SI8310997A - Activator of a plasminogen of human tissue. - Google Patents

Abstract

Using recombinant techniques, it is produced human tissue plasminogen activator (t-PA) v usable quantities. This invention also makes it possible the production of t-PA which is contaminant free by they monitor in his natural cellular environment. Also described are methods, carriers for expression and various host cells useful in manufacturing.

Description

FIELD OF THE INVENTION

The invention is in the field of genetic engineering.

A technical problem

The present invention relates to a human tissue plasminogen activator corresponding to that in human serum and / or tissues, as well as a process for its production.

The state of the art

The present invention is made by working on the basis of the discovery of a DNA sequence and a derived amino acid sequence of a human plasminogen activator. This discovery made it possible to produce a human plasminogen activator using recombinant DNA technology, which again allows the production of material of adequate quality and quantity to initiate and perform animal testing and clinical testing as necessary conditions for marketing approval, which are not impeded by mandatory restrictions monitor the isolation processes used in the production and extraction of existing cell culture so far. The present invention is directed to these accompanying embodiments in all aspects.

Publications and other materials used herein to illuminate the theoretical basis of the invention and, in certain cases, to provide additional details pertaining to the use of the invention, are incorporated by reference and prepared for the purpose of being hereinafter numerically stated and appropriately grouped in the attached bibliography.

A. Human tissue plasminogen activator

The fibrinolytic system is in dynamic balance with the coagulation system while maintaining an intact, protected vascular layer. The coagulation system deposits fibrin as a matrix that serves to restore the hemostatic state. The fibrinolytic system removes the fibrin network after the haemostatic state is reached. The fibrinolytic system is activated by a proteolytic enzyme generated from the precursor plasminogen of the plasma protein. Plasminogen is translated into plasmin via activation by an activator.

There are currently two activators available, streptokinase and urokinase. Both are indicated for the treatment of acute vascular diseases such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial inclusions, and other venous thromboses. Together, these diseases are major health hazards.

The causal etiologic basis for these diseases indicates either partial or, in some cases, complete occlusion of the blood vessel with a blood clot - thrombus or thromboembolus. Traditional anticoagulation therapy, such as with heparin or coumarin, does nothing to directly increase the breakdown of the thrombus or thromboembolus. The previously cited thrombolytic agents, streptokinase and urokinase, have had practical and effective use. However, each has serious limitations. Neither of them has a high affinity for fibrin; as a result, both activate circulation and plasminogen bound to fibrin, relatively indistinguishably. Plasmin, which forms in circulating blood, neutralizes relatively quickly and is lost to beneficial atrombolysis. Residual plasmin will degrade some protein blockers, such as fibrinogen, factor V, and factor VIII, causing the potential for bleeding. Furthermore, streptokinase is strongly antigenic and patients with high antibody titers respond ineffectively to treatment and cannot be sustained on continuous treatment. Urokinase therapy is expensive, because of its complex isolation from human urine or tissue culture, and therefore is not generally accepted for clinical practice. Urokinase has been the subject of much research - see e.g. references 1-6.

The so-called plasminogen activators were isolated from various human tissues, e.g., uterine tissue, blood, serum - see mainly references 7-11 and from cell culture (reference 94). Their preparations are also described - see references 12-13. See also references 14-18. Plasminogen activators derived from these sources are always classified into two main groups: urokinase-type plasminogen activators (u-PA) and tissue-type plasminogen activators (t-PA) based on differences in their immune properties. (The abbreviations t-PA and u-PA are as proposed at the XXVIII Meeting of the International Committee on Thrombosis and Hemostasis, Bergamo, Italy, 27 July 1982).

The human melanoma lineage that secretes t-PA has recently been identified. Characterization of this plasminogen activator melanoma has shown that it is indistinguishable, neither immunologically nor in amino acid composition, from a plasminogen activator isolated from normal human tissue (reference 19,88).

The product was isolated in relatively pure form, characterized and found to be a highly active fibrinolytic agent (20).

Few studies (e.g., references 95 to 98) using t-PA purified from the melanoma cell line have shown its high affinity for fibrin compared to urokinase-type plasminogen activators. However, intensive study of human t-PA as a potential thrombolytic agent has been hampered by its extremely low concentration in blood, tissue extracts, vascular perfusion and cell cultures.

They have found that the use of recombinant DNA and associated technologies will be the most effective way of providing the large quantities of high quality plasminogen activator (formerly referred to as human plasminogen activator), essentially free of other human protein, in the required amount. Such materials are likely to express bioactivity that will allow their clinical use in the treatment of various cardiovascular conditions and diseases.

B. Recombinant DNA technology

Recombinant DNA technology has achieved a state of certain sophistication. Molecular biologists are able to recombine different DNA sequences with some ease, creating new DNA species that can produce copious amounts of exogenous protein product in transformed germs and cell cultures. General means and methods for in vitro ligation of various DNA fragments with blunt or sticky ends are at hand, so that strong expression carriers are produced that are useful in transforming particular organisms by directing their efficient synthesis of the desired exogenous product. However, on a product-by-product basis, the path is to some extent painstaking and science has not yet progressed to a stage where regular predictions for success could be made. Indeed, those who achieve successful results without a well-founded experimental basis work with a significant risk of inoperability.

DNA recombination of essential elements i.e. replication sources, one or more phenotypic selection characteristics, expression promoters, heterologous gene insertions, and the rest of the vector are mainly performed outside the host cell. The resulting recombinant replicable expression carrier or plasmid is introduced into cells by transformation, and with the growth of the transformant large amounts of the recombinant carrier are obtained. When the gene is inserted correctly with respect to the parts that control the transcription and translation of the encoded DNA message, the resulting expression carrier is useful for the actual production of the polypeptide sequence encoded by the inserted portion, a process known as expression. The resultant product can be obtained by cleavage, if necessary, of the host cell in microbial systems and regeneration of the product by appropriate purification from other proteins.

In practice, the use of recombinant DNA technology may express entirely heterologous polypeptides - so-called direct expression - or alternatively, may express a heterologous polypeptide that is fused to a portion of the amino acid sequence of a homologous polypeptide. In the latter cases, the desired bioactive product is sometimes made bio-inactive in a fused homologous / heterologous polypeptide until cleaved in the extracellular environment. See references (21) and (22).

In a similar way, the science of cell and tissue culture is well developed to study the genetics and physiology of the cell. The means and procedures for maintaining permanent cell lines made by successive serial transfers from normal cell isolate are at hand. For research use, they maintain such cell lines on a solid support in a liquid medium, or by growing in suspension containing nutrient carriers. It seems that, for large preparations, proportions only cause mechanical problems. For further basis, we turn our attention to references (23) and (24).

In a similar way, protein biochemistry is a useful, essentially necessary complement, in biotechnology. The cells that produce the desired protein also produce hundreds of other proteins, endogenous products of cell metabolism. These contaminating proteins, as well as other compounds, may, if not separated from the desired protein, prove to be toxic if given to an animal or a human during therapeutic treatment with the desired protein. For this reason, protein biochemistry techniques allow the design of separation processes that are appropriate for the particular system being observed and for providing a homogeneous product that is safe for its intended use. Protein biochemistry also proves the identity of the desired product, characterizing it and ensuring that cells produce it faithfully, without alterations and mutations. This branch of science is also involved in biotest design, stability studies, and other procedures that must be used before successful clinical research and marketing are conducted.

Description of a solution to a technical case problem

The present invention is based on the discovery that recombinant DNA technology can be successfully used to produce human tissue plasminogen activator (tPA), preferably in direct form, and in sufficient quantities to initiate and perform animal and clinical testing as prerequisites for market acceptance. The product, human t-PA, is suitable for use in all its forms, in the prophylactic and therapeutic treatment of humans for various cardiovascular conditions or diseases. In one important aspect, the present invention is directed to methods for treating vascular disorders in human patients using t-PA and its suitable pharmaceutical preparations.

