТТТСАСАСТО их 16. САСССАСАСТСТСААССАСАСОС

Table of KhMUIIiY about part 2 BSERO ast

F "Krpi village" EsoKI r

A ATTSbSTASsS АСАСАССААС bСSATСС tetetostt 55 strvyasttst ASbSTTOSAA vsstatodya sttostEAAs AAOSTSTTSA o SA ASA toSbAVSSTT TOSATA SAASSASTTS TTSSASAAST o ASTETOSSAD osttteosst TeTTATSTS doststttte ASASSTSSADS

TSAKAETr SSAVASSSSSA they koe TSTesASTte 0 60 11 Y with sstksttost TAASTSTTTST SAASSATooS DasslttosA AttosASotS

Sat. SA AtteAsAAt SstTosTASSS ASST tAdestesAye

В9111 Vatni satvAAsss TSTstosttt SASATSTo 65 stAeree ASASASSAMA STSTASASSTA b

Table XIX

Oligonucleotide BSERO part 3 1. САТССАСАСТТТСАСТАСТТТСТТ 2. ТСТСАДСААДАСТАСТСАААСАСТСТС 0 z. САСАССТTTSSSTOSTСААААССААС a. ATSOSTTSSTTTTSASSASSASSADASS 5. SSATTTSSSSSASSASSASOSTOSTTT and 6. SSASAASSASSSTSTOSTOSSSAA 7. STSSSSSSSSATTSASAASSATS c. САСТСАТОСТТСТСААТОoСАбСО 9. ASTOSTSATASSTTTSASADASTTT 10. SADTAASTTTSTSDASSSTATSAS se )1. АТТСАСАСТТТАСТССААСТТСТ о 12. СТСДДАСААСТТССАСТАААСТСТ 15. ТСАСАССТАААТТСААСТТСТАСАС о 0 16, АССССТСТАСААСТТСААТТТАССТ й 15. ССПСТСАДСССТСТАСААСТОСТ со 16. СТСТСАССАСТТСТАСАСССТТС «І ою 17. САСАСАТАДСССССАСТСАТАА 18. СТТСТТАТСАСТСССОСТТАТ « ло 19. САДСАСТСТАСАТСТААСАААб - - 20, ТССАСТТТСТТАСАТСТАСАСТ :» Таблица ХХ часть З 5БСЕРО і-й JSC t ABVTSTtTE ASTASTTTST Trustees

STSTASAAAS TSATSALASA A SADA shk 2

Mami ossestsyaA passvasroya toskno AsASestTost tstossesTe

FO 50 sssAssAstt ttsstte shi TSToSsASSsSA ABASSSSSSAS s» sAttsAYAas SATeVSTOST SATAYSTTSSA SADASTTTATTSSATttTAS staAAsttte stor saAera STATOSADST stslAAH WustslatAt - - 12 59 TesAASTTST o ASASSTAZH ATTSAASTTS tASSASTSSTE AABSSTOTAS

ASSTTSAdSA KetvrssAtt TAASTTSAAAS Kern TTSbSASATS o g sa o AASTSSTBAS ASATAdsSSs SASTSATAAG. And, on Friday, SAT ВСАА 60 ate hares

ATSTAASAAA S

TASATTSTTT SASSST

20 b5

Table XXI

Gene B5SERO -1-41 nipbItii AgoA1v

АБСТТССАТА АААСАССТСС АСАААSATTS АТСТСТСАСТ ССАСАСТTTTT

ASSTAT TTTTSTSASS TSSTTSTAAS TASASASTSA bSTSTSAAAAA

SSADASATAS TTSTTSSAAS STADASASAS TSADAASATS ASSASTSSTT

SSTTTSTATS AASAASSTTS SATTTSTTSS ASTTTTTOTAS TOSTSASSAA

70 stostsdasA ststtsttts AASSAAAAASA TTASSSSASS ASASASSAAS

САССАСТСТСАСАСАДАС ТТССТТТТtсТ ААТеССАТОС TСТСТОСТТС

СТТААСТТСТ АСССТТССАА АССТСТСАА СТТбСТСААС DASSTSTTSA

SAATTSADSA TOSSAASSTT TSSATASSTT SAASSASTTS TTSSASAAST

85

TSAAASSSSTT SSAAASSSSSA DSAATASAST TSSASAAAAS TSToSASTTS

SSTTSTTbST TAASTSTTST SADSSATSSS AASSATTSSSA ATTOSASSTS

ССААСАДССА АТТСАСААСА СТТССТАССС TTSSTAАССТ TAАССТСССАС bATAААСССССТСТСТССТTT САСАСТТТС ASTASTTTST TSASASSTTT

STATTTSSOS ASASASSAAA STSTASADAS TSATSAAASA ASTSTSSADA bbSTSSTSAAA AASSAASSSA TTTSSSSSASS ASASoSToST TstTosseste

SSSASSASTTT TTSSTTSSST ADASSSSToS TSTOSSASSA ASASSSSSSAS s

SATTSASADS SATSASTOST SATSTTSA BAAASTTATT SASSASTTTAS

StTADSTSTTS STATSSASVA STATSSAAST STTTSAATAA STSTSAAATS (5)

