BG60770B2 - Reduced human tissue plasminogenous activator - Google Patents

Abstract

Truncated human tissue plasminogen activator (t-pa), consisting primarily of amino acids 69-527, produced in significant quantities by recombinant DNA (rDNA) technology. The invention relates to a method for the production of t-pa without the contaminants with which it is normally associated in its natural cellular environment, for the preparation of expression vectors and various host cells. 1 claim

Description

The present invention is for a human plasminogen activator corresponding to that found in human serum and / or tissues and for compounds and novel forms as substitutes, as well as for methods and methods for its homogeneous production in therapeutically significant amounts.

The invention stems in part from the discovery of DNA sequencing and, subsequently, the amino acids of a human plasminogen activator. This finding made it possible to reproduce a human plasminogen activator through the application of recombinant DNA technology, providing conditions for obtaining sufficient quantities of material to initiate and conduct trials on experimental animals and in clinical conditions, as a necessary condition for obtaining a marketing authorization, unimpeded of the necessary limitations inherent in the isolation methods used so far, including extraction and extraction from existing cell cultures.

Publications and other materials used to illuminate the origin of the invention, as well as in individual cases to provide additional details for its operation, are enclosed in the bibliographic reference and are conveniently numbered and grouped in the attached bibliography.

A. Human tissue plasminogen activator

The body's fibrinoliotic system is in dynamic balance with the coagulation system and both maintain the normal state of the bloodstream. The coagulation system deposits fibrin, as a matrix serving to restore hemostatic status. The fibrinolytic system removes the fibrin network after the haemostatic state is reached. The fibrinolytic process is carried out by the proteolytic enzyme plasmin, which is obtained from the plasma protein precursor plasminogen. Plasminogen is converted to plasmin by activation through an activator.

Currently, two activators are available on the pharmacy network, streptokinase and urokinase. Both are indicated for the treatment of acute vascular diseases such as myocardial infarction, pulmonary embolism, deep vein thrombosis, peripheral artery obstruction and other venous thrombosis. These diseases pose a great danger to human health.

The etiological basis of these diseases shows, in some cases, partial and most often complete obstruction of a blood vessel from a blood clot - a thrombus or thromboembolism. Traditional anticoagulant therapy, such as heparin and coumarin, does not directly increase the dissolution of thrombi or thromboembolism. The thrombolytic agents mentioned above, streptokinase and urokinase, have wide and effective application. But each of them has severe contraindications. They have no high affinity for fibrin; therefore, both antico20 aggregates activate circulating and fibrin-bound plasminogen relatively indiscriminately. The plasmin (or more fibrinolysin) formed in the circulating blood is neutralized too quickly and loses its thrombo25 lytic properties. Residual plasmin reduces some protein coagulating factors, such as fibrinogen, Factor V, and Factor VIII, which make up the hemorrhagic potential. In addition, streptokinase is a potent antigen and patients with high antibody titers respond ineffectively to treatment and cannot remain on long-term treatment. Urokinase therapy is costly due to its isolation from human urine or tissue cultures and this is the reason why it is not universally accepted in clinical practice. Urokinase has been the subject of much research - see references 1-6.

The so-called plasminogen activators have been isolated from various human tissues, e.g. from uterine tissue, blood, serum - see references 7-11, and from cell culture ref. 94. The compounds obtained are described (refs. 12, 13, 14-18). The plasminogen activators derived from these sources were classified into two large groups: urokinase type plasminogen activators (UPA) and tissue type plasminogen activators (t-PA), based on differences in their50 immunological properties. (Abbreviations y-PA and t-PA were proposed at the XXVIII Congress of the International Committee on Thrombosis and

I hemostasis, Bergamo, Italy, July 27, 1982).

Recently, a human melanoma cell line was identified that secretes m-PA. The study of this melanoma plasminogen activator showed that it did not differ immunologically in the composition of the amino acid compounds from the plasminogen activator isolated from normal tissue cells (references 19, 88).

The product was isolated in relatively pure form, studied and found to have the properties of a highly active fibrinolytic agent (20).

Some studies (refs 95-98) using m-PA purified from a melanoma cell line have demonstrated its high affinity for fibrin compared to the urokinase-type plasminogen activators. However, intensive studies of human t-PA as a potential thrombolytic agent have been hampered by its extremely low concentration in blood, tissue extracts, vascular perfusers, and cell cultures.

It has been recognized that the use of recombinant DNA and related technologies is the most effective way of providing the required large quantities of high quality human tissue plasminogen activator, mainly purified from other human proteins. Such materials will likely demonstrate bioactivity giving access to clinical use in the treatment of various cardiovascular conditions or diseases.

B. Recombination technology for

DNA

DNA recombination technology has reached high levels. Molecular biologists are now able to recombine different DNA sequences with ease, creating new DNA molecules capable of producing copied amounts of exogenous protein products in transformed microorganisms or cell cultures. Common methods and methods are expressed in the ligation of the "sticky" end fragments of DNA producing efficient expression carriers suitable for the partial transformation of microorganisms, thereby directing their synthetic ability to the desired exogenous product. However, due to the individual characteristics of the starting products, the path remains wandering and science has not yet made significant progress. In fact, the one who marks successful results without a solid experimental basis runs the risk of failure.

Recombination of DNA from essential elements, i. the replication locus, of one or more phenotypic selection traits, the expression promoter, the heterologous gene insertor, and the residual vector are typically performed outside the host cell. The resulting recombinant replicant expression carrier or plasmid is introduced into the cells by transformation, and large amounts of the recombinant carrier are recovered from the cultured transformant. Where the gene is correctly inserted with respect to the segments that regulate the transcription and translation of DNA-encoded information, the resulting gene expressor is capable of actually producing a polypeptide sequence for which the inserted gene gives the code, and the process is referred to as 25 gene expression. The resultant product can be obtained by lysis, if necessary, of the host cell, the microbial system and subsequent appropriate purification from other proteins.

In practice, fully heterologous polypeptides can be expressed through the use of DNA recombination technology. direct expression - or alternatively, a heterologous polypeptide synthesized as a segment of an amino acid sequence of a homologous polypeptide can be expressed. In the latter cases, the expected bioactive product is sometimes found to be bioinactivated upon synthesis, the homologous / heterologous polypeptide is fragmented into the extracellular space. See references (21) and (22).

In the same way, the rules of the art have been established for the study of ge45 netics and cell physiology for both cell and tissue cultures. Methods and methods for maintaining permanent cell lines prepared from successful serial transfers from isolated normal cells are known. For application in the studies, these cell lines are maintained on a solid support in liquid medium or through

I growth in a suspension containing nutrients. Increasing the yield for large-scale industries seems to solve only mechanical problems. See references (23) and (24). 5

Similarly, the biochemical properties of proteins are exploited by being applied in biotechnology. The cells producing the desired protein also produce hundreds of other proteins, endogenous products of 10 cell metabolism. These contaminating proteins, as well as other compounds, if not removed from the desired protein, may manifest themselves as toxins if administered to animals or humans during the course of the therapeutic course of the desired protein 15. Protein biochemistry technology offers the possibility of separation procedures suitable for the respective systems to produce homogeneous, usable products. Protein biochemistry confirms the identity of the desired product and ensures the reliability of production without alterations or mutations. This branch of science also includes the creation of medicines, preliminary studies and other necessary procedures for implementation prior to successful clinical trials.

The present invention is based on the discovery that DNA recombination technology can be successfully used for the production of human tissue plasminogen activator (m-PA), preferably in a direct manner, and in amounts sufficient to initiate and conduct experimental testing animals, as well as clinical 35 tests, as mandatory prior to sale. The resulting human t-PA is suitable for administration, in all its forms, for the prophylaxis or treatment of human beings in various cardiovascular conditions or diseases. Therefore, the present invention, in a very important aspect, has been proposed as a method of treating vascular disease in humans by administering tPA and its corresponding pharmaceutical forms.

The present invention encompasses an essentially pure human tissue plasminogen activator. The product is produced by genetic engineering of microorganisms or cell culture systems and provides opportunities for the production of human tissue plasminogen activator in a highly efficient manner. In addition, depending on the host cell, the human tissue plasminogen activator may contain associated glycosylates to a lesser or greater extent than the natural product. In any case, T-PA does not contain the contaminants it is normally associated with in the cellular environment.

The present invention also relates to replicating DNA expressing carriers carrying gene structures for the human tissue plasminogen activator code in expressive form, for colonies of microorganisms or cell cultures transformed with them, or for microbial or cell cultures from the transformed colonies or cultures capable of produce a human tissue plasminogen activator. In these aspects, the present invention is directed to various processes for producing gene sequences, DNA expression carriers, colonies of microorganismic strains and cell cultures and specific formations. The invention also includes the production of fermentation cultures from said microorganisms or cell cultures.

Figure 1 shows a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresed from ³¹ 8-methionine-labeled proteins precipitated with anti m-PA immunoglobulin O secreted by melanoma cells with or without a protease inhibitor.

Figure 2 shows electrophoresis of immunoprecipitated translation products of matrix ribonucleic acid (mP Η K) fractions obtained from melanoma cells.

Figure 3 shows a hybridization model of 96 bacterial colonies transformed with DNA using a depot of ³² Marked 14-tag as a probe made mainly from 5 amino acid sequences of human m-PA.

Figure 4 is a full-length endonuclease restriction map of human tPA cDNA.

Figures 5a, 5b and 5c show the nucleotide sequence and the extracted full-length amino acid sequence of human m-PA cDNA.

I

Figure 6 is a schematic construction of the expression plasmid rbekaK1PA ”.

Figure 7 shows the results of the fibrin layer assay for fibrinolytic activity of E.c.HH cells transformed with pbKAPA.

Figure 8 is a TXVH (high pressure liquid chromatography) record of the human t-PA peptides from the digestive enzyme trypsin.

Figure 9 shows the construction of a plasmid encoded for the direct expression of mature human m-PA in (E.c.OH).

Figure 10 shows the results of a sample fibrin layer tested for the fibrinolytic activity of human m-PA produced by E.c.OH.

Figure 11 shows the construction of DHFR (mutant / or wild-type unaffected) / mPA encoded plasmid suitable for transformation to mammalian tissue cell culture.

Figure 12 is a schematic diagram of a human tissue plasminogen activator obtained by the illustrated method in E. 1.

A. Definitions

As used herein, "human tissue plasminogen activator" or "human t-PA" or "t-PA" means an intrinsic human (tissue type) plasminogen activator produced by microbial or cell culture systems in bioactive forms comprising protease moieties and corresponding to these tissue plasminogen activators, otherwise natural to human tissue. The human tissue plasminogen activator protein obtained in this invention has been determined by a deterministic DNA gene and the related amino acid sequence. It is understood that natural allelic variations exist and fall from individual to individual. These variations can be demonstrated by the amino acid (amino acid difference) in the universal sequence or by deletions, substitutions, insertions, inversions or addition of amino acid / and in the above mentioned sequence. In addition, the location and degree of glycosylation will depend on the nature of the environment around the host cell.