The present invention further comprises a substantially pure activator of human tissue plasminogen. A product produced with genetically engineered micro-organisms or cell culture systems provides the opportunity to produce a plasminogen activator in a much more efficient way than has been possible so far, allowing commercial exploitation that has not been possible so far. Further, depending on the host cell, the plasminogen activator of human tissue may contain accompanying glycosylation to a greater or lesser extent compared to the native material. In any case, t-PA will be free of contaminants, which are usually accompanied by its non-recombinant cellular environment.

The present invention also relates to replicable DNA expression carriers that produce gene sequences that encode a human tissue plasminogen activator in a form that can be expressed, to soybeans of microorganisms or cell cultures that are transformed and to microbial or cell cultures of such transformed strains or cultures capable of producing plasminogen activator of human tissue. In further aspects, the present invention relates to various methods useful for the preparation of said gene sequences, to carriers for DNA expression, to soybean microorganisms and cell cultures, and to their specific embodiments. Further, the present invention relates to the preparation of cultures of said microorganisms for fermentation and to cell cultures.

Brief description of the drawings

Figure 1 shows 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with 35 _s- methionine labeled proteins that can be precipitated with anti t-PA secreted from melanoma cells with or without a protease inhibitor.

Figure 2 shows the electrophoresis of immunoprecipitated translational products of mRNA fractions derived from melanoma cells.

Figure 3 shows a hybridization scheme of 96 bacterial colonies transformed with cDNA using a ³² P-labeled 14-mer as a probe made based on a sequence of 5 amino acids of human t-PA.

Figure 4 is a full-length restriction endonuclease blueprint of the human t-PA human t-PA.

Figures 5a, 5b and 5c show the nucleotide sequence and the derived full-length t-PA cDNA amino acid sequence.

Figure 6 is a schematic diagram of the construction of the pdeltaRIPA * expression plasmid.

Figure 7 shows the results of a plate fibrin assay for fibrinolytic activity of E. coli cells transformed with pdeltaRIPA *.

Figure 8 is a HPLC trace of the peptide from the trypsin secretion of human t-PA.

Figure 9 shows the construction of a plasmid encoding for direct expression of the mature

Ί Human t-PA in E. coli.

Figure 10 shows the results of a plate fibrin assay on the fibrinolytic activity of human t-PA produced by E.coli transformed with pt-PAtrpl2.

Figure 11 shows the construction of DHFR (mutant or wild type) / t-PA coding plasmids that are suitable for transformation in mammalian tissue culture cells.

12 is a schematic diagram of a human tissue plasminogen activator as prepared by the procedure illustrated in E.l.

A precise description

A. Definitions

As used herein, human tissue plasminogen activator ah human t-PA ah t-PA denotes a human external (tissue type) plasminogen activator produced by microbial ah cell culture systems, in bioactive forms comprising a protease moiety and corresponding to those plasminogen activators, which are otherwise native to human tissue. The human tissue plasminogen activator protein produced here was defined using determined DNA gene and deductive amino acid sequencing. Clearly, there are natural allelic variations and they occur from individual to individual. These variations can be illustrated by amino acid differences in the total sequence ah by omissions, substitutions, substitutions, inversions or addition of amino acids (e) in said sequence. Furthermore, the location and degree of glycosylation depends on the nature of the cellular environment of the host.

In the use of recombinant DNA technology, there is the potential for the preparation of various plasminogen activator derivatives of human tissue, variously modified by single or multiple substitutions, omissions, addition or replacement of amino acids, e.g. by direct site mutagenesis in basic DNA. The preparation of derivatives containing the essential kringle region and the serine protease region, which are mainly characteristic of plasminogen activator of human tissue, will be included but otherwise modified as described above. All such allelic variations and modifications occurring in human tissue plasminogen activator derivatives are within the scope of the present invention, as well as other related human external (tissue type) plasminogen activators, which are physically and biologically similar, as long as they remain essential, characteristic the activity of human tissue plasminogen activator in its species is intact.

We have prepared a human tissue plasminogen activator 1) having methionine as the first amino acid (present on the basis of insertion of the ATG start signal codon before the structural gene) or 2) having, where methionine is intra- or extracellularly cleaved, its normal first amino acid, or 3) or together with its signal polypeptide or conjugated protein, which is different from the conventional signal polypeptide, wherein the signal polypeptide or conjugate can be specifically cleaved in an intra- or extra-cellular environment (see reference 21), or 4) by direct expression in the mature form without the need for cleavage of any foreign, redundant polypeptide. The latter is particularly important when a given host is unable or unable to efficiently release the signal peptide when the expression carrier is intended to express a human tissue plasminogen activator together with its signal peptide. In each case, the human t-PA thus produced, in its various forms, is regenerated and purified to a level suitable for use in the treatment of various vascular conditions or diseases.

It further has t-PA forms, which also include single-stranded protein (1-strand) and 2-strand protein. The latter is proteolytically derived from 2-chain compounds. It is theorized that the 2-strand protein is associated with the fibrin produced and that the proteolytic conversion of the 1- to 2-strand material takes place at the site of plasminogen conversion to plasmin. The present invention provides the administration of a 1-chain protein for in vivo conversion exactly as described or provides the administration of a 2-chain protein, which has also been shown to be active. 2-strand protein can be prepared by in vitro proteolytic conversion after 1-strand material is produced. The so-called kringle surface is positioned upstream of the serine protease and is believed to play an important function in binding its plasminogen activator tissue to the fibrin matrix; therefore, we observe the specific activity of the present plasminogen tissue activator against sensitive, existing thrombus. Here, a plasminogen tissue activator is produced that contains an enzymatically active moiety corresponding to the native material, and the term human tissue plasminogen activator defines products that comprise such moiety alone or together with complementary amino acid sequences up to the full length of the molecule.

To summarize the present invention, therefore, human t-PA has a functional definition; it can catalyze the conversion of plasminogen to plasmin, bind to fibrin and, based on its immunological properties, classify it as t-PA, as shown above.

In essence, pure form as used to describe the state of human t-PA produced by the invention means that it is free of proteins or other materials that normally accompany human t-PA when produced with the help of non-recombinant cells, i.e. in his native surroundings.

DHFR protein refers to a protein that may have an activity that accompanies dihydrofolate reductase (DHFR) and which must therefore be produced by viable cells on a non-hypoxanthine, glycine and tinidine basis. (-HGT substrate). In general, cells lacking DHFR protein are unable to grow on this basis, and cells containing DHFR protein do so successfully.

Občut Cells sensitive to ΜΤΧ are cells that cannot grow on substrates containing the DHFR inhibitor methotrexate (ΜΤΧ). So cells are susceptible to ΜΤΧ cells that, unless genetically modified or otherwise supplemented, will not grow under normal conditions on a basis appropriate for the cell type when the concentration is ΜΤΧ 0.2 / xg / ml or greater . Some cells, such as bacteria, do not express sensitivity to zato because they do not allow it to enter within the cell boundary, even if they contain DHFR, which is otherwise sensitive to this drug. Basically, cells containing as their DHFR wild-type DHFR protein will be sensitive to methotrexate if they are not permeable or consumable according to ΜΤΧ.

Wild-type DHFR refers to dihydrofolate reductase, as is commonly found in the particular organism in question. Wild-type DHFR is mainly in vitro sensitive to low concentrations of methotrexate.

DHFR with low affinity for binding to ΜΤΧ has a functional definition. It is a DHFR protein that, when generated in cells, will allow rast sensitive cells to grow in a substrate containing 0.2 gg / ml or more ΜΤΧ. Such a functional definition is understood to depend on the ease with which the organism produces a low-affinity DHFR protein for binding to ΜΤΧ as well as the protein itself. However, as used in the context of the present invention, such a balance between these two mechanisms need not be confused. The invention functions in relation to imparting viability against these levels ΜΤΧ and is not a consequence of whether the ability to do so is enhanced by expression in addition to the intrinsic nature of the DHFR produced. A suitable DHFR protein that falls within this definition is described in U.S. Pat.

report serial no. No. 459,151, filed Jan. 19, 1983, and incorporated herein by reference.