TSSAASTTTST TSASASSTAAA ATTSAASTTo TASSASSOSTS AASSSTOoTAS

АССТТСААСА АСТСТССАТ ТААСТТСААС АТСТОСССАС ТТССбАСАТС

ААСТССТСАС АСАТАДСССС САСТСАТААС AАСАСТСТАС i. тТтеАССАСТО ТСТАТСОбо СТСАСАСТО ТТСТСАСАТС so zaii

ATSTAASADA S p

TASATTSTTT SASSST

The collected parts of ZSERO are arranged in a row in M1IZ and Parts 1, 2 and Z are isolated from the phage as "I fragments of NipapaKrpi, Krpi/Vai! and Vopuzai!. oh

The currently preferred expression system for the products of the ZCERO gene is a secretion system based on the secretion of o-factor 5. segemisiae, as described in co-pending US patent application No. 487753, filed on April 22, 1983 by Ogapi A. Vitseg, published on October 31, 1984 as European patent application 0123294. In short, the system includes structures where DNA, "

The leading sequence of the os-factor gene of yeast is located immediately 5 k from the coding region of the expressed zxogenic gene. As a result, the translatable gene product includes a leading or signal sequence, which is "processed" by an endogenous yeast enzyme in the direction of secretion of the remaining part of the product. Since the design makes it possible to use the o-factor translation initiation codon (ATO), there was no need to create such a codon in Position -4 of the ZSERO gene. As can be concluded from Table XXI, alanine (y41) encoding the r sequence is behind the linker sequence, which allows direct insertion into the plasmid, including DNA for the first 80 residues of the y-factor conductor following the t-promoter of the o-factor. The specific preferred structure for the expression of the ZSERO gene contains a four-way crosslinking, including the indication of a fragment of the ZSERO part and a large fragment

F 50 of Nipai's/Zai's split! reaSZ3 plasmids. The promoter and leader sequences of the o-factor, as well as the 5SERO gene, are isolated from the resulting plasmid rSZ/5SERO by cleaving WatNi and splicing this cleaved Watni into the rUE plasmid with the formation of the rUE/5SERO zxpression plasmid.

EXAMPLE 12

This example refers to the expression of recombinant products of ESERO and ZSERO genes within systems! Expressions of Example 11. o When using the system! of expression designed for use in E. soy host cells), the ro3b6 plasmid of Example 11 is transformed in E. soy AM7 cells, previously transformed with a suitable plasmid, i.e.) RMUMI, which has adopted the Svsu gene. Culture! cells in B broth (ampicillin 5 µg/ml and kanamycin 5 µg/ml, preferably with 10 mM MazO)) are kept at a temperature of 28"C and during the growth of cells in the culture to bo O.O.600 5 01, EPO expression is induced by increasing the temperature up to 42"C. The cells grow to about 40

O.0., providing ERO (estimated by gel) about 5 mg/O0 l.

Cells are collected, lysed, crushed with a French press (10,000 rpm) and treated with lysozyme and MP-40 detergent. The sediment obtained as a result of centrifugation at 24,000 khad is dissolved in

NS guanidine and subjected to further one-time purification with the help of C; (Mudas) VZLC with reversed 65 phase (EN, 0 - 8095, 5B0OMM MNAAs, pH 4.5). Determining the sequence based on the protein shows that the product is more than 9595 pure, and the product obtained! introduce two different amino ends, А-Р-Р-К... and Р-Р-К...,

with a relative quantitative relationship equal to Z : 1. That is the last observation of PERO and (dec

Aa |PNERO products mean that the "processing" of amino ends within host cells serves to remove the final omethionine and, in some cases, the initial alanine. Activity determined by radioimmunoassay for isolates is at the level of 150,000 - 160,000 Sh/mg; the activity revealed by ip migo is about 30,000 - 62,000 U/mg; and the activity revealed by IP MIMO varies from 120 to 720 SHO/mg. (Compared to the standard of human urinary isolate - 70,000 IU/mg, in each test).

The dose-effect curve for the recombinant product in the tested IP is significantly different from the curve of the human urinary EPO standard. 70 EPO-analog plasmids, formed in parts A and B of Example 11, are each transformed into rMUMI-transformed E. soy AM7 cells, and the cells are cultivated as described above. Cleaning is isolated! examines both KIA and ip migo analyses. The values obtained in the KIA and ip myo tests for the expression products (Azp2, dev-Rgo?2 according to IeChPERO, are approximately 11,000 U/mg and 6,000 U/mg protein, respectively, while the analysis values for |Niv CyERO are about 41,000 Sh/mg and 14000 U/mg of protein, respectively, 75. This means that the analogue products are recognized as 1/4 - 1/10 "active", as well as the "parent" expression product in the tests.