The potential behind the application of DNA recombination technology to produce derivatives of various human tissue plasminogen activators may be modified differently as a result of single or multiple amino acid substitutions, deletions, additions or substitutions, for example, by site with managed DNA mutagenesis. This can be accomplished by preparing derivatives storing an essential "kp £ 1e" region or from a serine region. All such allelic variations or changes leading to human tissue plasminogen activator derivatives are within the scope of this invention, as well as other similar human non-tissue (tissue type) plasminogen activators, with similar physical and biological properties. The human tissue plasminogen activator is prepared (1) using for its first amino acid the methionine or (2) where the methionine is intracellularly degraded to use its first amino acid, or (3) together with another characteristic polypeptide thereof. or a conjugated protein instead of the conventional characteristic polypeptide, the latter and the conjugated specifically being degraded into the intra- or extracellular space (see reference 21), or (4) by direct expression of the mature form without the need for erodable every extraneous and superfluous polypeptide. The latter method is particularly important when the host cell cannot or cannot efficiently remove the characteristic peptide when the expression carrier is directed to express the tissue plasminogen activator together with its characteristic peptide. In any case, the human t-PA produced in this way, in its various forms, is recovered and purified to a level suitable for use in the treatment of many vascular conditions and diseases.

In addition, m-PA possesses forms that include single-stranded protein (1-strand) and double-stranded protein (2-strand). The latter is proteolytically derived from a single chain compound. It is known from theory that two-stranded protein is associated with fibrin produced, and this proteolytic conversion from single-stranded to two-stranded component takes place at the locus of plasminogen to plasmin conversion. The present invention proceeds from the proposal for implementation5

I of 1-stranded protein as described or for the implementation of 2-stranded protein as shown to be possible. The 2-stranded protein can be obtained in vitro by conversion after production of the 1-stranded protein product. The so-called “Κιϊηβίε” region is positioned opposite to the serine region and is thought to play an important role in the binding of the tissue plasminogen activator to the fibrin matrix; therefore, the observed specific activity of the present plasminogen activator present is actually existing with respect to blood clots. The tissue plasminogen activator contains enzymatically active regions corresponding to the natural product, and the term human tissue plasminogen activator defines products including such single regions or together with additional amino acid sequences throughout the molecule.

Generally speaking, the human t-PA represented by this invention has a functional definition; this is the ability to catalyze the conversion from plasminogen to plasmin, to bind fibrin, and is qualified as a mpA based on the immunological properties mentioned above.

"Essential pure form" is used to describe this condition of m-PA obtained by the methods of the invention and purified by a protein or substances typically associated with m-PA when produced by non-recombinant cells, i. in its "natural" environment.

"DHFR protein" means a protein (protein) having active association properties with Di-Hydro-Folate Reductase and which is produced by cells capable of living in a hypoxanthine, glycine, and thymidine-deficient medium (-XGT medium). Generally, cells that are deficient in DHFR protein are unable to grow in such an environment, and cells containing DHFR proteins produce growth.

"MTX-sensitive cells" refers to cells that are unable to grow in a medium containing the DHFR methotrexate inhibitor (MTX). Thus, “MTX-sensitive cells” are cells that, except for genetically modified or other additives, will not sprout in a growth-friendly medium when the MTX concentration is 0.2 μβ / ιηΐ or greater. Some cells, such as bacteria, do not exhibit MTX sensitivity due to their impermeability to MTX across the cell membrane, although they contain DHFR 5 protein. Generally, cells containing a similar DHFR protein will be sensitive to methotrexate if they are permeable to MTX.

Wild-type DHFR refers to dihydrofolate reductase, which is usually found in individual organisms. Wild type DHFR is usually sensitive to low concentrations of methotrexate (MTX).

"DHFR protein with low affinity for MTX binding" has a functional 15 definition. It is a DHFR protein that, when generated in cells, enables the growth of MTX-sensitive cells in an environment containing at least 0.2 μβ / πιΐ or more of MTX. Such a definition is known to depend on the ability of the body to produce DHFR protein "with low binding affinity for MTX". However, as used in the context of this invention, as a balance between the two mechanisms, there will be no concern. The invention works according to the determined levels of MTX and is not effective when influenced by the increased expression and in addition to the innate nature of the produced DHFR. The conventional DHFR protein that is suitable for this definition is described in i.5. 45ο 459 151, filed Jan. 19, 1983.

An "expression vector" refers to vectors that are capable of expressing DNA sequences, where the latter are functionally linked to other sequences capable of selecting their expression. It has been suggested, although it has not been precisely established that these expression vectors can replicate in the host organism as episodes or as an integral part of chromosomal DNA. A clear inability to replicate will present them as effectively inactive. In short, an "expression vector" has a functional definition, and any DNA sequence that is capable of efficiently expressing a particular DNA code configuration is included in that term, applying to a specific sequence. Generally speaking, expression vectors when applying the techniques for

DNA recombination is often in the form of "plasmids", which refers to double-stranded DNA structures that are unrelated to the chromosome in their vector forms. In the present specification, "plasmid" and "vector" are used interchangeably, the plasmid being the more commonly used form of vector. However, the invention is intended to include such other forms of expression vectors that provide equivalent functions and which are already available for use.

"Recombinant host cells" refers to cells that have been transformed by construct vectors with the application of a DNA recombination technique. As defined, t-PA is produced in amounts achieved through this transformation, significantly less than the amounts that would be obtained from untransformed hosts. m-PA derived from such cells can be defined as "recombinant m-PA".

B. Host and vector cell cultures

The vectors and methods of the invention are suitable for use in host cells from a wide variety of prokaryotic and eukaryotic organisms.

Generally, prokaryotes are preferably due to cloning of DNA sequences in the construction of the vectors used in the invention. For example, E.sOH K12 strain 294 (ATCC 31ο 31446) has been specifically applied. Other microbial strains that may be used include E.coneH strains, such as E.coneB, E.cone X1776 (ATCC 31537). These examples, of course, are intended to illustrate rather than limit.

Prokaryotes can also be used for expression. The above strains, as well as E.c.H.N. 3110 (Ρ-, λ prototropic, ATCC 27ο 27325) bacilli such as your 5inH1 and $, and other enterobacteria such as 5a1 top11a 1ur1ptipit or ZeggaNa tagsezepsis, can also be used in different species.

Plasmids generally contain replicon and control structures, are derived from species compatible with the host cell and used in conjunction with these hosts. The vector typically carries a replication region, just like the tagged structures that are capable of providing phenotypic selection in the transformed cells. For example, E. colo is typically transformed using pBK322, and the plasmid is derived from a variant of E. colo (VoPuag, e1 al., Sep 2: 95 (1977). PBK322 contains genes resistant to ampicillin and tetracycline, thus providing all methods for identification of transformed cells Plasmid pBK322 or other microbial plasmid must also contain, or be modified to contain, promoters that can be used by the microbial organism to express its own proteins. These promoters are most commonly used in recombinant DNA with conventions including β-lactamase (penicillinase) and lactose promoter systems (Cbp £ e1 ak, No. 1ige, 275: 617 (1978); Waqiga eak, 5> ceps, 198: 1056 (1977); (Οοεάάεΐ, e (ak, EWS 281: 544 (1979)) and the tryptophanan (trp) promoter system (ΟοεάάεΙ, e1 ak Νυείεϊε Αεΐάδ Kez., 8: 4057 (1980); EPO Arr1, Rik 00ο 0036776). , other microbial promoters have been discovered and used, and details of their nucleotide structures have been published, enabling skilled specialists to bind them functionally to plasmid vectors (δϊ εΠεηΙίδΐ, E1 ak, CE 20: 269 (1980).

In addition, in addition to prokaryotic and eukaryotic germs, yeast cultures can also be used. Seagulls are now the most commonly used species among eukaryotic microorganisms, although many other strains are commonly available. For expression in Sassagotusek, the plasmid ΥΕρ7, for example, (8PpsPsotb, e (a1, IaShge, 282: 39 (1979); Kschehtap e! A1, Sepe, 7: 141 (1979); Tsshsetreg, e1 a1, Sepe, 10: 157 ( This plasmid contains the τρπΐ gene, which provides a selection marker for a mutant yeast strain lacking the ability to grow in tryptophan, for example, ATCC 440ο 44076 or PEP4-1 Sopech, Sephex, 85:12 (1977)). The presence of a τρπΐ lesion as a characteristic of the yeast host genome provides an effective medium for detecting growth transformations in

And the absence of tryptophan.

Suitable structures in yeast vectors include the 3-phosphoglycerate kinase promoters (Nkhetap, e! Ak, 1. VyukSet, 255: 12073 (1980) or other glycolytic enzymes (Nex. E. (Ak.A.άά. Epgut Ke§. 7 : 148 (1968): No11aps1, e1ac, Vusepteptu, 17: 4900 (1978) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, hexokinase, pyruvate decoxoxylase, glycoso-6phosphaturate, triosophosphate isomerase, phosphoglucose isomerase, glucokinase In the appropriate expression plasmids created, the identified structures are ace These promoters that are linked to expression vector 3 of the expression sequence to achieve polyadenylation of mRNAs Other promoters that have additional advantages such as transcription controlling growth conditions are the alcohol dehydrogenase 2 promoter regions, isocytochrome C , acid phosphatase, degrading enzymes relevant to nitrogen metabolism, and the above-mentioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the digestion of maltose and galactose (NoNapb). Each plasmid vector containing a yeast promoter, a replication locus and a termination end of the sequence is suitable for use.

In addition to microorganisms, cell cultures derived from multicellular organisms can also be used for hosts. In principle, any such cell culture is appropriate, depending on whether it is a vertebral or invertebral culture. However, interest would be of greatest interest in vertebral cells and the propagation of such cells in culture (tissue culture) has become a routine procedure in recent years (Τίκδίιε Si1Sge, Asabeps Rhekch, Kgize RaIgzop, Ebiogz (1973)). Examples of such usable host cell lines are UECO and HEbA cell lines, Chinese hamster ovary (CHO) cell lines and O138, BHK, CO8-7 and ΜϋΟΚ cell lines. Expression vectors for such cells typically include (if necessary) a replication locus, a promoter located against the expression gene, binding sites for the corresponding ribosome, RNA binding sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the 5 control functions of expression vectors are often provided by viral material. For example, the commonly used promoters are derived from Rooshua, Αάεηονϊηιχ 2 and most commonly 31ppas Weise 40 (8U40). The early 10 and late promoters of the 8U40 virus are specifically used because they are readily extracted from the virus as a fragment that also contains the 8U40 viral replication locus (Pegs, E1, K1ige, 273: 113 (1978). larger fragments of 8U40 can also be used, the resulting ones thus including approximately 250 bp sequences extended from the Ηΐηά III region against B§1 I localized at the viral replication locus, and it is also possible, and often desirable, to use a promoter or control sequence usually associated with the desired gene c control sequences thus obtained are compatible with the host cell systems.