The expression vector includes vectors that can express DNA sequences contained therein when such sequences are operatively linked to other sequences that may affect their expression. It is implicit, although it is not always specifically stated that these expression vectors must replicate in host organisms, either as episodes or as an integral part of chromosomal DNA. Clearly, lack of replication capability will make them operationally ineffective. In summary, the expression vector is given a functional definition and any DNA sequence that can express the specific DNA code contained therein is included in that term when used for a specific sequence. Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids that refer to double-stranded circular DNA strands that are unrelated to their chromosome in the form of their vectors.

In the present application, the plasmid and the vector are used interchangeably because the plasmid is the most commonly used form of the vector. However, it is an object of the invention to also include such other forms of vectors having equivalent functions and which will be recognized in the technique described later.

Recombinant host cells are cells that have been transformed with vectors that are constructed using recombinant DNA techniques. As defined herein, t-PA is produced in quantities obtained on the basis of this transformation, rather than in such smaller quantities or, as is customary, in quantities less than detectable than can be produced. with the help of an untransformed host. t-PA produced by such cells can be called recombinant t-PA.

B. Host cell cultures and vectors

The vectors and procedures described herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms.

Of course, prokaryotes for cloning DNA sequences in the construction of the vectors useful in the invention are generally desired. For example, the E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains that can be used include E. coli soybeans, such as E. coli B and E. coli Χ17776 (ATCC No. 31537). These examples are, of course, intended to be illustrative and not limiting.

Prokaryotes may also be used for expression. The aforementioned soybeans can be used as well as E. coli W3110 (F, lambda ', prototrophic, ATCC No. 27325), such bacilli as Bacillus subtilis, and other enterobacteria such as Salmonella tvphimurium or Serratia marcesans and various pseudomonas species.

Generally, plasmid vectors containing replicon and control sequences derived from species compatible with host cells are used in connection with these hosts. The vector typically carries a replication site as well as marker sequences that can provide phenotypic selection in transformed cells. E.g. E. coli is typically transformed using pBR 322, a plasmid derived from E. coli species (Bolivar, et al. Gene 2: 95 (1977)). pBR322 contains genes for resistance to ampicillin and tetracycline, thus providing free agents for the identification of transformed cells. The pBR322 plasmid 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. Promoters most commonly used in the construction of recombinant DNA include promoter systems for β-lactamase (penicillanase) and lactose (Chang et al. Nature, 275: 615 (1978); Itakura, et al., Science, 198: 1056 ( 1977); (Goeddel, et al Nature 281: 544 (1979)) and the tryptophan (trp) promoter system (Goeddel, et al. Nucleic Acids Res. 8: 4057 (1980); EPO Appl. Publ. No. 0036776 Although most commonly used, other microbial promoters have been discovered and used, and details pertaining to their nucleotide sequences have been published, allowing an experienced researcher to operatively link them to plasmid vectors (Siebenlist, et al.) Ecclesiastes 20: 269 (1980) .Eurokiotic microbes such as yeast cultures may also be used for polio prokaryotes, most commonly used in eukaryotic organisms are Saccharomvces cerevisiae or common baker's yeast, although a large number of other strains are also available. in Saccha romvces commonly used plasmid YRp7, e.g. (Stinchcomb, et al, Nature, 282: 39 (1979); Kingsman et al, Gene, 7: 141 (1979); Tschemper, et al, Gene, 10: 157 (1980)). This plasmid already contains a trump gene that provides a selection marker for a mutant yeast strain lacking the ability to grow in tryptophan, e.g. ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)). Presence suffers damage as a characteristic of the host cell genome, the yeast then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable sequences for promotion in yeast vectors include promoters for

3-phosphoglycerad kinase (Hitzeman, et al., J. Biol. Chem., 255: 2073 (1980)) or other glycolytic systems (Hess, et al., J. Adv. Enzymes Reg. 7: 149 (1968) Holland et al., Biochemistry, 17: 4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerad mutase, pyruvate ketosephosphate, pyruvate ketosephosphate isomerase, phosphoglucose isomerase and glucokinase. In constructing suitable expression plasmids, the terminal sequences accompanying these genes are also ligated into the 3 'carrier to express the desired sequence, which must be expressed to provide polyadenylation of mRNA and termination. Other promoters that have the additional ability of controlled transcription by growth condition are the promoter regions for alcoholdehydrogenase 2, isocitochrome C, acid phosphatase, degradative enzymes that accompany nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase and the enzymes responsible for the exploitation of maltose and galactose (Holland, ibid.). Any plasmid vector containing a yeast compatible promoter, replication origin and terminal sequences is suitable.

In addition to microorganisms, cell cultures derived from multicellular organisms may also be used as hosts. In principle, any such cell culture functions, whether it is from vertebrates or invertebrates. Of course, the greatest interest in vertebrate cells and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture Academic Press, Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 and MDCK cell lines. Expression vectors for such cells typically include (as appropriate) the origin of replication, a promoter located in front of the gene to be expressed, along with any ribosome binding sites, RNA slicing sites, polyadenylation sites, and transcriptional terminal sequences.

For use in mammalian cells, control functions on expression vectors are often provided by viral material. For example, promoters commonly used are derived from polio, Adenovirus 2, and most commonly from Simian Virus 40 (SV40). Previous and later SV40 promoters of the virus are particularly useful because they can be obtained from the virus as a fragment also containing the SV40 viral replication origin (Fiers, et al, Nature, 273: 113 (1978), incorporated herein by reference ). Smaller and larger SV40 fragments may also be used, provided that a sequence of about 250 bp extending from the Hind III site to the Bgl I site located in the viral replication source is included. Further, it is also possible, and often desirable, to use promoter or control sequences that typically accompany the desired gene sequence, provided that such control sequences are compatible with host cell systems.

The origin of replication can be provided either by constructing a vector to include an exogenous source, such as one derived from SV40 or another viral (e.g., polio, Adeno, VSV, BPV, etc.) source, or can be provided. through a mechanism for chromosomal replication of host cells. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

When selecting the desired host cells for vector transfection according to the invention comprising DNA sequences encoding both the t-PA and the DHFR protein, it is appropriate to select the host according to the type of DHFR protein used. When using wild-type DHFR protein, it is desirable to select DHFR-deficient host cells by enabling the use of the DHFR encoding sequence as a marker for successful transfection in a selective basis lacking hypoxanthine, glycine and thymidine. A suitable host cell in this case is a Chinese hamster ovary (CHO) cell line defective by DHFR activity, made and paraphrased, as described in Utlaub and Chasin, Away. Natl. Acad. Sci. (USA) 77: 4216 (1980), which is incorporated herein by reference. On the other hand, if DHFR protein with low affinity for ΜΤΧ is used as a control sequence, it is not necessary to use DHFR deficit cells. Because the mutant DHFR is methotrexate resistant, substrates containing ΜΤΧ can be used as a selection agent, provided that the host cells themselves are not sensitive to methotrexate. However, many EQ cells capable of absorption appear to be sensitive to methotrexate. One such cell line is the CHO line, CHO-K1ATCC no. CCL 61.

The examples shown below describe the use of E. coli using the lac i promoter system and the use of CHO cells as host cells and the use of expression vectors that include the SV40 origin of replication as a promoter. Of course, it is within the knowledge of those skilled in the art that they use analog techniques to construct expression vectors for the expression of a desired protein sequence in alternative prokaryotic or eukaryotic cultures of host cells.

Satisfactory amounts of human t-PA are produced by cell cultures, and subsequent purification using a secondary coding sequence enhances even higher levels of production. The secondary coding sequence comprises dehydrofolate reductase (DHFR), which is influenced by an externally controlled parameter such as methotrexate by allowing expression control by controlling methotrexate concentration (ΜΤΧ).

C. Procedures used

When cells without strong cell membrane barriers are used as host cells, transfection is performed by a calcium sulfate staining process, as described in Graham and Van der Eb, Virologv, 52: 546 (1978). However, other methods for introducing DNA into cells may also be used, such as by nuclear injection or by condensation of the protoplast.