In an expression system intended for use in host cells 5. segemisiae, plasmid

RUE/5SERO are transformed into two different strains, U5ORA (genotype o, rep4-3 gri) and KKV1 (genotype so, rep4-3 gri). Transformed hosts of U5ORA are cultured in medium 50 (Method! genetics of yeast, Soyd Zriipud Nagbrog I aroghaiog, Soyd Zrgipd Nagrog, M.M., p. 62 (1983)), supplemented with acids sazatipo 0.596, pH 6.5 at a temperature of 30"С The medium collected when the cells are grown to 36 O.O. contains the product!EPO at a level of about 244 U/ml (97 μ/O.O.L., which is detected by KIA). Transformation of KKa1 cells grown to 6.5 O. O. or 60 O.0., provide the creation of media with EPO concentrations of about 80 - 90 O/ml (34 μ/OO l, as revealed by KIA). Preliminary analyzes show significant heterogeneity in the products obtained by using zxpression systems, which apparently, due to changes in glycosylated proteins, as well as a relatively high content of mannose attached carbohydrate.

Plasmid! RoSZ and ruUE in E. soybean HB101 cells were submitted for registration in accordance with the Rules of Production of the United States Patent Office dated September 27, 1984, by the American Collection of Type Cultures, 12301 Ragkiamp Ogime, CosCampe, Maguiapa, under deposit numbers A.T. .S.S. 39881 and A.T.S.S. 39882 "I respectively. Plasmids RSEM526 in AM7 cells, pRSEM536 in /M103 cells and rmMUMI in /M103 cells were registered in a similar way on November 21, 1984 as A.T.S.S. 33932, 33934 and 33933, respectively. and,

Strain! У5РО4 and КК81 Zasspagotusez segemiside were registered on November 21, 1984 as A.T.S.S. 20734 and 20733, respectively.

It should become obvious from the consideration of the above illustrative examples that the present invention in its many aspects offers an exceptionally valuable product! and the way

The proposed polypeptides are markedly useful materials, whether they are microbially expressed products or synthetic products, the primary, secondary or tertiary conformation of which has become known for the first time thanks to the present invention.

As noted above, recombinantly produced and synthetic products! The inventions have different degrees of biological activity compared to ERO isolates from natural sources and therefore are designed to be suitable as substitutes for ERO isolates in culture media used for the growth of erythropoietin cells in culture. Also, because it is a polypeptide product! of the present invention have an activity similar to that of natural isolates of ERO, they are clearly suitable for use in zritroloztine therapy methods applied to mammals, including humans, for the development of any or all of the effects inherent in ERO, for example, their stimulation of the reticulocyte response, development of ferrokinetic effects (such as the effect of iron circulation in the plasma and the effect of the transition time into the brain substance), mass exchange! erythrocyte, o stimulation of hemoglobin C synthesis (see Ezspbasy, ei ai!., above) and, as noted in Example 10, an increase

Ge" 20 levels of hematocrit in mammals. The group of people treated with the proposed products included patients, as a rule, in need of blood transfusions, including trauma victims, surgical patients, kidney patients, including dialysis patients, and patients with a variety of blood composition, vision disorders, such as hemophilia, sickle cell disease, physiological anemia, and the like. Minimizing the need for blood transfusion therapy due to the use of 22 zritroposine therapy can be expected to lead to reduced transfer of infectious agents

HF) diseases. We offer a product! thanks to their production using recombinant methods, it should be assumed that they do not contain pyrogens, natural depressants and the like, and therefore it is undoubtedly a duty! to ensure increased overall return in therapeutic methods in relation to natural products. Zritropoztinovaya therapy using the proposed products should also be useful in increasing the oxygen-permeable capacity of individuals facing hypoxic conditions in the external environment and, possibly, in providing favorable cardiovascular effects.

The preferred method of administration of the proposed polypeptide products is parenteral (for example, intravenous, intramuscular, subcutaneous or intraperitoneal) routes, and the administration of the composition should, as usual, contain a therapeutically effective amount of the product in combination with acceptable diluents, carriers and/or stimulants. Preliminary pharmacokinetic studies note a longer half-life for monkey EPO products when administered intramuscularly rather than intravenously. As it is assumed, effective dosages can vary greatly depending on conditions, however, therapeutic doses, as expected, should be! vary from 0.1 (-7U) to 100 (-7000)) μg/kg body weight of the active material. Standard diluents, such as human serum albumin, are intended for use in the pharmaceutical compositions of the present invention along with standard carriers such as saline.