A replication locus may also be provided by constructing a vector with the inclusion of an exogenous vector, such as may be derived from 8U40 or another viral (Ro1wota, Αάεηο UZU VRU, etc.) source, or may be obtained from a host cell with a chromosomal replication mechanism. If the vector is integrated into the chromosome of the host cell, the latter is often appropriate.

When selecting a preferred host cell for transfection from vectors of the invention that comprise a DNA sequence encoding the two proteins m-PA and DHFR, it is appropriate to select the host according to the type of protein DHFR used. If a "wild-type" DHFR protein is used, it is preferable to select a host cell that is non-DHFR inactivated, which allows the use of the DHFR code sequence as a marker for successful transfection in a selected medium lacking hypoxanthin, glycine and thymidine. A suitable host cell in these cases is a Chinese hamster ovary (CHO) ovarian cell line deficient in DHFR, made and distributed as

The one described in (1) 1aBi and CbA $ 1n (Rg.Man. Aad. Asp. And $ A 77: 4216 1980), is inserted in the bibliographic reference.

On the other hand, if DHFR protein with low affinity for binding to MTX is used as a control sequence, there is no need to apply cells that are deficient in DHFR. Because the DHFR mutant is resistant to methotraxate, MTX containing medium can be used as a selection agent selected so that the host cells are themselves sensitive to methotraxate. Many eukaryotic cells that are able to absorb MTX are sensitive to methotrexate. One such cell line is that of CHO, CHO-K1 ATCC Сο CCB 61.

The experiments described below describe the use of E.c.OH with lac and trp promoter systems and the use of CHO cells as host cells and expression vectors that include the 8Y40 replication locus as a promoter. However, it would be advantageous to use analogous techniques for constructing expression vectors to express desired protein structures in alternative prokaryotic or eukaryotic host cell cultures.

Satisfactory amounts of t-PA are produced from cell cultures, but later improvements using secondary code sequences suggest an increase in production levels. The secondary code sequence includes dihydrofolate reductase (DHFR), which is treated with an external control parameter such as methotrexate, and this allows expression to be controlled by controlling methotrexate concentration (MTX).

C. Methods used

If the host cells used are free of permeable cell barrier membranes, transfection is carried out by the calcium phosphate precipitation method as described by CgAnate and Wap de Eb, 52: 456 (1973). However, other methods of introducing DNA, such as nuclear injection or protoplastic fusion, can be applied.

If prokaryotic cells or cells containing a substantially cell wall are used, the preferred method of transfection is calcium treatment with calcium chloride as described by SoBep, E. f! a1. Ργοο.ΝηΙΙ. Assad. 5α. (ΙΙ5Α), 69: 2110 (1972).

A standard ligation technique is used to construct suitable vectors containing the desired code and control sequences. Isolated plasmids or DNA fragments are digested, anchored and re-ligated into desired forms to those imposed by plasmids. The digestion is carried out with a restriction enzyme (s) in a suitable buffer. In general, about 1 μ% of plasmids or DNA fragments are applied with 1 unit of enzyme in about 20 μl of buffer solution. (Manufacturers offer suitable buffers and substrates for special restriction enzymes). The incubation time is assumed to be 1 hour at 37 ° C. After incubation is complete, the protein is removed by extraction with phenol and chloroform, and the nucleic acid is recovered from the aqueous fraction by precipitation with ethanol.

If "L1ip1" ends are required, the preparation is treated for 15 minutes at 15 ° C with 10 units of polymerase I (Κίεηονν), phenol-chloroform for extraction and ethanol for precipitation.

The separation dimensions of the degraded fragments were carried out with the 6 percent polyacrylamide gel described by Soedde1, ϋ. it is! a1, Аυείείε Astz Kez, 8: 4057 (1980), and is noted in the bibliography.

For ligation of approximately equimolar amounts of the desired components, compatible to ensure accurate compliance, are treated with about 10 units of T4 DNA ligase at 0.5 μ ' DNA. (When split vectors are used as components, it is possible to use the split vectors to prevent re-ligation by pre-treatment with bacterial alkaline phosphatase).

For the assay, to confirm the correctness of the plasmid sequence, the ligation mixtures are applied for transformation of E.sOH K12 strain 294 (ATCC 31446) and transformants selected for atrCNHp or 1E1gasNpe resistance, where appropriate. Plasmids from transformants obtained by restriction and / or combination of the Mez51P8 method were obtained (α1, ΝικΙείε

I

Ααάχ. Ke $. 9: 309 (1981) or by the Mahat method, is! ak ΜείΗοόκ ΐη by Egguta ^ y, 65: 499 (1980).

The amplification of DHFR protein coding sequences is accomplished by growing host cell cultures in an environment of approximately 20-500,000 nM concentration of methotrexate, a suitable inhibitor of DHFR activity. The effective concentration range is highly dependent, of course, on the nature of the DHFR gene, the protein, and the characteristics of the host cell. It is clear that universally defined upper or lower limits cannot be established. Appropriate concentrations of other folic acid analogues or other compounds that inhibit DHFR may also be administered. However, MTX is convenient and effective to use.

D. General description of preferred embodiments of the invention

Human tissue plasminogen activator was prepared according to the following protocol:

1. Human melanoma cells actively producing tissue plasminogen activator were cultured by fusion.

2. Cell granules from such cell cultures were extracted in the presence of ribonuclease inhibitors to isolate all cytoplasmic RNA.

3. An oligo-άΤ column isolates all matrix (information) RNA (mRNA) in polyadenyl form. This mRNA was size fractionated by acid urea agarose gel electrophoresis.

4. The gel fraction containing specific RNA for the tissue plasminogen activator was identified as follows: The RNA of each of the gel fractions was translated into rabbit reticulocyte lysate ΐη νΐίτο by the addition of canine pancreatic microsomes. The resulting translation products were immunoprecipitated with a specific ΙβΟ antibody.

5. Appropriate RNA (21 to 248) was converted to the corresponding single strand complementary DNA (cDNA) from which double stranded cDNA was obtained. After poly-6C addition, the latter was inserted into a vector as a plasmid carrying one or more phenotype markers.

6. The vectors thus obtained were used to transform bacterial cells providing a clonal cDNA library. A pool of radiolabeled synthetic 5 deoxy oligonucleotides complementary to codons for known amino acid sequences in m-PA, such as the 814-tag5 pool.

A A C

5 '-dTS () CA () AND () TCSS-3 "

A C T (complementary to sequences coding for a known n-1n1-amino acid sequence: tryptophan-glutamic acid-tyrosine-cysteine-aspartic acid (No. E-Υ-Οϋ) was obtained and used to probe the colony library.

7. From positive cDNA clones, plasmid DNA was isolated and structured.

8. Structured DNA encoding m-PA was then added ϊη νΐίτο for insertion to a suitable expression vehicle, which was used to transform a suitable host cell to grow into cell culture and produce the desired human tissue plasminogen activator.

9. Thus produced, the human tissue plasminogen activator has 251 amino acids in its enzyme series - protease region and a "kp £ 1e" containing a structure that is thought to be related to fibrin binding.

The above procedure is effective in obtaining pure t-PA. Methods of the invention employing additional methotrexate-sensitive coding sequence allow the production in cultures of host cells of m-PA with antigenic activity in amounts greater than 0.1 µg per cell per day. With appropriate application of boosting conditions, yields greater than 20 pound per day per cell can be achieved.

Alternatively, gene expression levels achieve a production of more than 9 x 10 ' ⁶ PE per cell per day, and with appropriate amplification, more than 18 x 10' ⁴ PE per cell day m- PA can be produced.

The advantages obtained in this aspect are in the invention of methotrexate, such as

I preparation, which is normally fatal for cellular uptake and enables cellular growth in an environment of controlled levels of MTX, by amplification of the gene encoded for the DHFR code structure (3Gtpke, CoeP T. e! A! ,, δεΐεηεε 202: 1051 (1978); Shesiyeg, L.E. e! A1, Sapseg Kez. 32: 153 (1972); Sap £, δ.E.E! A1, Sep 7: 391 (1976).

The importance of this aspect of the invention shows that amplification of the DHFR gene can cause amplification of associated sequences that encode other proteins. This occurs when the associated protein is hepatitis B surface antigen (HBxA®) (Cbhpap.L.La1, Rgos.

AsoO. 5c, 79: 1815 (1982); a protein of E.c.O. and XRDCT (Kdp0oM, SogPop, e1a1, 1.Moes.ap <1 ArrCep., 1: 165 (1981)); and endogenous sequences of DHFR / 30X40 plasmid combination (CaiRtap, KR is! a1, ί.ΜοΙεσ. ΒίοΙ. 159: 601 (1982)).

Other mechanisms related to methotrexate resistance include a decrease in the binding affinity of the DHFR protein so that it is less susceptible to methotrexate (ΡΗηίοίί. \ Ν.Ρ. E! A !, 8 so Sep Sep 2: 245 ( 1976).

Both the wild-type DHFR genes and the MTX-resistant protein DHFR have been found to be amplified by the presence of MTX due to their own reduced binding capacity. In principle, this aspect of the invention relates to the use of the amplification effect of DHFR structure on associated protein coding sequences to provide a control mechanism that allows the expression levels of m-PA sequences to be increased, in the presence of MTX or due to prior treatment of transformed cells with MTX.

F. Examples

The following examples are intended to illustrate, but not limit, the invention. In these experiments, cultures of E.coI and CHO cell lines suitable for the protein type DHFR code sequence were used as host cells. However, other eukaryotic and prokaryotic cells are also suitable for the methods of the invention.

F.1 Human m50 gene expression

PA in E.soI

F.1.A Legend to the figures

FIG. 1 is an autoradiogram of δϋδ RAOE - 10% sodium dodecyl sulfate polyacryl 5 lamide electrophoresed gel expressed by immunoprecipitated [ ³⁵ δ] - methionine - tagged protein (s) secreted by human melanoma cells for 3 hours νη ν in the presence (lane b) or in the absence (lane a) of the protease inhibitor aprotinin. After immunoprecipitation with tissue plasminogen activator specific ΙβΟ, three bands white observed (band a), with molecular weights of approximately ca.