When prokaryotic cells or cells containing substantially cell membrane constructions are used, the desired transfection process is calcium treatment using calcium chloride as described in Cohen, F.N. et al. Natl. Acad. Sci. (USA), 69: 2110 (1972).

The construction of suitable vectors containing the desired sequences for encoding and control is performed using standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, cut and ligated to the desired shape to form the required plasmids.

The cleavage is carried out by treating the restriction enzyme (or several enzymes) in suitable buffer. Mostly, 1gg of plasmid or DNA fragment with about 1 unit of enzyme in about 20g of buffer solution is used. (Appropriate amounts of buffer and substrate for specific restriction enzymes are specified by the manufacturer). The incubation operative time is about 1 hour at 37 ° C. After incubation, the protein is separated by extraction with phenol and chloroform, and the nucleic acid is isolated from the aqueous fraction by ethanol precipitation.

If blunt ends are required, the preparation is treated for 15 minutes at 15 ° with 10 units of polymerase I (Klenow), extracted with phenol-chloroform and precipitated with ethanol.

Separation of cleaved fragments by size is performed using a 6% polyac15 rilamide gel as described in Goeddel, D., et al, Nucleic Acids Res., 8: 4057 (1980), which is incorporated herein by reference.

For the ligation of approximately equimolar amounts of desired components, which are suitably tailored at the end to ensure correct matching, they are treated with about 10 units of T4 DNA ligase per 0.5 μ-g DNA (when splitting vectors are used as components, it may be useful, to prevent split vector religion by pretreatment with bacterial alkaline phosphatase).

For analysis, to confirm the exact sequence in the engineered plasmids, ligation mixtures were used to transform E. coli Ki2 strain 294 (ATCC 31446), and successful transformants were selected based on ampicillin or tetracycline resistance when needed. Plasmids from transformants are prepared, analyzed by restriction and / or sequencing using the procedure of Messing, et al, Nucleic Acids Res., 9: 309 (1981) or Maxam, et al, Methods and Enzymology, 65: 499 (1980).

Amplification of DHFR protein coding sequences is performed by growth of host cell cultures in the presence of about 20-500,000 nM concentration of methotrexate, a competitive inhibitor of DHFR activity. Of course, the effective concentration interval depends very much on the nature of the DHFR gene, protein, and host properties. Clearly, there is no way to identify the generally defined upper and lower limits. Appropriate concentrations of other folic acid analogues or other DHFR inhibiting compounds may also be used. Of course, it is convenient, easily accessible and efficient.

D. General description of preferred embodiments

Human tissue plasminogen activator is obtained according to the following protocol:

1. Human melanoma cells producing plasminogen activator tissue are cultured until they mature.

2. Cell granules from such cell cultures are extracted in the presence of ribonuclease inhibitors by isolating all RNA cytoplasm.

3. The oligo-dT column isolates the entire information RNA (mRNA) in polyadenylated form. Such mRNA is further fractionated by acid-urea gel agarose gel electrophoresis.

4. The gel fraction containing the specific plasmidogen activator tissue RNA is identified as follows:

RNA from all gel fractions is translated into an in vitro rabbit reticulocyte lysate system supplemented with dog pancreatic microsomes. The resulting translation products are then immuno-precipitated with a specific human tissue plasminogen activator IgG antibody.

5. The corresponding RNA (21 to 24S) is converted to the corresponding complementary single stranded DNA (cDNA) from which the double stranded cDNA is produced. After poly-dC tailoring, it is inserted into a vector such as a plasmid carrying one or more phenotypic markers.

6. The vectors thus prepared are used to transform bacterial cells by providing a library of cloned DNA. We make a group of radiomarked synthetic deoxyoligonuleotides that are complementary to codons for known amino acid sequences in t-PA, such as e.g. group 8 14-mer, 5'-dTC (Q) CA (g) TA (^) TCCCA-3 'complementary to sequences encoding known - see below - amino acid sequence: tryptophan glutamic acid - tyrosine - cysteine - aspartic acid (WEYCD) and is used to probe a library of colonies.

7. Plasmid DNA is isolated from the positive cDNA clones and sequenced.

8. The sequenced DNA encoding t-PA is then digested in vitro for insertion into a suitable expression carrier, which is used to transform a suitable host cell that is allowed to grow in culture and produce the desired plasminogen activator of human tissue.

9. The human tissue plasminogen activator thus produced has approximately 251 amino acids in its enzymatic serine protease moiety and a kringle containing an upstream sequence which is currently believed to be responsible for fibrin binding. The mature protein plus its signal sequence has a total of 562 amino acids.

The pre-process is itself successful in producing pure t-PA. The methods of the invention used for the methotrexate-sensitive complementary coding sequence allow the production of antigenically active t-PA protein in cell cultures of greater than 0.1 pg per cell per day. Appropriate amplification conditions can yield greater than 20 pg per cell per day. Under the changed conditions, gene expression levels leading to the production of more than 9 x 10 ' ⁶ Plow units per cell per day or, with appropriate amplification, more than 18 x 10 ^-4 Plow units of t-PA activity can be achieved.

In this aspect of the invention, we use the advantage of methotrexate as a drug that, although normally fatal to cells that can use it, allows cells to grow in the presence of controlled levels ΜΤΧ by amplifying a gene encoding a DHFR coding sequence (Schimke, Robert T. et al, Science 202: 1051 (1978); Biedler, JL et al, Cancer Res. 32: 153 (1972); Chang, SE, et al, Celi, 7: 391 (1976).

For this aspect of the invention, it is an important demonstration that amplification of the DHFR gene can induce amplification of the accompanying sequences encoding other proteins. This seems to be the case where the accompanying protein is the hepatitis B surface antigen (HBsAg) (Christman, J Et al, Proc. Nat. Acad. Sci., 79: 1815 (1982): E. coli protein XGPRT (Tingold, Gordon. et al, J. Molec, and Appl. Gen. 1: 165 (1981); and the endogenous sequence of the DHFR / SV40 plasmid combination (Kaufman, RF et al. J. Molec. Biol., 159: 601 (1982)) .

Other mechanisms for providing methotrexate resistance include reducing the affinity for binding of DHFR protein so that it is less sensitive to methotrexate (Flintoff, WF et al. Somat. Full Genet. 2: 245 (1976), but even in this case it appears to occur amplification.

Genes for both wild-type DHFR and ΜΤΧ-resistant DHFR appear to be amplified in the presence of ΜΤΧ, based on their reduced binding capacity. Therefore, in principle, this aspect of the invention relates to the use of the effect of DHFR sequence amplification on sequences to encode a flanking protein, allowing increased levels of expression of the t-PA sequence in the presence of ΜΤΧ, or based on the pre-treatment of transformed cells with ΜΤΧ.

E. Examples

The following examples are intended to illustrate and not limit the invention. In the case of the host cell cultures, we used E. coli host cultures and a CHO cell line, which is suitable for the DHFR encoding sequence of the type of protein to be introduced. Of course, other eukaryotic and prokaryotic cells are also suitable for the process of the invention.

E.l Expression of the human t-PA gene in E. coli

E.l.A Legends of Figures

Figure 1 is an autoradiogram of 10% SDS PAGE showing immunoprecipitated ( ³⁵ S) -methionine-labeled protein (s) secreted from human melanoma cells during a 3-hour pulse in vivo, in the presence of (line b) or the absence (line a) of the protease inhibitor aprotinin. After immunoprecipitation with a specific IgG plasminogen activator, three bands (line a) having molecular weights of about 65,000, 63,000 and 35,000 were observed. In the presence of the protease inhibitor, however, no molecular weight type of 35,000 was observed. There are no immunoprecipitated products when using preimmune serum (line c). The migration and molecular weights of the ¹⁴ C-labeled protein standards are shown to the left of line a.

Figure 2 describes the gel electrophoresis of immunoprecipitated translation products of RNA fractions isolated from an acid-urea agarose gel. The major band is observed in fractional numbers 7 and 8 after translation in the presence of the dog's pancreatic microsome and then immunologically precipitating with a specific plasminogen tissue IgG activator. This strip has a molecular weight of about 63,000 daltons. The size of the mRNA migrating to fractions 7 and 8 is approximately 21 to 24 S. The positions of ribosomal RNA markers, determined by electrophoresis on an RNA carbamide gel and visualized by ethidium bromide staining, are marked above the corresponding gel lines.