Stimulating materials suitable for use in the proposed compositions include 7/0 compounds, indicated independently for zytroposic stimulating effects, such as testosterone, stimulating precursor cells, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin and triiodothyronine, as well as agents , commonly used in the treatment of aplastic anemia, such as methenolen, stanozol and nandrolone | (see, for example, Kezedoyi, ei ai.,

Raptipegula Meadis, 23, 243 - 248 (1981); Msbopidie, ei ai., Kidpeu !pi, 25 (2), 437 - 444 (1984); /5 Ramiomis-Kapiega, hey! Ehri. Netayo!., 8(Zirr. 8), 283 - 291 (1980); and Kypg, REVZ I etsegv, 14a(1), 105 - 108 (1982)). It is assumed that as stimulants, substances that increase or synergize zritropostin or asialo-ERO, such as adrenergic agonists, thyroid hormones, androgens and VRAs, are used as stimulants. Yuipp, "Scitepi Sopseriv ip Egu(Pgoroyeviv), doip UUyPeu apa Bopz (Spispezieg, Epadiapa, 1983); MUeyapa, ef ai!.,, Visb 44(3), 173 - 175 (1982); Kaitapii, Kiadpeu Ipi, 22 , 383 - 391 (1982); ZPanpiai,

Mem. Epo. U. Mei., 289, 72 - 80 (1973), Rivpeg, ei aiYi., Eiegoiidz, 30(6), 833 - 845 (1977); Ogabre, ei ai., 9.

Ex. Mea., 149, 1314 - 1325 (1979); and ViMyai ei aiYi., Ehri. Netaiyo!., 10(1), 133 - 140 (1982)) as well as classes of compounds denoting "hepatic zritropozticheskihe factor" | see. Matsdniop, ei ai., Asia.

Naetai., 69, 171 - 179 (1983)) and "zritrotropin" |as described by Sopodoie, ei aiYi., ip Arzigashi 364, Rgoseedipdov 7 |pigepayopa! Sopdgevv oi Epdoshypoyudu (SOceres Su, Smeres, OshShu 7 - 7, 1984); Sopdoye, Viospet. Ha

Viorpuv. Kev. Sott., 115(2), 447 - 483 (1983) and Sopodoie, Apa)Yi. Viospet., 140, 428 - 433 (19843) and "zrytrogeny" |as described in the work of Koiptanp, ei ai., U. Zy!ygo. Opsoi., 20, 105 - 108 (1982))Y. Preliminary and 9) screenings intended to measure zritropozticheskih responses of co-hypoxic polycythemic mice pre-treated with either 5-5-dihydrotestosterone or nandrolone, and then with zritropoztin, showed questionable results. co

The diagnostic uses of the proposed polypeptides are also extensive and include the use of labeled and unlabeled forms in a variety of immunoassay techniques, including KIA, EGISA, and the like, as well as in a variety of analyzes performed to detect IP myo and IP mimo activity. See, for example, the works of Juipp, ei ai., Ehri. Netaiyo!., 11(7), 590 - 600 (1983); (Zirzop, ei ai., RaiyoIodu, 16, 155 - 156 (1984); KguzviaI, Ehri. Netaiyo!., 11(7), 649 - 660 (1983); Zayo, ei aiY., dar. 9. Mea. , 23(1), 16 - 21 Z (1984); Maiyap, ei aI., Mem. Epa. U). Mea., 308(9), 520 - 522 (1983); and various rural materials related to the research mentioned in those works. The proposed polypeltides), which include synthetic peptides that contain the sequences of the residues of the first-discovered EPO, also provide extremely useful pure materials for the production of polyclonal antibodies and "banks" of monoclonal antibodies specific for the different continuous and prairie-type 470 EPO subtypes. As one example, a preliminary analysis of the amino acid sequences of Table 1 in a water curability mixture according to Knorr, et al., R.M.A.5. (O.5.A.), 78, pp. 3824 - 3828 (1981) and secondary structures in accordance with Spo, ei aiY.,, App. To whom Viospet., 47, p. 251 (1978) showed that synthetic "" peptides duplicating a continuous sequence of residues in positions 41 - 57, inclusive, 116 - 118, inclusive, and 144 - 166, inclusive, should probably be produced!

Monoclonal and polyclonal antibodies, 1 immunoreactive both with a synthetic peptide and with a whole protein, are extremely useful to stimulate an antigenic reaction. Such materials, as expected, should be useful in the detection and related purification of ERO and related products. e Illustratively, three synthetic peptides were obtained: c (1) VERO 41-57, M-P-0-T-K-U-M-P-T-A-M-K- bu 70 v-m-E-U -6; (23 VERO 116-128, К-Е-А-И-5-Р-Р-0-А-А-5-А-А; p" (3) VERO 144-166, У-У-5-М -Е-И.-8-6-К--К--- т-6-Е-4-С-8-Т-6-0-8.

Preliminary studies of immunization using multi-specific polypeptides revealed 99 a relatively weak positive reaction to PERO 41 - 57, an unassessable reaction to PERO 116 -

HF) 128 and a strong positive reaction to PERO 144 - 166, as determined by the ability of serum d) rabbit antibodies to immunoprecipitate human urinary EPO isolates, labeled 1292I, Preliminary studies of the activity of ip mimo, carried out on three peptides, did not show significant activity as before /Each in individually or in combination.