65000, 63000 and 35000. However, in the presence of a protease inhibitor, species with 35,000 molecular weight were not observed. When predominant serum (band c) was used, the products were not immunoprecipitated. To the left of lane a are the transitions and molecular weights of protein standards labeled with ^I C.

Figure 2 depicts gel immunophoresis of immunoprecipitated translation products from RNA fractions isolated from acidic urea agarose gel. A large band was observed in fractions of numbers 7 and 8 after translation in the presence of canine pancreatic microsomes, followed by immunobuptake 30 with tissue plasminogen activator specific ΙβΟ. This band has a molecular weight of 63,000 daltons. The size of the mRNA migrating in fractions 7 and 8 is approximately 21 to 248. The positions of ribose35 RNA markers that were determined after RNA urea electrophoresis and visualized by ethanide bromide are indicated above the corresponding gel band.

FIG. 3 presents the hybridization model of 96 bacterial colonies with

A A C

P-cGGS () CA () TA () TCSSA (\ UEU-C-O) probe CCT individual transformants sprouted in microtiter medium, a microorganism imprint on a nitrocellulose membrane. The colonies were then lysed, bacterial DNA fixed, and filters were hybridized with ³² P-14-tag probes (\ ν-Ε-Υ-Ό-ϋ). The filters were pro11

I myths to remove non-hybridized sample and exposed on x-ray film. This autoradiogram is representative of the configurations obtained with 48 individual filters (4600 individual colonies). An example of a positive tissue plasminogen activator cDNA clone on filter number 25 is designated E10 (arrow).

Figure 4 is a full-length map of endonuclease restriction of m-PA cDNA. The number and fragments obtained from cleavage by endonuclease restriction were determined by electrophoresis on a 6 percent acrylamide gel. The positions of the regions were confirmed by a nucleic acid sequence (presented in Fig. 5). The coding region of the largest open frame is the sequence of the putative signal peptide, while the dotted region represents the sequence of the putative mature tissue plasminogen activator (527 amino acids). The 5 'end of the mRNA is on the left and the 3' end is on the right.

Figures 5a, 5b, 5c illustrate the nucleotide sequence and the extracted full-length amino acid sequence of a human tissue plasminogen activator cDNA. A record is shown as a continuous sequence of 35 amino acids (-35 to -1), representing their mature configuration. This 35-amino acid sequence is assumed to consist of a hydrophilic "pr" sequence, recording the series (+1) of the mature protein, from about 12 to 15 amino acids, in order preceded by a "conventional" hydrophobic signal (extending from 5 'to -35). This type of pre-sequestration of secreted proteins has been described previously, for example with preproalbumin. According to this theory, all secreted tissue plasminogen activator molecules will start with serine (+1) as the amino terminal. Another theory suggests that the hydrophilic sequence may be involved in the function of tissue plasminogen activator in a similar manner to that observed with plasminogen, where a 10,000-daltone peptide can be degraded by the amino-terminal portion of a natural plasminogen (C1-plasminogen) into smaller molecules, with a new amino terminal, modeled LUH-plasminogen. The Luz-plasminogen is more readily activated to plasmin and also has a greater affinity for fibrin than the Cl-5 plasminogen. Plasmin has been shown to catalyze the conversion of Cl-plasminogen to Luz-plasminogen. This type of control mechanism results in a “feedback positive” mechanism. The first amounts of 10 plasmins form degraded fibrin and also result in the generation of plasminogen and molecules that are easier to activate and bind more closely to their substance than natural plasminogen. The result 15 is faster degradation of fibrin. The hydrophilic peptide of the tissue plasminogen activator may be involved in a similar mechanism, its degradation is the result of a modified binding of the enzyme to fibrin. In each case, the 35 amino acid sequence is regarded as a sequencing of the mature protein.

Figure 6 is a schematic view of the tissue plasmidogen activator expression plasmid pc1ekK1PA. The starting plasmid pbECA25E10 was first cleaved with P3H to an isolate 376 bp fragment, which was then digested as shown in the figure.

Figure 7 shows the results of the analysis of the fibrin layer for the fibrinolytic activity of the expression product obtained U1a pbECKRA in transformed cells.

Figure 8 is a TXVH record (high pressure liquid chromatography) of human tissue plasminogen activator peptides from the digestive enzyme trypsin (Absorption at 210 pt). The arrow identifies the peak corresponding to the peptide used to construct the nucleotide probe provided by the colony library. The peptide represented by this peak was digested with respect to its entire sequence: b-T-λΥ-Ε Υ-C O-Y-P-5-C-5-T-C-C-b.

The sequences of the other large peaks were also sequenced and it was found to confirm the amino-sequence correctness of the human tissue plasminogen activator. The one-letter peptide code for amino acids is the following:

I

Ahr ϋ Asparagine to No I Isoleucine Tgh T. Threonine Yeah B Levcin 5g 5 Series Tug Υ Tyrosine C1i Well Glutamine to-on Rue Well Phenylalanine Rgo R. Proline Ηίχ B Histidine C1u P Glycine Buh K. Lysine A1a A Alanine Ατβ K. Arginine Dry P Cysteine Tr \ ν Tryptophan ν ₃ ι V Valine C1p Ω Glutamine Me! m Methionine Ahh N Asparagine

Figure 9 shows the construction of a plasmid encoded for the direct expression of mature human m-PA in E.coH. 50 μ% of plasmid pPA17 were digested with 8aI3A1, ΗϊικΙΙ and Hbα1 and electrophoresed on 6 percent polyacrylamide gel. Approximately 0.5 μ &apos; of the 55 bp 8aa3A1-HbA1 fragment were reproduced. Similarly, about 3μβ of the 263 bp Hba1-No.1 fragment was purified from 80 µ§ by clone pPA25E10 by first isolating the 300 bp ΡχίΙ - Jag1 fragment. All decompositions were carried out at 37 ° C for one hour and the reaction products were dissolved and electroeluted from 6% polyacrylamide gel. The two indicated deoxyoligonucleotides 5 'OAATTSTATTSTASTAST (I) and 5' SATSATTTATTATASASTATO (I) were synthesized by solid phase phosphotriester (51). 100 µl of oligonucleotide II were phosphorylated in a 30 μl reaction mixture containing 60 mM Tnx (pH 8), 10 mM ΜβΟ ₂ , 15 mM β-mercaptoethanol and 50 μ Οί [at ³² P] ATP (AteghNat 5000 SP ttoR ¹ ). , 12 units of T4 polynucleotide kinase were added and the reaction continued at 37 ° C for 15 minutes, 1 μΐ of 10 mM ATP, and 12 units of T4 kinase were added and the reaction continued for an additional 30 minutes. After extraction with phenol / CHCl1, the ₃ phosphorylating oligomer II and the 5 'hydroxyl oligomer I were combined with 0.5 μβ eluted 55 bp 8a1A1-HbA1 fragment and precipitated with ethanol. These fragments were ligated at room temperature for 4 hours in 60 μΐ of 20 mM Tpx-HCl (pH 7.5), 10 mM M ₈ Cl ₂ 10 mM dithiothreitol, 0.5 mM ATP, and 1000 units of T4 DNA ligase. The mixture was dissolved for 1 hour with 48 units of No. 1, 20 units of EcoK1, and 40 units of ΒβΙΙΙ (to eliminate polymerization by ligation of the coupled 8aI3A1 terms) and electrophoresed on a 6% gel. 338 bp product (approximately 0.1 μβ) was recovered by electroelution. The residues of the m-PA code structures (amino acids 111-528) were isolated on a 1645 bp fragment using the plasmid pPA25E10 of No. 1 and ΒβΙΙΙ. The plasmid pE1PA1g103 is a derivative of the plasmid pE1PA25 (52) in which the EcoK1 region distal to the LE1P A gene has been removed (53). Three μβ of pBe1RA1gp103 were digested with 20 units of EcoK1 and 20 units of ΒβΙΙΙ for 90 minutes at 37 ° C, electrophoresed on a 6 percent polyacrylamide gel, and the large (4200 bp) vector fragment was recovered by electroelution. For the final construct, 80 ηβ of ΕεοΚΙ-ΒβΙΙΙ pBe1PA1g103 was ligated with 100 ηβ of 1645 bp ΝαΓί-ΒβΙΙΙ fragment and 20 ηβ 338 bp EcoK1-No1 fragment for 10 hours at room temperature. Plasmin produces a clean lysed zone in the fibrin layer and the area of this zone may correlate with the amount of tissue plasminogen activator in the sample.

E.1.B. Tissue plasminogen activator mRNA source

Human melanoma cells (Βοχνεχ Me1apota) cell line, ATCC number SKB9607) were used. The melanoma cells were cultured on a confluent monolayer culture in 100 µg Eaglec M1hta Exhepb1 Messia with sodium bicarbonate added (0.12 percent final concentration), 2 mM glutamine and 10 percent heat inactivated fetal calf serum. To confirm that melanoma cells actively produce human tissue plasminogen activator, human melanoma cells were cultured until confluent in 24 well microtiter plates. Depending on the presence or absence of 0.33 μΜ protease inhibitor aprotinin, the cells were washed once with phosphate buffered saline and 0.3 ml of sulfur-purified methionine. 75 μ Οί of [ ³⁵ 8] methionine were added and cells were labeled for 3 hours at 37 ° C. At the end of the three hour marking period, the medium was removed from the cells and treated with either tissue plasminogen activator specific ββΟς or pre-immune immunoprecipitation serum (54). Immunoprecipitation products were shown by electrophoresis on 10 percent 5C5-acrylamide gel. The layer was fixed, dried and subjected to fluorography.

F.1.C Isolation of RNA and size fractionation.

All RNA from melanoma cell cultures was extracted according to the ν / agd e) ak method (55). The cells were precipitated by centrifugation then resuspended in 10 mM No. Cl, 10 mM Tn3-HCl pH 7.5,

1.5 mM M £ Cl ₂ . Cells were lysed by adding ΝΡ-40 (1 percent final concentration) and nuclei were precipitated by centrifugation. The supernatant contained all the RNA, which was further purified by repeated extraction with phenol and chloroform. The aqueous phase was made 0.2 M in NaCl and then all RNA was precipitated by the addition of 2 volumes of ethanol. Oligo - άΤ cellulose chromatography has been used to purify mRNA from all RNA obtained (54). Typical yields of 10 grams of cultured melanoma cells were 5 to 10 milligrams of total RNA and 50-200 micrograms of Ro1u (A) p1g mRNA.