Figure 3 shows a hybridization scheme of 96 colonies with ³² P-dTC ( _G ) CA (£) TA (£) TCCCA (WEYCD) probe. 96 individual transformants were grown on a microtiter plate, replicants were applied to the plates, and grown on a nitrocellulose membrane. The colonies were then cleaved, bacterial DNA was filtered, and the filters were hybridized with ³² P-14 mer (WEYCD) probes. The filters are washed to separate the non-hybridized probe and exposed to the X-ray film. This autoradiogram represents the schemes obtained with 48 individual filters (4600 independent colonies). One example of a positive plasminogen activator clone activator cDNA on filter no. 25 is designated E10 (arrow).

Figure 4 is a full-length restriction endonuclease blueprint of the human tissue plasminogen activator cDNA. The number and size of fragments produced by restriction endonuclease cleavage were evaluated by electrophoresis through 6% acrylamide gels. The positions of the sites are confirmed by the nucleic acid sequence (presented in Figure 5). The region for encoding the largest open reading frame is in the box, and the shaded region represents the putative sequence of the signal polypeptide, while the dotted region represents the putative sequence of the mature plasminogen tissue activator (527 amino acids). The 5 'end of the mRNA is to the left, while the 3' end is to the right.

Figures 5A, 5B and 5C illustrate the nucleotide sequence and derived amino acid sequence of a full-length plasminogen activator of human tissue plasminogen. The 35 amino acids (-35 to - 1) that precede the mature sequence are described as a continuous sequence. This 35-amino acid sequence is thought to consist of a hydrophilic pro sequence preceding the serine (+1) of the mature protein, with about 12 to 15 amino acids, which is again preceded by a conventional hydrophobic signal (which propagates 5 'to -35). This type of pre-pro structure on secreted proteins has been described previously, e.g. with preproalbumin. Assuming this theory, all molecules of the secreted plasminogen tissue activator begin with serine (+1) as the amino terminus. Another theory is that the hydrophilic sequence may be involved with the function of plasminogen tissue activator in an analogous manner, such as that observed with plasminogen, where a 10,000 daltons peptide can be separated from the amino terminal portion of the native plasminogen (Glu-plasminogen named after the amino terminal residue). leading to a smaller molecule with a new aminoterminus labeled Lys-plasminogen. Lys-plasminogen is more readily activated into plasmin and also has a greater affinity for fibrin than Glu-plasminogen. Plasmin have been shown to catalyze the conversion of Glu- to Lys-plasminogen. This type of control mechanism leads to a positive feedback control mechanism. The first amounts of plasmin formed, in addition to fibrin degradation, also lead to the generation of plasminogen molecules that are more readily activated and also more firmly bound to their substrate than native plasminogen. The result is faster plasmin degradation. The plasminogen activator hydrophilic peptide of the tissue could be involved in a similar mechanism, with its cleavage leading to a modified binding of the enzyme to fibrin. In each case, a 35 amino acid sequence is considered to be a sequence of a mature protein.

6 is a schematic diagram of the construction of the plasmid pdeltaRIPA ⁰ for expression of a plasminogen activator of tissue. The starting plasmid pPA25E10 was first digested with Pstl to isolate the 376 bp fragment (base pairs), which was then digested as shown.

Figure 7 shows the result of a plate fibrin assay on the fibrinolytic activity of an expression product obtained via pdeltaRIPA ⁰ in transformed cells.

Figure 8 is an HPLC trace of peptides from trypsin secretion (their plasminogen tissue activator) (absorbance at 210 nm). The arrow indicates the dot corresponding to the peptide used to construct the nucleotide probe used with the colonies library. The peptide represented by this dot was found to have an entire sequence: L-T-W-E-Y-C-D-V-P-S-C-S-T-C-G-L. Similarly, the other major dots were sequenced and were found to confirm the correct amino sequence of the human tissue plasminogen activator. The one-letter peptide code that refers to amino acid labels is as follows:

Asp D aspartic acid Ile I isoleucine Thr T threonine Leu L leucine Sir S serine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly Mr glycine Lys K lysine Ala A alanine Arg R arginine Cys C cysteine Trp W tryptophan Val V valine Gin Q glutamine Met M methionine Asn N asparagine

Figure 9 describes the construction of a plasmid encoding for direct expression of a mature human tissue plasminogen activator in E. coli. 50 μ-g of plasmid pPA17 was digested with Sau3AI HincII and Hhal and electrophoresed on a 6% polyacrylamide gel. We regenerate approximately 0.5 μ-g of the 55 bp Sau3AI-HhaI fragment. In a similar manner, approximately 3 p, g of the 263 bp Hhal-Narl fragment was purified from 80 / xg of the clone pPA25E10 first by isolating the 300 bp Pstl-Narl fragment and then digesting this fragment with Hhal. All digerations were carried out at 37 ° C for 1 hour and the reaction products were separated and electroeluted from 6 percent polyacrylamide gels. The two labeled deoxyoligonucleotides 5 'dAATTCATGTCTTATCAAGT (I) and 5' GATCACTTGATAAGACATG (II) were synthesized by a solid phase phosphotriester process (51). 100 pmoles of oligonucleotide II are phosphorylated in a 30 gl reaction containing 60 mM Tris (pH 8), 10 mM MgCl ₂ , 15 mM beta-mercaptoethanol and 50 gCi / gamma ³² P / ATP (Amersham 5,000 Ci mmol · ¹ ), 12 are added of T4 polynucleotide kinase units and the reaction was allowed to run at 37 ° C for 15 min. Then 1 g of 10 mM ATP and 12 units of T4 kinase were added and the reaction was allowed to proceed for another 30 minutes. After phenol / CHCl ₃ extraction, phosphorylated oligomer II and 5 'hydroxyl oligomer I were combined with 0.5 μ% eluted 55 bp Sau3AI-HhaI fragment and 2 μ% 263 bp Hhal-Narl fragment and ethanol precipitated. These fragments were ligated at room temperature for 4 hours in 60 μ \ 20 mM TrisHC1 (pH 7.5), 10 mM MgCl ₂ , 10 mM dithiothreitol, 0.5 mM ATP, and 1000 units of T4 DNA ligase. The mixture was digested for 1 hour with 48 Nar I units, 20 EcoRI units, and 40 Bgl II units (to eliminate polymerization via ligation of cohesive Sau3AI terminuses) and electrophoresed on a 6% gel. The 338 bp product (approximately 0.1 gg) is regenerated by electroelution. The remaining sequences for the coding of t-PA (amino acids

111-528) was isolated on a 1645 bp fragment by digesting plasmid pPA25E10 with Narl and Bglll. Plasmid pLeIFAtrplO3 is a derivative of plasmid pLeIFA25 (52), in which the EcoRI site is removed from the LeIF A gene 53). Three gg of pLeIFAtrpl03 were digested with 20 EcoRI units and 20 Bglll units for 90 minutes at 37 ° C, electrophoresed on a 6% polyacrylamide gel, and a large vector fragment (about 4200 bp) was regenerated by electroelution. For the final construction, 80 ng of EcoRI-Bglll pLeIFAtrpl03 was ligated with 100 ng of a 1645 bp Narl-Bglll fragment in 20 ng 338 bp of EcoRI-Narl fragment for 10 hours at room temperature. This ligation mixture was used to transform E. coli K-12 strain 294. Plasmid DNA was made from 38 of these transformants and digested with EcoRI. Of these plasmids, ten contained the desired 600 bp and 472 bp EcoRI fragments. DNA sequence analysis confirmed that none of these plasmids (pt-PAtrpl2) had the desired nucleotide sequence at the junctions between the trp promoter, synthetic DNA and cDNA.