Despite the fact that the introduction of the sequence of amino acid residues of mammalian EPO presented in the illustrative examples essentially determines the primary structural conformation of mature EPO, it should be understood that the specific sequence of the 165 amino acid residues of monkey EPO in Table M and the 166 residues of human EPO in Table MI is not limiting the amount of useful polypeptides proposed by the invention. The various freely occurring allelic forms are covered by the present invention! EPO, which in the past was included in research on biologically active mammalian polypeptides, such as human interferon. (Compare, for example, the human immune interferon having an arginine residue at position Mo140 in EPO, which was reported in published patent application 0077670, and the species having glutamine at position Mo140, as reported in Ogau et al., Maishge, 295, pp. 503 - 508 (1982). Both species differ in that they constitute the "mature" sequences of human interferon). Allelic forms! mature EPO polypeptides may differ from each other and from the sequences presented in Tables M and M, in terms of length and sequence and/or in terms of deletions, substituted, inserted or added amino acids in the sequence along with logically consequent variations in the ability to glycosylate. As noted above, one 70 allelic form of human EPO, as it seems, should include a methionine residue in the proposed 126. Presumably, allelic forms that occur in nature! cDNA sequences and

ERO-encoding genomic DNA is probably also due! to meet, and those encoding multiple types of allelic polypeptides or simply using different codons! for designation of the same polypeptides as indicated.

In addition to naturally occurring allelic forms of mature EPO, the present invention also includes other EPO "products", such as polypeptide analogues of EPO and fragments of "mature" EPO. Following the methods of the already published application for the Ayop patent, it a). (MUO/83/04053), it is possible to easily calculate and produce genes encoding microbial expression of polypeptides having a primary conformation that differs from the conformation specified here for mature EPO in terms of the identity or location of one or more residues (for example, substitution , final or intermediate additions and deletions). Alternatively, modifications of cDNA and genomic EPO genes can be easily implemented by means of well-known methods of site-directed mutagenesis and use! for the generation of analogues and derivative EROs. Such products should be ERO! possess at least one of the biological properties of EPO, but may differ in other properties. For example, the designed products of the ERO of the present invention include those that are abbreviated! by, for example, deletions (Azp2, dev-Rgo? according to Pe"ypERO, Idev-Tig193 according to Ago YANERO and "d27-55PERO", and the latter has residues coded by the remote Zkzone; or which is more stable! Kk hydrolysis (and , therefore, may have more pronounced or longer-lasting effects than naturally occurring EPO); or which have been modified to remove one or more potential sites for co-glycosylation (which may lead to high activity for products produced by yeast); or which have one or more cysteine residues removed or replaced, for example, with histidine or serine residues (such as analog (Niv PERO), and potentially more easily excreted in an active form from microbial systems; or which have one or more tyrosine residues replaced with phenylalanine (such as analogues (Rpe "PERO, |Rpe""yERO and |Rpe 7YAPERO) and can bind more or less easily to EPO receptors on target cells. Also, coverage fields peptide fragments duplicating only a part of a continuous amino acid sequence or secondary conformations within the mature EPO, and the fragments may have one EPO activity (for example, receptor binding) and not the other (for example, zritropostichesky activity). Especially important! in that "relationship" is a potential fragment of ERO, which is explained when considering the sequence of human genomic DNA, presented in Table Mi, i.e. "Ffragment" of a complete continuous EPO sequence, which is delineated by intron sequences and which can form

From" is a sharp expression of the "domain" of biological activity. It is noteworthy that the lack of activity of ip mimo for any one or more "ERO products" of the present invention in general does not preclude therapeutic utility (see Mueyapa, e(a., above)) or utility in other contexts, such as ERO tests or

ERO antagonism. Antagonist! zritropoztina can be very useful in the treatment of polycythemia or in cases of overproduction of ERO | see, for example, Adatvzop, Nozr. Rgasiise, 18(12), 49 - 57 (1983) and Neitapp, ei "iz" a!., Siip. the grove Naetai., 5, 335 - 342 (1983)).

Another feature of the present invention is the cloning of the DNA sequence described here, which encodes a polypeptide! Human and monkey erotica, very valuable! for information, b 20 which they offer in relation to the amino acid sequence of mammalian zrithropostin, which is still unavailable, despite decades of analytical processing of isolates of naturally occurring products. DNA sequences are also very valuable as a product suitable for large-scale microbial synthesis of zrithropostin using a variety of recombinant techniques. In addition, the DNA sequence proposal is useful! 25 new and useful viral and circular plasmid DNA vectors, new and useful

GF) of transformed and transfected microbial prokaryotic and zukaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), as well as a new and convenient method of cultured growth of such microbial host cells capable of expressing EPO and EPO products. The DNA sequences of the present invention are also very suitable materials for use as labeling probes in the whitened EPO and the protein associated with it, encoding cDNA sequences and genomic DNA of mammals, other than humans and monkeys, specifically illustrated here. Degree of possible use

DNA sequences in various alternative methods of protein synthesis (for example, in insect cells) or in genetic therapy of humans and other mammals are still impossible to determine. Because we offer DNA sequences, as expected! become useful in the development of transgenic species of mammals, which can serve as zukaryotic "hosts" for the production of zritropostine and products of zritropostine in large quantities. See, mainly, the work of Raiti (eg, ei ai., Zsiepse, 222(4625), 809 - 814 (1983).