Fractionation from Po1uA ⁺ mRNA (200 μβ) (56) was performed by urea-agarose gel electrophoresis. The agarose gel layer (57, 58) was composed of 1.75 percent agarose, 0.025 M sodium citrate, pH 3.8, and 6 M urea. Electrophoresis was performed for 7 hours at 25 milliamps and 4 ° C. The gel was then fractionated with a razor blade. The individual layers were melted at 70 ° and extracted twice with phenol and once with chloroform. The fractions were then precipitated with ethanol and subsequently analyzed by ΐη νΐίΐΌ translation into rabbit reticulocyte lysate systems, VeFehba KeeseagN EaB. (59, 60) mixed with canine pancreatic microsomes as follows: The translations were performed using 25 μ <E of [ ³⁵ 5] methionine 500 nanograms of each RNA gel layer in a final volume of 30 μ1 containing 25 mM HEEP5, 43 , 8 mM potassium chloride, 10 mM creatinine phosphate. 19 amino acids 50 mM each, 1.1 mM magnesium chloride, 16.6 mM ethylenediaminetetraacetic acid (EDTA), 0.16 mM dithiothreitol, 8.3 mM chemin, 16.6 μβ / ηιΐ creatinine kinase, 0.33 mM calcium chloride , 0.66 mM ESTA, 23.3 mM sodium chloride.

The incubation was carried out at 30 ° C for 90 minutes. Canine pancreatic microsomal membranes obtained from crude microsomes using ΕϋΤΑ to remove ribosomes (61) and treated with nuclease as described previously (62) and placed in translation medicine at a final concentration of 7 A ₂₆₀ units / ml. The translation products of the immunoprecipitated translation products were analyzed by electrophoresis on a 10 percent polyacrylamide gel with sodium dodecyl sulfate as described previously (63). The discolored gel layers were fixed, dried and subjected to fluorography (64).

The resulting translation products from each gel fraction were immunoprecipitated with rabbit anti-human tissue plasminogen activator specific ΙβΟ. One major immunoprecipitated polypeptide bond was observed in the translation of RNA fractional numbers 7 and 8 (migrates 21 to 245) having a molecular weight of approximately 63,000 daltons. This relationship was not observed when pre-priming Ιβ за was used for immunoprecipitation, suggesting that these polypeptides were a specific tissue plasminogen activator.

E.G. Obtaining a library of colonies containing tissue plasminogen activator sequences.

Five μβ of gel fractionation mRNA (gel layer 7 mRNA) was used to produce double-stranded cDNA by standard procedures (52, 65, 66). The cDNA was fractionated on a 6 percent polyacrylic amide gel. cDNAs larger than 350 base pairs in length (125 ηβ) were electroeluted. 30 µl of cDNA was expanded with deoxy (C) residues using terminal deoxynucleotidyl transferase (67) and renatured with 300 µl of plasmid pBK322 (68), which is similarly bound to deoxy (C) residues in the ΡχΙ region I (67). The reconstituted mixture was then transformed into E.c.H.K12 strain 294 (ATCC 31ο 31446). Approximately 4,600 transformants were obtained.

E.1.D. Obtaining a DNA probe

Purified human tissue plasminogen activator was obtained using the procedures described in the accompanying literature (19, 20).

The molecule was scanned to locate the regions most suitable for synthetic sampling as follows:

To make the protein suitable for trypsin degradation, it was reduced and carboxymethylated. 2 µg of tissue plasminogen activator were first dialyzed in 0.01 percent Tuep 80 overnight at room temperature. The lyophilized protein was then dissolved in 12µl of 0.56M TP8-HCl buffer (pH 8.6), 8 molar in urea and 5mM ΕϋΤΑ. The disulfide bonds were reduced by the addition of 0.1µl of D-mercaptoethanol. This reaction was carried out under nitrogen for 2 hours at 45 ° C. The reduced disulfides were alkylated to the carboxymethyl derivative with the addition of 1.0 µl of 1.4M iodoacetic acid in 1Ν CaOH. After 20 minutes at room temperature, the reaction was quenched by dialysis at 0.01 percent Tdaep 80, for 18 hours at room temperature and lyophilized.

The resulting lyophilized carboxymethylated protein was redissolved in 3 m! 0.1M sodium phosphate buffer (pH 7.5). Trypsin (TPCC) (ratio 1 to 50) was added and dissolved at 37 ° C. Multiple amounts (0.1 ml) were taken at 3 hours, 6 hours and 12 hours. A second trypsin addition was carried out at 12 hours. After 24 hours, the reaction was stopped by freezing the sample until it could be injected with HPLC. The degradation progress was determined by 808 gel folds. All gels were empty except for the poorly expressed 3-hour band. This indicates that the 24 h degradation is complete and no major peptides remain.

A sample (0.5 m1) was injected into the high resolution ABEX C-8 ultrasphere 5 μ with two runs. An acetonitrile gradient was prepared in a stepwise fashion (1 to 5 percent in 5 minutes, 5 to 35 percent in 100 minutes, 3550 percent in 30 minutes). In one of the two preparation runs, the eluant was monitored at two wavelengths (210 ps and 280 ps). The extent of absorption at both wavelengths was used to indicate tryptophan-containing peptides.

The peptide peaks, most probably because of their tryptophan content or because of their use for other reasons, were first sequenced. This allowed the determination of the sequence of most of the tryptophan peptides. After sequencing of about 25 of the most probable peptide peaks, all sequentially regulated data were pooled to obtain a preliminary model of the primary tissue plasminogen activator structure. From this data and this model, many possible probes were located.

E.1.E. Identification of bacterial clones containing cDNA sequences of a tissue plasminogen activator.

The colonies were individually inoculated into the wells of microtiter plates containing bB (93) + 5 μl / πι1 tetracycline and stored at -20 ° C after the addition of ϋΜ8Ο to 7 percent. Two copies of the colony library were grown on nitrocellulose filters and DNA from each colony fixed on the filter, according to the method of Wohh Pei85 (69).

The probe

A A C

P-labeled-TC () CA () TA () TCCCA was obtained from (synthetic oligomer) OU-E-Y-C-P) 14-tag stock as described above. Filters containing 4600 transformants were re-hybridized for 2 hours at room temperature in 50 mt sodium phosphate pH 6.8, 5 x 88C (80), 150 lb. salmon sperm DNA, 5 x solution

I of Odepagsk (85) 10 percent formamide and then hybridized with 50 χ 10 ⁶ per minute of the indicated sample in the same solution. After overnight incubation at room temperature at 6 x 88C, 0.1 percent 8ϋδ for 30 minutes, 5 once in 2 x 88C and then exposed to ΚΟϋΑΚ X-5 X-ray film with ϋιιροηΐ ίίβΗΐηίηβ Ρΐιιχ amplifying screens for 16 hours.

The plasmid DNA was isolated by rapid method (71) from all colonies to show a positive hybridization reaction. cDNA inserts from these clones were then sequenced after subcloned fragments in M13 vector tr 7 (73) and according to the Mahat OP'egG chemical procedure (74). Fig. 3 shows a filter of No. 25 filter showing the hybridization configuration of a positive tissue plasminogen activator clone. the cDNA insert in clone 25E10 was demonstrated as DNA encoded for tissue plasminogen activator by comparing its amino acid sequence with a peptide sequence (see above) obtained from purified tissue plasminogen activator and by expressing the product produced, as described below in more detail below. The cDNA insert of clone 25E10 (plasmid pPA25E10) had 2304 base pairs in length with the longest open reading frame encoding a protein of 508 amino acids (M> ¥ 56 756) and containing 772 bp 30 3 'untranslated regions. This cDNA clone lacks the Ν-terminal coding sequence.

Bacterial clone E.soI (pPA25A10), and also as a sample of plasmid pPA25E10, were deposited in Atepsap Ture SiGiGe 35 SoIsIop and obtained Nos. 67587 and 40401, respectively.

E.1.J. Direct expression of human tissue plasminogen activator clone in E.coI.

As in FIG. 6.50µ &apos; of pPA25E10 were digested with P &lt; 1 &gt; I and a 376 bp fragment isolated by electrophoresis on a 6 percent polyacrylamide gel solution. Approximately 3 μ &apos; of this fragment were 45 isolated from the gel by electroelution, digested with 30 units of ϋάε I for 1 hour at 37 ° C, extracted with phenol and chloroform and precipitated with ethanol. The obtained ϋάε I adhesive ends were expanded to 50 Ь1 (ends) with the addition of 5 units of DNA polymerase I (fragment of

)Ίεηοχν) and 0.1 mM for each of άΑΤΡ, dSTR, άΟΤΡ, άΤΤΡ to obtain the reaction mixture and subsequent incubation at 4 ° C for 8 hours. After extraction with phenol and chloroform, the DNA was digested with 15 units of H2O 1 for 2 hours and the reaction mixture was electrophoresed on a 6 percent polyacrylamide gel. Approximately 0.5 μ &apos; s of the desired 125 Lp &lt; RTI ID = 0.0 &gt; &lt; / RTI &gt; This fragment encodes amino acids numbered 69 to 110 in the mature full-length tissue plasminogen activator protein.

For the isolation of 1645 bp # 2 I - B§1 II fragment, 30 μ &apos; of pPA25E10 were digested with 30 units of # I and 35 units of Ββΐ II for 2 hours at 37 ° C and the reaction mixture electrophoresed on a 6 percent polyacrylamide gel. . Approximately 6 μ &apos; of the desired 1645 bp Hag - ΒβΙ II fragment were recovered.

The plasmid pdecK18KC, which is derivative of plasmid p8KCeh16 (79), in which the Eco-1 region is proximal to the 1g promoter and distal to the 8KC gene, was removed by DNA polymerase I repair (28), and complementary The oligodeoxynucleotide AATTATSAATTSAT (synthesized by the phosphotriester method) was inserted into the residual Eco непосред site immediately adjacent to the Xaa I site, 20 μ% of p0eKK18KS was digested with Eco K1, extracted with chloroform and extracted with chloroform. The plasmid was then digested with 100 units of nuclease 81 at 16 ° C for 30 minutes in 25 µM sodium acetate (pH 4.6) 1 µM Ζηα ₂ and 0.3 M NaCl 1 to create the 'b1ip1' end with ATC sequence. Following extraction with phenol and chloroform and ethanol precipitation, the DNA was digested with Wt, electrophoresed on a 6 percent polyacrylamide gel, and the large (4300 bp) vector fragment was extracted by electroelution.