Figure 10 shows the result of a plate fibrin assay on the fibrinolytic activity of a plasminogen activator tissue expression product. Night culture of E. coli W3110 / pt-PAtrpl in Luria broth containing 5 gg ml · ¹ tetracycline was diluted 1: 100 in M9 substrate containing 0.2% glucose, 0.5% casamino acids and 5 gg ml ' ¹ tetracycline. Cells were grown at 37 ° C to Α _55θ 0.2 and _indolacrylic acid was added to a final concentration of 20 gg / ml. Samples were collected by centrifugation at Α _55θ = 0.5-0.6 (about 2xl0 ⁸ cells ml ⁴ ) and immediately frozen. The cell granules were suspended in 6M guanidine hydrochloride at 5x10 ⁸ cells / ml, sonicated for 10 seconds, incubated at 24 ° C for 30 min, and then dialyzed for 4 h against 25 mMTris-HCl pH 8.0, 250 mM NaCl, 0.25 mM EDTA and 0.01 percent Tween 80. After dialysis, the samples were centrifuged at 13,000 rpm for 2 minutes and 10 g of the supernatant were analyzed for plasminogen activator activity. Following the procedure from Granelli-Piperno and Reich (87), the plate was incubated for 3.5 hours at 37 ° C and the vaccination zones were measured. Quantification is achieved by comparison with the dilutions of the purified plasminogen activator of melanoma tissue activator.

E.l.B. Plasminogen activator activator mRNA source

Human melanoma (Bowes) cells were used. Melanoma cells were cultured to cast monolayers in 100 ml Earles minimal base medium supplemented with sodium bicarbonate (0.12% final concentration), 2 mM glutamine and 10% heat-inactivated calf fetal serum. To confirm that melanoma cells are actively produced by a human tissue plasminogen activator, human melanoma cells are cultured until cast in a 24-well microtiter cup. Either in the presence or in the absence of 0.33 μΜ protease inhibitor aprotinin, the cells were washed once with phosphate-buffered saline and 0.3 ml of methionine-free serum was added. 75 gCi ( ³⁵ S) -methionine was added and the cells were labeled at 37 ° C for 3 hours. Once the 3-hour marking is complete, the substrates are separated from the cells and treated with a specific human tissue plasminogen activator IgG, or pre-immunostained immunoprecipitation serum (54). Immunoprecipitation products were displayed by electrophoresis on a 10% SDS-acrylamide gel. The gel was fixed, dried and subjected to fluorography.

E.l.C. Isolation of information RNA and fractionation by size

Total RNA from melanoma cell cultures was extracted essentially as reported in Ward et al. 55). Cells were granulated by centrifugation and then resuspended in 10 mM NaCl, 10 mM Tris-HCl pH 7.5,1.5 mM MgCl ₂ . Cells were digested by addition of NP-40 (final concentration 1 percent) and the nuclei were granulated by centrifugation. The supernatant contains total RNA which is further purified by repeated extractions with phenol and chloroform. The aqueous phase was made 0.2 M relative to NaCl and then the entire RNA was precipitated by the addition of 2 volumes of ethanol. Oligo-D-Cellulose chromatography (54) is used to purify the mRNA from whole RNA preparations. Typical yields of 10 g of cultured melanoma cells are 5 to 10 mg of total RNA and 50-20 gg of Poly (A) plus mRNA.

Fractionation of PolyA ⁺ (200 / tg) (56) was performed by electrophoresis through carbamidagarase gels. The agarose gel slice (57, 58) consisted of 1.75% agarose, 0.025 M sodium citrate, pH 3.8, and 6 M carbamide. Electrophoresis was performed for 7 minutes at 25 milliamps and 4 ° C. The gel was then fractionated with a razor blade. The individual slices were dissolved at 70 ° C and extracted twice with phenol and once with chloroform. The fractions were precipitated with ethanol and then tested by in vitro translation in the Bethesda Research Lab rabbit reticulocyte lysate system. (59, 60) supplemented with dog pancreatic microsomes as follows: translation is performed using 25 / tCi ( ³⁵ S) methionine and 500 nanograms of each slice of RNA gel in a final volume of 30 / tl containing 25 mM HEPES, 48 , 3 mM potassium chloride, 10 mM creatine phosphate, 50 mM each of 19 amino acids, 1.1 mM magnesium chloride,

16.6 mM EDTA, 0.16 mM dithiothreitol, 8.3 mM hemin, 16.6 / tg / ml creatine kinase, 0.33 mM calcium chloride, 0.66 mM EGTA, 23.3 mM sodium chloride.

Incubations were performed at 30 ° C for 90 minutes. The pancreatic microsomal membranes of the dog are prepared from coarse microsomes using EDTA to remove ribosomes (61) and treated with nuclease as described (62) and were present at a final concentration of 7 Α ^ θ units / ml in the translation mixture. Translational products or immunoprecipitated translational products were analyzed by electrophoresis on 10% polyacrylamide gels in sodium dodecyl sulfate as described previously (63). Unpainted gel pieces were fixed, dried and subjected to fluorography (64).

The resulting translational products from each gel fraction were immunoprecipitated with specific IgG activators of rabbit anti-human tissue plasminogen. One major immunoprecipitated polypeptide band is observed in the translation of RNA fractions of numbers 7 and 8 (migration from 21 to 24 S) having a molecular weight of approximately 63,000 daltons. We did not observe this band when preimmunological IgG was used for immunoprecipitation, suggesting that these polypeptides are specific for plasminogen activator of tissues.

E.l.D. The preparation of a colony library containing the activator frequencies of plasminogen tissue / tg mRNA fractionated on gel (slice of mRNA 7) was used to prepare double stranded cDNA using standard procedures (52, 65, 66). cDNA was fractionated by size on a 6% polyacrylamide gel. cDNAs greater than 350 base pairs in length (125 ng) are electroelected. 30 ng of cDNA was expanded with deoxy (C) residues using terminal deoxynucleotidyl transferase (67) and germinated with 300 ng of plasmid pBR322 (68), which was similarly tailored to deoxy (G) residues at the Pst1 site (67). The quenched mixture was then transformed into E. coli K12 strain 294 (ATCC No. 31446). We get about 4,600 transformants.

E.l.E Preparation of DNA probe

Purified human tissue plasminogen activator is obtained by the procedure described in references (19, 20).

The molecule is scanned to locate the regions most suitable for the preparation of synthetic probes as follows:

Reduction and carboxymethylation were performed to prepare trypsin-digestible proteins. A sample of 2 mg plasminogen activator was first dialyzed against 0.01% Tween 80 overnight at room temperature. The lyophilized protein was then dissolved in 12 ml of 0.56 M Tris-HCl buffer (pH 8.6), 8 molar with respect to the urea and 5 mM EDTA. Disulfide bonds were reduced by the addition of 0.1 ml / 3-mercapto ethanol. This reaction was carried out under nitrogen for 2 hours at 45 ° C. The reduced disulfides were alkylated into a carboxymethyl derivative by the addition of 1.0 ml of 1.4 M iodoacetic acid in 1 N NaOH. After 20 minutes at room temperature, the reaction was stopped by dialysis against 0.01% Tween 80 for 80 hours at room temperature and lyophilization was performed.

The resulting lyophilized carboxymethylated protein was redissolved in 3 ml of 0.1 M sodium phosphate buffer (pH 7.5). Trypsin (TP CK) (ratio 1 to 50) was added and digested at 37 ° C. Aliquots (0.1 ml) were withdrawn after 3 hours, 6 hours and 12 hours. A second trypsin supplement was performed after 12 hours. After 24 hours, the reaction was stopped by freezing the sample until it could still be injected with HPLC. Digerization progress was determined using an SDS gel on aliquots. All gels were empty except for a bad strip on an aliquot after 3 hours. This suggested that the digestion within 24 hours was complete and that no large peptides remained.

The sample (approximately 0.5 ml) was injected into an Altex C-8 ultrasphere 5 μm high resolution column in two portions. A gradual acetonitrile gradient was used (1 percent to 5 percent for 5 minutes, 5 percent to 35 percent for 100 minutes, 35-50 percent for 30 minutes). In one of the two preparative experiments, the eluent was monitored at two wavelengths (210 nm and 280 nm). The absorption ratio at two wavelengths was used to indicate peptides containing tryptophan.