In light of what has been discussed here, the specific description of the illustrative examples certainly does not limit the scope of the present invention, and numerous modifications and variations in its implementation will be apparent to those skilled in the art. As one example, though

DNA sequences offered by illustrative examples include cDNA sequences and genomic DNA, this application provides information about the amino acid sequence, 7/0 Essential for the construction of a DNA sequence, this invention involves the production of a DNA sequence that can be constructed based on knowledge of amino acid EPO sequences. They can encode EPO (as in Example 12), as well as a fragment!

EPO and polypeptide analogs of EPO (ie, "products of EPO"), which may possess one or more of the biological properties of EPO occurring in nature, however, do not possess the others (or possess the others to a varying extent).

The DNA sequence proposed by the present invention, thus, must! cover everything

DNA sequences suitable for use in providing expression in a prokaryotic or zukaryotic host cell of a polypeptide product having at least part of the primary structural conformation and one or more of the biological properties of zytroposin, and are selected from: (a)

DNA sequences presented in Tables M and M; (B) DNA sequences that hybridize to DNA sequences defined in (a) or their fragments; (with)

DNA sequences that, due to the degeneration of the genetic code, must be hybridized to

DNA sequences defined in (a) and (b). In this regard, it deserves attention, for example, that the existing gene sequences of the allelic EPO of humans and monkeys and the gene sequences of other species of mammals, as expected, should be! hybridize to sequences in Tables M and

MI or their fragments. In addition, due to the degeneration of the genetic code of the ZSERO and ESERO genes, i) or mutagenization of cDNA or genomic DNA sequences encoding different fragments

EPO and its analogues should also hybridize to the instructions of more DNA sequences. Such hybridizations can easily be carried out under the hybridization conditions described here in relation to the initial isolation of DNA encoding human and monkey EPO, or more stringent conditions if it is desirable to reduce the background hybridization. X,

Similarly, although the above examples illustrate the invention in relation to microbial co-expression of EPO products in the context of mammalian cell DNA expression inserted into a hybrid vector of bacterial plasmid and viral genomic species, the scope of the invention includes a wide variety of expression systems. , which include a vector! of homogeneous species related to a variety of bacterial, yeast, and mammalian cells in culture, as well as to expression systems that do not include a vector (such as transfection of cells with calcium phosphate).

In this regard, it will become clear that the expression of, for example, the DNA of monkeys! in monkey host cells in culture and human host cells in culture is indeed an example of the expression of "xogenic" DNA, since DNAERO, whose high-level expression is sought, will not have its sources in the host's genome. in the System! The expressions of the present invention, in addition, assume that these actions flow into the cytoplasmic formation of EPO products and the accumulation of glycosylated and non-glycosylated ones; of EPO products in the host cell cytoplasm or membranes (for example, accumulation in bacterial periplasmic spaces!), or in supernatants of culture media, as explained above, or in

Quite rare systems, such as, for example, the expression systems of R. aegidipoza (described in the work of Ogau, ei a|., s Viogiespoiodu, 2, pp. 161 - 165 (1984).

The improvement of the hybridization methodology of the present invention, although it was illustrative in application more to DNA screening/DNA-hybridization, is equally applicable! to RNA/RNA screening and 2) RNA/DNA. The methods of mixed probes, as illustrated here, mainly constitute a series of improvements in hybridization processes, allowing for faster and more reliable isolation

Me. polynucleotides. Many separate processing improvements include: improved colony transfer and conservation techniques; the use of nylon-based filters, such as Sepebsgeep and

Sepebsgeep Riy, allowing repeated sounding with the same filters and repeated use of the filter, the use of new methods of treatment with protease | compare, for example, with Taishb, ei ai!., Apai. Viospet., 126, two years 222 - 230 (1982)); the use of very low individual concentrations (about 0.025 picomoles) of a large number of mixed probes (for example, more than 32); and carrying out hybridization and post-hybridization cycles in (F, strict temperature intervals, closely approaching (that is, within 4"С and, preferably, within 27"С) to the lowest calculated dissociation temperature of any of the used mixed probes. This improvement This is fully explained by the fact that mixed probe techniques, which include 4 times the number of probes, which, as previously reported, have always been successfully used even in cDNA screens on relatively small numbers of informative DNA types , were successfully applied to the whitening of a gene of a unique sequence in the screening of a genomic library of 1,500,000 sterile spots. This was easily carried out, essentially, simultaneously with the publication of the opinion of Apdegsop, her aY,, more than the ability of screening with the use of b5 mixed probes of whiteness ". ..impractical for the isolation of mammalian protein genes, when the corresponding DNA is not step down!".

Distinctive features of the invention, disclosed in the preceding description, in the following claims and/or in the attached drawings, can separately and in any combination become material for the implementation of the invention in its various forms.