The expression plasmid was assembled by ligation together with 0.2 of μ &apos; s, 0.06 μ &apos; of 125 bp "L1ipG" end - No. I fragment and 0.6 μ% of 1645 Ip No. 1 - Ββΐ II fragment with 10 units of T4 DNA ligase for 7 hours at room temperature and when used to transform E. coli strain 294

I (ATCC 31ο 31446) for ampicillin resistance. DNA plasmid was obtained from 26 colonies and digested with Xaa I and Eco ΚΙ. 12 of these plasmids contain the desired 415 bp Xba-Eco-E and 472 bp Eco-E fragments. DNA sequence analysis verified that several of these plasmids had an ATC (Anti-Thymocyte Globulin) stimulation code correctly set at the start of amino acid number 69 (serine). One of these plasmids, p (1eK1PAKa) was tested and produced the desired tissue plasminogen activator (Figure 7) .The bacterial clone E.cOP (LE11aK1RA) and also a sample of plasmid pBe11aK1RA were deposited in Sytepsap75 and 85 and 40400, respectively.

E. 1.3. Full-length tissue plasminogen activator cDNA

a) Obtaining a library of colonies containing N-terminal tissue plasminogen activator sequences: 0.4μς from the synthetic oligonucleotide

5 'TTSTASSASSASSESS 3' was used for priming a 7.5μς gel fraction of 8 mRNA (above) to obtain double stranded cDNA using standard procedures (65, 66). The cDNA was size fractionated on a 6 percent polyacrylamide gel. A fraction larger than 300 base pairs (36 n & a) was electroeluted. 5 ng cDNA was elongated with deoxy (C) radicals using terminal deoxycytidyl transferase (67) and renaturation with 50 ng of plasmid pBK322 (68), which was similarly linked to deoxy (O) radicals in the ΡχΙ region I (67). The reconstituted potion was then transformed into E.cOPP12 strain 294. Approximately 1500 transformants were obtained.

b) Human genomic DNA hybridization: Since the cDNA priming reaction was performed using a synthetic fragment that hybridized 13 base pairs from the N-terminal of clone pPA25E10, an inappropriate restriction fragment became available in this region with 29 base pairs 16-tag sequence) for cDNA clone screening. Therefore, it was necessary to isolate a human tissue plasminogen activator genomic clone to identify all initially expanded cDNA clones containing code sequences of a tissue plasminogen activator with a Ν-terminal.

The first step in this process involves establishing that a single homologous tissue plasminogen activator gene is present in human genomic DNA. To determine this, 8oi1Ngp hybridization was performed. In this procedure (77), 5 μς of human high molecular weight lymphocyte DNA (obtained as in 80) was completely digested with various restriction endonucleases, electrophoresed on a 1.0 percent agarose gel (81) and dried on a nitrocellulose filter ( 77). A ³² P-labeled DNA probe was prepared (76) from the 5 'end of the cDNA insert of pPA25E10 (230 bp Hra II - Kza 1 fragment) and hybridized (82) with a nitrocellulose filter. 35 χ 10 ⁶ per minute of sample were hybridized for 40 hours and then washed as described (82). Two endonuclease degradation structures provide only one hybridizing DNA fragment: B ₈ 1 H (5.7 Kp) and Ρνυ II (4.2 Kp). Two hybridizing DNA fragments were observed with Htc II (5.1 Kp and 4.3 Kp). Taken together, these data suggest the presence of a single tissue plasminogen activator gene in the human genome, and that this gene contains at least one intervening sequence.

c) Screening of a human lambda phage library for tissue plasminogen activator genes.

The strategy used to identify lambda phage recombinants carrying tissue plasminogen activator genes consists of detecting a homologous nucleotide with a probe prepared from the tissue plasminogen activator of cDNA from pPA25E10. One million recombinant phage lambda were seeded on a Petri dish with ϋΡ 50 8 µR P at a density of 10000 µg / 15 cm, and a nitrocellulose filter was prepared for each plate by the method of Veppup ap OaU15 (78). By standard procedure (83). ^{A 32} P labeled DNA probe was obtained from 230 base pairs of HPA II - Kza I fragment localizing 34 base pairs from the 5 'end of plasmid pPA25E10. Each nitrocellulose filter was re-hybridized at 42 ° C for 2 hours in 50 mM sodium phosphate (pH 6.5), 5 x 85C (77), 0.05 ητβ / πιΐ ultrasound, salmon DNA, 5 x solution on Pepaggs (84),

I percent formamide and then hybridized with 50 χ 10 ⁶ per minute from the labeled probe in the same solution containing 10 percent sodium dextran sulfate (85). After overnight incubation at 42 ° C, the filters were washed 5 times 4 times at 50 ° C in 0.2 x 88C, 0.1 percent 5ϋδ for 30 minutes, once in 2 x 58C at room temperature and then exposed to COAac HC-5, an X-ray movie with Tsyrop! I crashed a boost screen overnight. 10 All 19 clones were harvested, which were hydrated with the probe. Phage DNA was obtained as previously described (86) by 6 recombinants. Lambda clone C was selected to obtain fragment Ρνυ 11 for 15 colony screening. 30 µg of DNA was digested with Ρνιι II for 1 hour at 37 ° C, electrophoresed on a 1.0 percent agarose gel. A 4.2 kilogram fragment with already proven content of tissue plasminogen activator sequences was electroeluted and purified. The ³² P marker probe was obtained by standard colonization hybridization procedures (83) as described below.

c) Screening a colony library 25 for 5 'tissue plasminogen activator sequences.

Colonies were transferred from the plates and seeded with nitrocellulose filters, and DNA from each colony fixed to the filter, 30 according to the Ноηιηδίείη - No ^ pezz procedure (69). ^{The 32} P-labeled sample was prepared from beef thymus priming (83) 4.2 kBp Ρνιι II fragment from an isolated tissue plasminogen activator lambda genomic clone. Filters containing 35 transformants were hybridized with 112 x 10 * cpm of ³² P-genomic PcII fragment. The hybridization was for 16 hours, using the conditions described by Pgc3b! a1. (82). The filters were extensively washed and 40 were then exposed to a Co <1K HC-5 X-ray film with ϋιιροηΐ ΕίβΗίηϊηβ - P1x amplification screens for 16-48 hours. 18 colonies clearly hybridized the genomic probe. DNA plasmid was isolated from each of these colonies and 45 was bound to nitrocellulose filters and hybridized with ³² P-labeled synthetic oligonucleotide (16-joke) used for the original priming reaction. Of the eighteen clones, seven hybridized with kinase 16. From the sec-50 assay after subcloned fragments in the γ13 vector tr ⁷ (73), one clone (pPA17) was shown to contain the correct 5 ′ N terminal region of a tissue plasminogen activator, a signal leading sequence, and 84 bp 5 ′ untranslated region. From both clones pPA25E10 and pPA17, the complete nucleotide sequence (Fig. 5) and the full-length restriction configuration (Fig. 4) of the tissue plasminogen activator clone were determined.

The bacterial clone E.soH (pPA17), as well as the probe of plasmid pPA17, were deposited in the Ashepsap Toure Cicige SoSesbop and received an inlet. 6ο 67586 and 40402 respectively.

The natural tissue plasminogen activator molecule has the potential to stabilize through 17 disulfide bridges based on homology to other protease series. There are four potential N - glycosylation regions, three localized "kp £ 1e" regions, azp ₁₁₇ , a8p _{) 84} , a8p ₂₁₈ regions, and one potential region of the light chain region, a5P ₄₄₈ . Variations in the structure of oligosaccharide ligands may be responsible for the different molecular forms (65,000 and 63,000 moles).

E.1.I. Direct expression in E.c.H of a full-length tissue plasminogen activator cDNA clone.

Reconstruction of the complete coding sequence was possible thanks to the use of a simple HbA1 restriction endonuclease region separated by the two partial clones pPA17 and pPA25E10. The restriction fragment 55 bp 8a and 3A1-HbA1, corresponding to amino acids 5-23, was isolated from plasmid pPA17. The 8aI3A1 restriction region was localized to a codon, four of the putative mature code sequences, and was applied to remove the signal peptide code region. The 263 bp Hbα1ΝηγΙ fragment (coding for amino acids 24-110) was also isolated from plasmid pP25E10. Two synthetic deoxyoligonucleotides were identified by the recovered amino acid codes 1-4, incorporated on the ATC (translational glob.) Translational stimulating codon and created by the EcoK1 binding term. These three fragments were then ligated together to form the 338 bp fragment encoding for amino acids 1-110. This fragment and the 1645 bp ΝβγΙ-Β ^ ΙΙΙ fragment from pPA25E10 were then ligated

I between the EcoK1 and Ββци regions of plasmid pBe1P1gr103 (53) to give the expression plasmid p (-PRA1p12). Oa1 Agpo sequence of 1 g leader peptide, but lacks the leader peptide ATC stimulus codon (52).

A sample of plasmid p1-PA1gr12 was deposited in Atepsap Ture Siigue SoIsIop and was assigned number 40404.

E.1.Y. Sequential analysis

Sequence analysis was based on Ebtap degradation (83b). The sample was placed in a glass of Vesktap 890B or 890C sequencer. RoLugepe TM (poly Ν, Ν, Ν ′, Ν ^1- tetramethyl- ^1H -trimethylenehexamethylene diammonium diacetate) was used as a carrier in the beaker (63C). The sequencer was modified with a cold trap and several program changes to reduce background peaks. The reagents were Vesectap Sequence 0.1 M 9aabgo1 buffer, phenylisothiocyanate and heptafluorobutanoic acid.

The selected Edman cycles were manually converted to 2-anilino-5-thiazolinone derivatives. 1-Chlorobutane was dried under nitrogen. Then 1.0 N hydrochloric acid in water was added to 2-anilino-5-thiazolinone and heated to 70 ° C for 10 minutes to convert to 3-phenyl-2-thiohydanthion (FTX derivative). The FTX amino acid residue was then dissolved in 50% acetone nitrile and water and injected into a high-pressure, reversed-phase liquid chromatograph. Each FTX amino acid was then identified by comparison with the retention times of the standard FTX amino acid mixture that were introduced into the conversion vial and treated in the same manner as the sequencer cycle.

E.1.K. Assays for detection of tissue plasminogen activator expression

1. direct analysis of plasmin formation

a) Theory

Sensitive tissue plasminogen activator assay can be obtained by monitoring the tissue plasminogen activator catalyzing the conversion of plasminogen to plasmin. Plasmin is an enzyme for which chromatographic analyzes exist. These assays are based on the proteolytic degradation of a chromophore group tripeptide. The degree of degradation of di5 directly depends on both the specificity and the concentration of the protease that has been tested. The basis of anilysis is the determination of the amount of plasmin formed by the incubation of a tissue plasminogen activator containing a solution with a plasminogen solution. The highest amount of activator forms the largest amount of plasmin. Plasmin was measured by monitoring its degradation by chromatogenic substrate 82251 (supplied by Kash Ogoir, Ιηί. Sheshuyub, ST.).

b) Procedure

Multiple samples were mixed with 0.10 mI of 0.7 ηΐβϊ / πιΐ plasminogen (in 0.05 M Tp 3 HCl, pH 7.4 containing 0.012 M MacI) and an additional volume of 0.15 m1. The mixture was incubated at 37 ° C for 10 minutes, 0.35 ml of 82251 (1.0 µM solution in the above buffer) was added, and the reaction continued for 30 minutes at 37 ° C. Glacial acetic acid (25 μΙ) was added to terminate the reaction. Samples were centrifuged and the absorbance measured at 405 nm. Activity quantification is achieved by comparison with a standard urokinase solution. The activity was recorded in P10 and P1b units, with 90000 P1 and P1b units equivalent to the activity shown by 1 pound of purified tissue plasminogen activator.