We first sequenced the peptide dots that were most likely to contain tryptophan, or those that we believed were useful for other reasons. This allowed the determination of the sequence around the largest number of tryptophan. After sequencing around the top 25 possible peptide peaks, we aligned all sequence-aligned sequence data to obtain a preliminary model of the primary structure of the plasminogen activator tissue. Based on these data and models, several possible probes were located.

E. 1 .F. Identification of bacterial clones containing plasmidogen activator tissue cDNA sequences

The colonies were individually inoculated into wells of microtiter plates containing LB (93) + 5 μg / ml tetracycline and stored at -20 ° C after the addition of DMSO. Two copies of the colony library were grown on nitrocellulose filters and DNA from each colony was fixed to the filter using the Grunstein Hogness procedure (69).

³² P-labeled - TC (^) CA (q) TA (^) TCCCA probe was prepared (from synthetic oligomer) (WEYCD) from 14 mer groups as described above. Filters containing 4,600 transformants were hybridized for 2 hours at room temperature in 50 mm sodium phosphate pH 6,8,5xSSC (80), 150 jttg / m DNA of sonicated salmon sperm, 5x Denhardt solution (85), 10 percent formamide and then hybridized with 50x 10 ⁶ counts per minute of the labeled probe in the same solution. After incubation at room temperature overnight, the filters at room temperature were washed 3 times in 6x SSC, 0.1% SDS for 30 minutes, once with 2x SDS and then exposed on a Kodak XR-5 film for Dupont Lightening X-rays Plus a reinforcement sieve for 16 hours.

Plasmid DNA was isolated by rapid procedure (71) from all colonies showing a positive hybridization reaction. The cDNA inserts from these clones are then sequenced after subcloning the fragments into the M13 vector mp 7 (73) and using the Maxam Gilbert chemical process (74). Figure 3 shows filter no. 25 showing a hybridization scheme of a plasminogen activator positive clone of a tissue. It is shown that the cDNA insert in clone 25E10 DNA encoding a plasminogen activator by comparing its amino acid sequence with a peptide sequence (see above) obtained from a purified plasminogen activator and using its expression product produced in E . coli as described below. The cDNA portion of clone 25E10 (plasmid pPA25E10) had 2304 base pairs in length with the longest open reading frame encoding a 508 amino acid protein (mol. 56.756) containing a 772 bp 3 'untranslated region. This cDNA clone had no N-terminal coding sequence.

E.l.G. Direct expression of human tissue plasminogen activator clone in

E. coli

With reference to Figure 6, 50 / xg pPA25E10 (above) was digested with Pati and the 376 bp fragment was isolated by electrophoresis on a 6% polyacrylamide gel. About 3 μ% of this fragment was isolated from the gel by electroelution, digested with 30 units of Dde I for 1 hour at 37 ° C, extracted with phenol and chloroform and ethanol precipitated. The obtained Dde I adhesive ends were extended to the warm ends by the addition of 5 units of DNA polymerase I (Klenow fragment) and 0.1 mM dATP, dCTP, dGTP, dTTP in the reaction mixture and incubation at 4 ° C for 8 hours. After extraction with phenol and chloroform, DNA was digested with 15 Narl units for 2 hours and the reaction mixture was electrophoresed on a 6% polyacrylamide gel. We regenerated approximately 0.5 μ-g of the desired 125 bp Narl fragment with a blunt end. This fragment encodes amino acids no. 69 to 110 mature full-length plasminogen activator protein.

To isolate the 1645 bp Nar I - Bgl II fragment, 30 μξ pPA25E10 was digested with 30 Nar I units and 35 Bgl II units for 2 hours at 37 ° C and subjected to electrophoresis on a 6% polyacrylamide gel. We regenerate approximately 6 / xg of the desired 1645 bp Nar Ι-Bgl II fragment.

The plasmid pdeltaRlSRC is a derivative of plasmid pSRCexl6 (79), in which Eco R1 sites close to the trp promoter and away from the SRS gene are removed by DNA polymerase I repair (28) and self-complementary oligodeoxynucleotide AATTATGAATTCAT (synthesized using (75)) is inserted into the remaining Eco RI site directly adjacent to the Xbal site. The 20 µg pdeltaRlSRC was digested until complete with Eco RI, extracted with phenol and chloroform and ethanol precipitated. The plasmid was then digested with 100 units of nuclease Sl at 16 ° C for 13 minutes in 25 mM sodium acetate (pH 6.4), 1 mM ZnCl ₂ and 0.3 M NaCl to create the top end with the ATG sequence. After extraction with phenol and chloroform and ethanol precipitation, DNA was digested with Bam HI, electrophoresed on a 6% polyacrylamide gel, and a large vector fragment (4,300 bp) was regenerated by electroelution.

The expression plasmid was coupled to a total ligation of 0.2 gg vector, 0.06 gg 125 bp Narl fragment with a blunt end, and 0.6 μξ Narl-Bglll fragment 1645 bp fragment with 10 units of T4 DNA ligase for 7 hours at room temperature and used to transform E. coli strain 294 (ATCC No. 31446) to ampicillin resistance. Plasmid DNA was made from 26 colonies and digested with Xbal and Eco RI. 12 of these plasmids contained the desired 415 bp Xbal-Eco RI and 462 bp Eco R1 fragments. DNA sequence analysis confirmed that some of these plasmids had the ATG initiation codon correctly positioned at the start of amino acid no. 69 (serine). One of these pdeltaRIPA ⁰ plasmids was tested and the desired plasminogen activator was produced (Fig. 7).

E.l.H. Full-length plasminogen activator activator cDNA

a. ) Preparation of a colony library containing N-terminal sequences of plasminogen tissue activator

0.4 gg of the synthetic oligonucleotide 5 'TTCTGAGCACAGGGCG 3' was used to primate 7.5 gg of the mRNA 8 gel fraction (above) to prepare double stranded cDNAs using standard procedures (65, 66). cDNA was fractionated by size on a 6% polyacrylamide gel. A fraction larger than 300 base pairs (36 ng) is electroelected. Expand 5 ng of deoxy (C) residues using terminal deoxynucleotidyl transferase (67) and germinate with 50 ng of plasmid pBR322 (68), which was similarly digested with deoxy (G) residues at the Pst I site (67). The tempered mixture was then transformed into E. coli K12 strain 294. About 1500 transformants were obtained.

b. ) Southern hybridization of human genomic DNA

Because the priming cDNA reaction was performed using a synthetic fragment that hybridized 13 base pairs from the N-terminal clone pPA25E10, no suitable restriction fragment was available in this region with 29 base pairs (which includes a 16-dimensional sequence) for cDNA clone testing. Therefore, it is imperative that we isolate the genomic clone of a plasminogen activator of human tissue in order to identify cDNA clones extended by a primer containing N-terminal sequences encoding a plasminogen activator of tissue.