Claims (26)

The formula of the invention 1. An isolated DNA molecule encoding human erythropoietin and having the following /0 CHUucleotide sequence: ОСС ССА СсСА СОС STO ATS tTOT САС AOS СбА sto STO bAb lob TAS STO tTTO САС ССС AАС o ОАО ССС СА AAT ATS dso dos o sosS tot ost САА SAS TOS AOS TT AAT SAS AAT ATS AST OTO SSA SAS ASS AAA STT AAT TTS TAT OSS TO AAS AS ATO SAS hundred bob SAS SAS OSS to STA SAA hundred tob SAB soS hundred SS hundred Hundred TOb sAA ost hundred hundred sob sosS SAS SSS Hundred TTOo STS AAS TST tTSS САС sob o tob o сАо ССС STO САС СТО SAT hundred SAT AAA ОСС Oto AST boss sStTtT SOS dos ste ASS ST ste stt sSo0 OST STO OBA ССС САС AАС o САА ССС ATO ToС 720 сСст o ссА сАТ bso ссС tTsA SST OST SSA STO SOA ASA ATS AST OST SAS AST TTS SOS AAA STO TTS SbA STS TAS TSS AAT tTtTS sto sa OSA AAS STO AAs Sto TAS ASA obo bab oso TOoS AS ASA sb SAS. 2. An isolated DNA molecule encoding human erythropoietin and having the following nucleotide sequence: o ССС ССА ССА СОС STO АТС TST САС dos СОА СТО СТО САС o АСО TАС STO tТtТОo САС ССС AAb САС ССС САС AAT АСС АСо сосС tot ost САА САС TOS AOS tTTO AAT SAS AAT ATS AST STO SSA SAS ASS AAA with STT AAT TIS TAT SSS TOb AAb Or ATO SAb hundred bob SAB SAS OSS STA SAA hundred tob SAS sooS hundred bSS hundred STO TO o bAA ost oto o SSO SOS SAS o sSS hundred TTOo OTO AAS o TST o ToS SAS sob o bdo ССС o STO SAS STO SAT STO SAT AAA boss sto AST SOS Stt o soS dos ste "EE VSS AST STO STT SOb OST STO OSA CSS SAC AAS SAA ss ATS TOS yu SST SSA SAT So SSS TSA OST SST SSA Sto SSA ASA ATS o AST ost SAS AST TTS SOS AAAA STO TTS SOA sto TAS TOS AAT ttTs sto sos SBA AAS o StTo Ada sto TAS ASA bas o sAs OSS tToS AS ASA sob BAS « 700 All - in the village 3. An isolated DNA molecule encoding human erythropoietin and having the following nucleotide sequence: SAA tot Ssst oss tos sto tob sttT sto sto tToS osto sto tob ste sst sto bos sto sSsA sto Sto bos OSS sСсSA SSA SOS sto ATS tot sl 75 САС АСС ССА STO STO САС АСС TАС СТО ОТТО САС ОСС AАС о сСАб ss САС AAT ATS АСО АСо сосС ТсТ ОСТ о САА САС TOS АОС OTTO AAT САС и AAT ATO AST STO ССА САС АСС dАА сОТТ AAT o TTS TAT ОСС tob AAb Mn ASO ATO SAb sto bo САС САС СС сTA САА sto tob САС boss sto (22) 20 ss hundred hundred TSOo baAA ost hundred hundred sob boss SA os sta tto sto se» AAS OTST tToS SAS SSO tTOoS SAS SSS Sto SAS STO SAT STO SAT AAA ss hundred AST soS sStTtT SOS dos Sto ACC ST hundred SstT person ost hundred OSA SSS САС AАС САА СОС АТС tToS SStT o ССА SAT oso ССС о тТсА ost 25 OST ССА СТО СОА АСА АТС AST OST САС AST OTTO SOS AAA sto tt F, SA STO TAS TOS AAT TT STS SO Both AAS STO AAS STO TAS ASA name) SSO SAS SSS TOS AOS ASA sob SAS AbA. 4. An isolated DNA molecule encoding human erythropoietin and having the following 60 nucleotide sequence: b5 ATO so STO SAS OSAA tot sstT o sss o tTos hundred tosostto hundred hundred tes hundred hundred toa hundred sstosto SOS hundred SSA hundred hundred pos ССС СсСА СсА СОС Sto ATS TT САС AOS SOA STO STO SAB As TAS STO STO TT SA 9 ССС AАС САС ССС САС о ААТ АТС АСО Со СОС TsT OST САА САС TOS АСС ТТсС AAT САС AAT ATS AST STO ССА САС АСС AAA СТТ AAT To TAT ССС tob AAS AOS ATO САС hundred bob САС САС ОСС STA САА ste tob САС boss hundred сСС hundred Sto TSO SAA ost sto sto ss o sos САС 70 SOS o sto tt STO AAS o tTstT o tToS o САС sso tob bas sss sto САС sto SAT STO SAT AAA soS sto AST o sSoS sStTtT SOS AOS STO ASS ASTT ste StTtT sob ost Sto SbA ССС SAS AL SAA OSS ATO TOS SST SSA SAT sSO SOS o tTsSA OST ssStT o SSA STO SOA ASA ATS AST OST SAS AST TT SOS AAA STO TTS SA ST TAS TOS AAT OTTO STO SOB SSA As sto AAS STO TAS ASA bob SAS SSS TOoS Or ASA Obb SAS AbA. 5. A DNA molecule complementary to the DNA sequence described in any of claims 1-4. 6. The DNA molecule according to claim 5, characterized by the fact that it is covalently bound to a defined label. 7. The DNA molecule according to claim 6, characterized by the fact that the defined label is a radioactive label. 8. The DNA molecule according to claim 6 or 7, characterized by the fact that it is single-stranded DNA. 9. A DNA molecule that hybridizes under harsh conditions with a protein-coding region of molecules! DNA characterized in any of claims 1-4. 