2. Indirect analysis of plasmin formation

a) Theory

A sensitive analysis of tissue plasminogen activator activity has been proposed (87). The assay is based on the determination of plasmin formation by measuring the amount of plasmin degradation from fibrin in agar medium containing fibrin and plasminogen. Plasmin produces a prominent lysis zone in the fibrin plaque. The area of this lysis zone may correlate with the amount of tissue plasminogen activator in the sample.

b) Procedure

Following the procedure of Sgapesh Iregpo and Kesch (87), the plates were incubated19

I wound 3.5 h at 37 ° C and the lysis zone was measured. Quantification was achieved by comparison with standard urokinase solution.

E.1.L. Determination of tissue plasminogen activator activity

1. Bacterial development and sample preparation.

A plasmid-containing E.cone colony (p <1e11aK1PA ”) was inoculated into a tube containing 5 mI of EB medium with 20 χζβ / ιηΐ ampicillin. The cells were left overnight at 37 ° C. A portion of this culture was diluted 1: 100 in 300 µl of M9 medium containing 20 μβ / πιΐ ampicillin. The cells were left in a flask at 37 ° C for 4 hours, yielding a 550 nm absorbance of 0.419. The tryptophan analog of indolacrylic acid was added to a concentration of 30 µg / ιηΐ. The cells were incubated for 90 minutes with an ongoing absorption of 550 ps of 0.628. The cells were separated by centrifugation and resuspended in 0.8 µl of 0.01 M Tx, pH 8.0 containing 0.01 Μ EFA. The resulting suspension was stirred at room temperature for 18 hours. The sample was centrifuged and the supernatant was analyzed for tissue plasminogen activator activity.

2. Establish activity

Table 1 shows the result of plasminogen activation by E.c.OH extract. The activity generated depends on the presence of plasminogen. This activity is not affected by the predominant rabbit serum but is inhibited by an antiserum that is produced against tissue plasminogen activator by melanoma cells. This demonstrates that E.c.OH extracts produce plasminogen, affecting activity that is inhibited by antibodies against tissue plasma5 nogenic activator.

FIG. 7 shows the result of the Petri fibrin assay for fibrinolytic activity. A standard amount of urokinase was added in the center row in con10 concentrations from left to right, of 0.24, 0.14, 0.10, 0.05, 0.02 ΗουβΗ units. The bottom row is from samples of a natural tissue plasminogen activator, with the same amount of enzyme in each well. The slots 15 comprise, from left to right, a tissue plasminogen activator, an antiplasminogen activator, plus tissue plasminogen activator antibodies. The top row wells contained 8 µl of recombinant tissue 20 plasminogen activator extracts from E.c.OH. In the first well there is only an extract, in the second there is preimmune serum added, and in the third there is tissue plasminogen attenuator antibody added. It is apparent that the pre25 immune serum does not affect the natural or recombinant tissue plasminogen activator, and that tissue plasminogen activator antibodies inhibit the activity of natural ones, such as E.c.OH extracts. Based on urokinase standards, extracts contain slightly less than 2.5 ΡΙοιίβΗ units per ml. This is favorable compared to the value obtained in Table 1 of 1.3 Rui ^ b units for 1.

Table 1 summarizes the results of the analyzes carried out as described above in F.1.K.1.6:

Table 1

E.cone from cultures containing rbeKASHRA

Plasminogen activation by extracts from

Sample A ⁴⁰⁵ Activity Calculated ΡΙοιίβΗ <%> units in t! Extract (without plasminogen) 0.043 (0) Extract 0,451 th most common (100) 1.3 Plus extract predominant serum 0,477 th most common 106 - Plus extract anti t-PA antibodies 0,079 9

I

Figure 10 presents the results of a sample analysis of a fibrin layer carried out with extracts of 10 b E.c.H fermentation cultures containing tissue plasminogen activator expression plasmid. The fibrinolytic activity of the tissue plasminogen activator containing the extract is shown in FIG. 10 (socket A). This fibrinolytic activity is inhibited by anti tPA ΙβΟ (socket C) but not by predominant ΙβΟ (socket B) or anti urokinase 1§C (socket ϋ) and no evidence of activity from the extract obtained from cells containing as control of leukocyte interferon plasmid pIE1RA »gr103 (Nest H).

F.2 Preparation of m-PA using DHFR protein with low MTX binding activity.

E.2.A Vector construction

The sequence encoding a human tissue plasminogen activator (m-PA) is inserted into an expression plasmid containing the mutant DHFR with low MTX binding activity described in the application, υ.δ. 55g. Νο. 459151, filed Jan. 19, 1983, corresponding to Eigoreap Ra1ep1 ArrPsaBop RiB1.

11ο 117060 (referenced in the Biblical reference) performed by the following procedure (see Fig. 11):

Three fragments of the partially matched plasmids, pPA25E10, and pPA17, and P1PA1P12 (above) were prepared as follows: Plasmid pPA17 was digested with ϋάε I using Κίεηοχν DNA polymerase 1, and cut into Ρ $ ί I; An approximately 200 bp fragment containing the 5'-terminal m-PA sequence was isolated. The second m-PA fragment was obtained by digesting pbe11aK1PA "with P1l1 and No. II, and isolating approximately 310 bp fragment. The third m-PA fragment was obtained by degradation of pPA25E10 being 40 Nag I or B &lt; 1 &gt; II and isolating approximately 1645 bp fragment containing further more than the m-PA code region, several 3 'untranslated sequences.

Plasmid pE342, which expresses HBU surface antigen (also referred to as pHB3348-E) has been described by Leuchzop e. A., Patent application 8eg. Νο. 326980, filed December 3, 1981, now abandoned because of continuation of application 5eg. Νο. 603539, more than 50 given on 24 April 1988, corresponding to Eigoreap Ra1ep1 ArrNsabop RiB1. Νο. 73656, which is mentioned in the bibliographic reference. (Briefly, the 5U40 virus locus at 5 »1 gigap was isolated by cleaving the 5> Y4O DNA with ΗΐηΟΙΙΙ, and converting the ends of УίηόΙΙΙ 5 to the EcoK1 ends by adding a converter (ACSTCAATTC). Following cleavage with EcoK1, the 348 bp fragment enclosing the locus, was isolated by electrophoresis and electroelution was polyacrylamide gel, and cloned into pBK322. from 15 HEIs (he patite B virus) (Ashta1 νίηικ Sepebsh. (Cb 5) Asad. Rheezz, Υ.Υ. 1980) (which encompasses the gene encoding ΗΒ $ Αβ) in the plasmid pMB (изizku e ак ak, IaTige. 293: 79 (1981 ) of the EcoKL and WatNk regions (pMb is a derivative of 20 pBK322, which has a deletion that eliminates sequences that are inhibitors of plasmid replication in monkey cells). , 25 representing the locus region of 8U40, a region of ρΚΙ-ΒβΙ was introduced into EcoK.1. The locus fragment can insert in another orientation. This fragment encodes both 8U40 promoters, the early and the late promoters, added to 30 replication loci. HBU genes can be expressed under the control of one of the promoters dependent on this orientation (pHB5348-E representing HB3 expressed under the control of the early promoter). pE342 was modified35 by partial cleavage with EcoK1, filling in the cleavage region using ΚΙεποχν DNA polymerase I, and ligation of the plasmid backbone, thereby removing the Eco KL region recording the 8U40 locus in pE42. The resulting plasmid, designing pE3420eIaK1, was cleaved with Eco ΚΙ, filled using Κίεηοχν DNA polymerase I and cut with your ΗΙ. After acrylamide gel electrophoresis, approximately 3500 45 bp, the fragment was electroeluted extracted with phenol-chloroform and precipitated with ethanol as above.

The p342E 3500 bp vector thus obtained, as well as the m-PA fragments described above, including approximately 2160 bp, were ligated together using standard techniques. A plasmid containing the three t-PA co21 tear fragments in its own orientation was isolated, characterized and modeled - pE342-t-PA. This plasmid was cleaved with 8ac II and treated with alkaline phosphatase (BKb). In order to achieve DHFR sequencing, approximately 1700 bp fragment was generated by 8ac11 cleavage by RENEC. (RENEC is a plasmid expressing the DHFR mutant described in i.8.8eg. .ο. 459151 (above). This fragment was ligated into a pE342-m-PA plasmid to generate pETRAEK400, and a plasmid that is analogous to RenEC except ββ the coding region was moved by cDNA sequences from m-PA.

E.2.B Expression and amplification of the tPA sequence pETRAEK400 (pETREK) was transfected into two (Shr-CHO-OX B11 cells and ONEC + CHO-K1 (ATCC CCB61) cells by the method of ClAat and Wap <1gEb. -cells were selected by sprouting in glycine, hypoxanthine and thymidine deficient medium. The transformed ϋΗΡΚ + cells were selected by growth in 100 ηΜ MTN. The colonies that grew in appropriately selected medium were isolated using cloning rings and propagated in the same medium. for several generations.

For amplification, colony cells were cleaved in medium containing 5 x ΙΟ ⁴ , 10 ⁵ ,

2.5 x 10 ⁵ , 5 x 10 ^5, and ΙΟ ⁶ ηΜ MTX and have been flown several times. The cells were seeded at a very low (10 ² - 10 ³ cell / medium) density in 10 cm plates and the resulting colonies were isolated.

F.2.B Analytical methods

The expression of m-PA in transfected amplified colonies can be conventionally analyzed by methods similar to those set out in F.1.K.1.6.

The co-amplification of DHFR and m-PA sequences was analyzed by isolating DNA from spliced monolayer colonies as follows: spliced monolayer colonies in 150 ml petri dishes were washed with 50 ml sterile PB8 (phosphate-buffered saline) and lysed by the addition of 5 ml from 0.1 percent δϋδ (sulfur-buffered saline), 0.4 M CaCl ₂ , 0.1 Μ ΕϋΤΑ, pH 8. After

For 5-10 minutes, the mixture was separated, extracted into phenol and chloroform, and precipitated into ethanol. DNA is resuspended in 1 ton! (for 150 mL of petri) 10 mM Tnp-HCl pH 8, 1 mM ΕϋΤΑ (TE), CuAhe added to 0.1 ιηβ / τηΐ, and the solution incubated for 30 minutes at 37 ° C. δϋδ is then added to 0.1 percent and pronase (at 8 µm) is also added to 0.5 πΐβ / ιηΐ. After incubation at 37 ° C for 3-16 hours, the solution was again extracted with phenol and chloroform, sequentially and precipitated with ethanol. The DNA pellet was resuspended in 0.5 ml water and cleaved with restriction enzymes. Approximately 5-10 μ% of the cleaved DNA was electrophoresed in agarose gel [1 percent agarose in Tpc-acetate buffer (40 µM Τγϊ5, 1 mM ΕϋΤΑ, adjusted to pH 8.2 with acetic acid)]; Sogiehe, it is! A1, I.ΒίοΙ.Сет. 257: 7887 (1982)). After the bromophenol blue dye migrated down the gel pathway, the gel was removed and stained with ethanide bromide. After visualization of the DNA with ultraviolet light, the DNA is transferred from the gel to the nitrocellulose filters, as in the procedure of 8o1bg (98: 503 (1975)). The filters were then hybridized with a labeled translation probe made of a 1700 bp 5c11 fragment of RENEC (prepared and hybridized as described above), or of approximately 1970 Bp B§1 II fragment of PETREK.

E. 3 Production of m-PA in a compound with wild-type DHFR protein

IS FOR. Vector construction In a manner analogous to the method used in constructing PETREK, a plasmid PETREK containing a DNA sequence encoding wild-type DHFR was constructed. The construction is similar to that described in Example E.2.A., except that the PENEC plasmid as a gene sequence source for DHPZ protein was replaced by plasmid ρΕ342.ΗΒν.Ε400.ϋ22 described in i.8.8 er. Νο. No. 459152, filed Jan. 19, 1983, now Rakio 4713339, issued Dec. 15, 1987, in correspondence with Eigoreap Rasho ArrNsaNop Rkakio. 117058 cited in the bibliographic reference. The plasmid ρΕ342.ΗΒν.Ε400.ϋ22 is the same as the RENEC, except for one only different base pair, as a mismatch between the wild type and the DHFR mutant. The thus obtained plasmid PETREK is analogous to PETREK, with 22

I note that the DNA sequence encoded by wild-type DHFR is substituted for that of the mutant. A sample of the plasmid retRRK was deposited in Atepsap Ture CyNige SoNesOop and received an incoming Νο 40403.

EZB Expression of the m-PA sequence RTTRK was used to transfect DHFR deficient CHO cells (IglAiB and Cbxl) using calcium phosphate precipitation by the method of CbaB and Wap deg Eb. Twenty-one colonies that grew on selective medium (HCT) were assayed by detecting plasmin formations resulting from the degradation of fibrin in agar plates containing fibrin and plasminogen, as described by Scape1P-P1regpo, e1 a1 . Tech. Med. 148: 223 (1978).

Four of the most positive clones were then quantified for plasmin formation according to the method set out in E.1.K.1.6.

From such quantitative determination, it was found that the four clones tested showed the same or comparable secretion ability of m-PA in the medium expressed in units / cell / day. Subclones were obtained by transferring an inoculum from two of the clones in separate plates containing -STT medium. Two of the resulting subclones, 18B and 1, were used for further analysis.

EZV Amplification and secretion levels of t-PA.

The aforementioned clones were seeded at 2 x 1ο ⁵ cells in 100 mt plates, in 50 nM MTX to aid amplification. The surviving cells, when analyzed by the aforementioned method, gave in all cases about 10 times the non-amplified amounts expressing the activity of the tissue plasminogen activator. Two of these clones were selected for further study and named 1-15 and 18B-9.

Further, subclone 1-15 was amplified by visiting 2 × 10 ⁵ cells in 100 mt plates containing 500 nM MTX. Analysis of the cells thus amplified showed a further increase in yield (from about 3 ranges) of m-PA when quantified according to the method in C.1.C, the levels were in the range of 7 x 10 "* units / cell / day. One portion of these amplified cells was then transferred and maintained the presence of 10,000 nM MTX. Subclones 1-15 and 18B-9 were then tested after being maintained for approximately 1-2 months under the conditions shown in Table 3.

Table 3

Cell line Growth conditions ηβ t-PA / cell / day * 1-15 _{5 (M} 500 nM MTX 28.5 x 10 ³ 1-15 ₅₀₀ 500 nM MTX 26.0 x 10 ³ _{B15 5y0} (-STT medium, without MTX) 8.3 x 10 ³ 1-15 ₅₀₀ (-STT medium, without MTX) 18.0 χ 10 ³ 1 ”1 ^ 10000 YuulM MTX 29.3 x 10 ³ 1 -15! ooooh Yu ^ M MTX 49.0 x 10 ³ 18B-9 50 nM MTX 14.3 χ 10 ³ 18B-9 50 nM MTX 14.4 x 10 ³ 18B-9 (-STT medium, without MTX) 14.3 χ 10 ³ 18B-9 (-NOT medium, without MTX) 14.4 x 10 ³ 1 (-STT medium, without MTX) 1.0 x 10 ³ 1 (-STT medium, without MTX) 0.7 x 10 ³

I * m-PA in the cell medium was quantitatively analyzed by radioimmunoassay as follows: Purified m-PA, and purified iodine m-PA marker derived from melanoma cells were diluted in series to include concentrations of 12.5 to 400 n§ / wl in buffer solution containing saline phosphate buffer, pH 7.3, 0.5 bovine serum albumin, 0.01 percent PM / een 80 and 0.02 percent ΝηΝ ₃ . Appropriate dilutions of the samples were added to the radiolabeling proteins for analysis. The antigens were allowed to incubate overnight at room temperature in the presence of a 1: 10,000 dilution of 1 & C fraction of rabbit anti t-PA antiserum. The antigen / antibody complex was precipitated by absorption with goat anti-rabbit Ιβгра immunogranules (ViKab) for two hours at room temperature. The granules were purified by the addition of brine, centrifuged for 10 minutes at 2000 χ β at 4 ° C. The supernatants were discarded and the radioactivity of the precipitates was measured. Concentrations were determined by comparison with a standard.

The cell lines are as follows: cell line “1” is a non-amplified clone from the original cluster of four clones. "115 _50θ " is an amplified subclone of cell line "1" that was amplified initially at 50 nM MTX to give 1-15 and then transferred for further amplification into 500 nM MTX. 1-15 _1θθθθ is a subclone of 1 - 15 _50θ which was subsequently amplified in the presence of 10000 nM MTX. Cell line 18B-9 is a subclone of the original cluster of four clones that was amplified with 50 nM MTX.

The sample with CHO cell line 1-15 _50θ was deposited in Atepsap Tuge Sikige SoiScope and received incoming Кο SKB 9606.

All of the amplified cells showed increased levels of m-PA secretion, well above those of the non-amplified cells. If a non-amplified culture secretes m-PA more than 0.5 ρβ / cell / day, amplification yields 50 p &lt; / cell / day.

G. Pharmacological structures

The compounds of the present invention can be formulated according to known methods for the preparation of pharmaceutically usable structures, wherein the product of the human tissue plasminogen activator is combined in the composition with a pharmaceutically acceptable carrier.

Suitable carriers and the technology for their preparation, incorporation of other human proteins, including human serum albumin, are described, for example, in the Kepp £ 1op'5 RNAKMASEiT1CaB δΟΙΕΝΟΕδ of the Eu / .MaSh mentioned in the incorporated bibliography. Such structures will contain an effective amount of protein, together with an appropriate amount of carrier, to produce pharmaceutically acceptable compositions suitable for effective administration.

For example, the human tissue plasminogen activator may be administered parenterally to individuals suffering from cardiovascular disease. Dosage and dosing may be paralleled with those in current use in the clinical trials of other cardiovascular agents thrombolytic agents, i. about 440

W / k® body weight, with the intravenous starting dose followed by a prolonged intravenous infusion of about 440 W / k # / hour for 12 hours in patients suffering from pulmonary embolism.

As an example of a suitable dose determination of the essential homogeneous human tissue plasminogen activator with paraenteric application, a vial containing 25,000 IU activity plasminogen activator, 25 IU and 45 IU No. C1 can be reconstituted with 5 ml of sterilization. and mixed with an appropriate volume of 0.9 percent sodium chloride or 5 percent beehose for intestinal administration.

3. Detailed description of recombinant human t-PA

The structure of the partial embodiment of human t-PA obtained in the experiments described45 has been subjected to investigation in some detail, both by illuminating the gene code sequence and by known biochemical techniques for protein analysis. A modern understanding of protein structure is illustrated in FIG. 12.

More Sopep et al. (88) demonstrated that double-stranded human m-PA is formed by the proteolytic degradation of a single-stranded molecule into two disulfide-linked polypeptides. The current level of knowledge allows us to conclude that the heavy chain (30882 moles) is derived from ΝΗ ₂ terminal part and that the light chain (28126 moles) involves the COOH-terminal region. The N-terminal, structured by a two-stranded molecule, suggests that the two-stranded form is generated by cleavage of a single arginyl-isoleucine bond (Fig. 12; drawn arrow).

The primary structure of the heavy chain portion of the human t-PA (Fig. 12) reveals a high degree of sequence homology with the so-called. "Kpk1e" regions of plasminogen (89) and prothrombin (40 and 41). The "Kpk1e" region refers to a characteristic triadic disulfide structure originally found in "προ" - a prothrombin fragment, first described in detail by Ma £ pi $$ op! a1. (91, 92). From the primary sequence of t-PA, two so-called The "kpk1e" regions, of 82 amino acids each, exhibit a high degree of homology to the 5 "kpk1e" plasminogen regions. The remaining лни-terminal 91 amino acids share a low degree of homology with the "conventional" "kpk1e" region. However, one may speculate that these areas may also adopt a structure containing multiple disulfide bonds, such as the 11 additional cysteine residues found here.

The light chain catalytic region of human m-PA, the so-called serine protease region, as in other serine enzymes, is most likely formed by histadine ₃₂₂ , asparagine _37] , and serine ₄₇₈ residues. Moreover, the amino acid sequences surrounding these residues are very homologous to the corresponding portions of other serine proteins, such as trypsin, prothrombin, and plasminogen.

Although reference is made to selected embodiments, it should be understood that the present invention is not limited to them but rather constitutes a substantiated list of appended claims.

Claims (1)

Claims A recombinant human tissue plasminogen activator consisting essentially of the amino acid sequence 69-527 depicted in Figures 5a, 5b and 5c.