10. The DNA molecule according to claim 9, characterized by the fact that it is covalently bound to a defined label. 11. The DNA molecule according to claim 10, characterized by the fact that the defined label is a radioactive label. with 12. The DNA molecule according to claim 10 or 11, characterized by the fact that it is single-stranded DNA. d) 13. Biologically functional circular plasmid or viral DNA vector containing the DNA molecule characterized in any of claims 1-4. 14. A strain of zukaryotic host cells, transformed or transfected with a DNA molecule, characterized in any of claims 1-4, in such a way that the host cell is capable of expressing the human erythropoietin encoded by the specified DNA molecule. "at 15. The strain according to claim 14, characterized by the fact that the indicated host cell is capable of glycosylating the indicated polypeptide. at 16. The strain according to claim 15, characterized by the fact that the specified host cell is a mammalian cell. Zo 17. The strain according to claim 16, characterized by the fact that the specified host cell is a CO5 cell. at 18. The strain according to claim 16, characterized by the fact that the specified host cell is a CHO cell. 19. A strain of zukaryotic host cells, stably transformed or transfected with a DNA vector, characterized in claim 13 in such a way that the host cell is capable of expressing the human zytroposin encoded by the specified DNA molecule. WITH 20. A polypeptide that includes part or all of the primary structural conformation of human erythropoietin, having the following amino acid sequence: s1u A1i1a Rgo Rko AkChch Petc I1e Sub Avr beht Akd Ua1! Pe Si Ak Tuk pe Petc b1y 1 A1i1a Ppuv bi Aia Si ABp o I1e Tk Tk siu Suv A1ia si niv Suv eg ey vp ts vp e va a who vr Э uv a vy e і5" 5 I A s1h A Iie tih ua1 R A trph chai A RO. s tTuh A1ia Ter Puv Akud Mee Si wai siu sip sip A1a Ua1 Si Uai1i F 50 Tkr bip siu Ge Ai1a Ppey Gep back bi Aza Uai! Pe Aku a1i1u sip A1i1a Pec Peip Ua1i Avp bek bek sip Rhgo Tkr bi Rko pe sip Gets m ; « Niv Ua1 Avr Guz A1ia Ua1i Bek s1u Pec Aka Bek Pec Tik Tit Pei Tech Ak A1a tep s1u Aia sip Guv Si Aia I1ie bek Rko Rko Avr A1ia Aia Bek A1i1a A1i1a Rko Petc Aku Tph I1e Tph A1ia ABr TPh RBe GF! AkCh o Ppuv Ppep Rre Ak Ua1! Tuk Bek ABp RPpe Pep Akd s1u Puv Pec t tsuv rep Tuk Tk Siu sich A1ia Sub Agu Tk b1u AB Yes, having the biological activity of increasing the production of reticulocytes and erythrocytes by the cells of the bone marrow and increasing the synthesis of hemoglobin or the consumption of iron, and characterized by the fact that it is a product of the expression of a xogenic DNA molecule according to any of claims 1-4 in zukaryotic cells, and which has a higher molecular weight according to ZOZ-RACE than zritropoztin, excreted from urine. 21. Polypeptide according to claim 20, characterized by the fact that it is a product of expression in mammalian cells. 65 22. Polypeptide according to claim 20, characterized by the fact that said polypeptide is a glycoprotein having an average carbohydrate content different from that of human erythropoietin isolated from mMO. 23. The polypeptide according to any one of claims 20-22, characterized by the fact that the specified xenogeneic DNA sequence is contained in an autonomously replicating circular DNA plasmid or a viral vector. 24. Polypeptide according to any of claims 20-23, characterized by the fact that it is a product of the expression of molecules! DNA, characterized in any of claims 1-4, in a mammalian cell. 25. The method for obtaining the polypeptide described in any of claims 20-24, which provides for cultivation in a suitable nutrient medium of a zukaryotic host cell characterized in any of claims 14-19, in such a way that the specified host cell is able to express the specified 7/0 polypeptide , and isolation of the indicated polypeptide. 26. A pharmaceutical composition containing the polypeptide described in any of claims 20-24 and a pharmaceutically acceptable diluent, adjuvant or carrier. c schi 6) (ze) (Se) (ze) « I c) - and? 1 sh" (95) (o) syu" name) 60 b5