The first phase in this process involved the identification of the presence of only one plasminogen activator gene of homologous tissue in human genomic DNA. In this procedure (77), 5 gg of high molecular weight human lymphocyte DNA (prepared as 80) was digested until complete with various restriction endonucleases, subjected to electrophoresis on 1.0 percent agarose gels (81) and deposited on a nitrocellulose filter (77). ^{A 32} P-tagged DNA sample was made (76) from the 5 'end of the cDNA insert of pPA25E10 (230 bp Hpall-Rsal fragment) and hybridized (82) with a nitrocellulose filter. 35xl0 ⁶ counts per minute of probe were hybridized for 40 hours and then washed as described (82). Two endonuclease digestion schemes provide only one hybridizing DNA fragment: Bgl II (5.7 Kbp) and Pvu II (4.2 Kbp). Two hybridizing DNA fragments were observed with Hinc II (5.1 Kbp and 4.3 Kbp). All these data collected suggest the presence of only one plasminogen activator tissue gene in the human genome and that this gene contains at least one intervention sequence.

c.) Human lambda phage library testing for plasminogen activator genes

The strategy used to identify lambda phage recombinants carrying plasminogen activator genes was the detection of nucleotide homology with a radioactive probe made with the plasminogen activator cDNA pPA25E10 cDNA. One million recombinant lambda phage was plated on a DP 50 Sup F plate with a density of 10,000 pfu / 15 cm plate by the procedure of Benton and Davis (78). ^{The 32} P-labeled DNA probe was prepared by standard procedures (83) from a 230 bp Hpall-Rsal fragment located on 34 base pairs from the 5 'end of plasmid pPA25E10. Each nitrocellulose filter was pre-hybridized at 42 ° C for 2 h in 50 mM sodium phosphate (pH 6.5), 5xSSC (77), 0.05 mg / ml DNA of sonicated salmon sperm, 5x Denhardt solution (84), 50% formamide and then hybridized with 50x10 ⁶ counts per minute of marked probe in the same solution containing 10 percent sodium dextran sulfate (85). After overnight incubation at 42 ° C, the filters are washed 4 times at 50 ° C in 0.2xSSC, 0.1% SDS for 30 minutes, once in 2xSSC at room temperature and then exposed to Kodak XR-5 film for X-rays with Dupont Cronex boosters overnight. A total of 19 clones are hybridized with the probe. The DNA phage is made of 6 recombinants as previously described (86). Lambda clone C was selected to prepare the PvuII fragment for colony testing. 30 g of DNA was digested with Pvu II1 for 37 hours and subjected to electrophoresis on 1.0 percent agarose gels. The 4.2-kilobase pair fragment, which was previously shown to contain plasminogen activator sequences, was electroelected and purified. ^The P-labeled probe is prepared by standard colonization hybridization procedures (83) as described below.

d.) Colony library testing for 5 'plasminogen tissue activator sequences

Colonies were transferred from the plates and cultured on nitrocellulose filters, and DNA from each colony was fixed to the filter using the Grunstein-Hogness procedure (69). ^{32 The} labeled probe was made by priming (83) a Pvu II fragment with 42 kilobase pairs of cow thymus from an isolated lambda genomic clone of a plasminogen activator. Filters containing 1500 transformants were hybridized with 112x10 ⁶ cpm ³² P-genomic Pvu II fragments.

Hybridization was performed for 16 hours using the conditions described in Fritsch et al (82). The filters were well washed and then exposed to a Kodak XR-5 film for X-rays with Dupont Lightnig Plus amplification screens for 16-48 hours. 18 colonies were clearly hybridized with the genomic probe. Plasmid DNA was isolated from each of these colonies and bound to nitrocellulose filters and hybridized with ³² P-labeled synthetic oligonucleotide (16-mer) used for the original priming reaction. Of the 18 clones, 7 were hybridized with a kinase 16-mer. After sequence analysis, after subcloning the fragment into ml3 vector mp ⁷ (73), one clone (pPA17) was shown to contain the correct 5 'N-terminal region of the plasminogen activator, the signaling leader sequence and the 84 bp 5' untranslated region. From the two clones pPA25E10 and pPA17, the complete nucleotide sequence of Figure 5 and the restriction scheme (Figure 4) of the full-length plasminogen activator clone were determined.

The native plasminogen activator molecule has the potential to stabilize with 17 disulfide bridges based on homology with other serine proteases. There are 4 potential sites for N-glycosylation, three are located in the kringle regions at asn ₁₁₇ , asn _lg4 , asn _21g and one potential site is in the light string region, at asn _44g . Variations in the structure of oligosaccharide ligands may be responsible for different molecular forms (types of molecular weights 65,000 and 63,000).

E. 1.L Direct expression of a full-length plasminogen activator clone cDNA in E. coli

Reconstruction of the entire coding sequence was possible using a common Hhal restriction endonuclease site shared by both the partial clones pPA17 and pPA25E10. A 55 bp Sau3AI-HhaI restriction fragment corresponding to amino acids 5-23 was isolated from plasmid pPA17. The Sau3AI restriction site was located on the codon of four putative mature coding sequences and used to separate the signal peptide encoding region. We also isolated a 263 bp Hhal-Narl fragment (encoding for amino acids 24-110) from plasmid pPA25E10. We have designed synthetic deoxynucleotides that renew codons for amino acids 1-4, incorporate the ATG translational initiation codon, and create the EcoRI cohesive terminus. These three fragments are then ligated together to form a 338 bp fragment encoding amino acids 1-110. This fragment and the 1645 bp Narl-Bglll fragment from pPA25E10 were then ligated between the EcoRI and Bglll sites of the plasmid pLeIFAtrplO3 (53) to obtain a plasmid for the expression of pt-PAtrpl2. The cloned t-PA gene is transcribed under the control of a 300 bp fragment of the E. coli trp operon containing the trp promoter operator and the Shine-Dalgar sequence of the trp leader peptide but lacks the ATG lead peptide initiation codon (52).

E. coli K12 strain W3110 (ATCC No. 27325) containing pt-PAtrpl2 was grown and extracts were prepared for testing fibrinolytic activity. One of the methods used to measure the activity of plasminogen activator is tissue fibrin assay (87). It measures the amount of your plasmin by measuring the extent of plasmin digestion of fibrin on an agarose plate containing plasminogen and fibrin. Plasmin produces a clear cleavage zone in the fibrin plate and the surface of this zone can be cholerated by the amount of plasminogen activator tissue in the sample. When the pt-PAtrpl2 clone extracts are tested for plasminogen activator activity using a plate fibrin assay, the cleavage zone is apparent. This fibrinolytic activity is inhibited by anti t-PA IgG, but not by preimmune IgG or anti-urokinase IgG, and no activity is seen from cells containing control leukocyte interferon plasmid pLeIFAtrpl03. Using the standard purified t-PA curve, it can be estimated that about 20 units of extracted activity are obtained per 10 ⁹ cells (for purified t-PA, 90,000 Plow units = 1 mg) (Figure 10).

E.l.J. Sequence analysis

Sequence analysis was based on Edman degradation (83b). The sample was introduced into a Beckman beaker 890B or 890C sequencer with a rotary beaker. Polybrene ™ (NjNjN ^ NMetramethyl-N-trimethylenehexamethylene diammonium diacetate) (63C) was used as the beaker. The sequencer is modified with a cold trap and some changes to the program so that the base dots (background dots) are reduced. The reagents were 0.1 molar Quadrol buffer, phenylisothiocyanate, and heptafluorobutyric acid of quality for the Beckman sequence.

The collected Edman cycles were manually converted into 2-anilino-5-thiazolinone derivatives.

1- Chlorobutane was dried under nitrogen. Then 1.0 N HCl in water is added

2- anilino-5-thiazolinone and heated at 70 ° C for 10 minutes to convert it to 3-phenyl-2-thiohydantoin (PTH derivative). The PTH amino acid residue was then dissolved in 50% acetonitrile in water and injected into a reversed-phase high-performance liquid chromatograph. Each PTH amino acid is then identified by comparing the retention times of a standard mixture of PTH amino acids, which is introduced into a conversion vial and processed in the same manner as a sequencer cycle.

E.l.K. Tests for the detection of plasminogen activator tissue activator expression

1. Direct test for plasmin formation

a. The theory

Sensitive analysis of plasminogen activator can be performed by tracing the conversion of plasminogen to plasmin catalyzed by the plasminogen activator. Plasmin is an enzyme for which chromogenic substrate assays exist. These tests are based on the proteolytic cleavage of tripeptides from the chromophore group. The rate of cleavage is directly related to both the specificity and the protease concentration being tested. The basis of the assay is the determination of the amount of plasmin generated after incubation of a plasminogen activator-containing solution with plasminogen solution. The greater the amount of activator, the greater the amount of plasmin generated. Plasmin was measured by monitoring its cleavage from chromogenic substrate S2251 (purchased from Kabi Group, Inc., Greenwich, CT).

b. Process

An aliquot of the sample was mixed with 0.10 ml 0.7 mg / ml plasminogen (in 0.05M Tris.HCl, pH 7.4 containing 0.012 M NaCl) and adjusted to 0.15 ml. The mixture was incubated for 10 minutes at 37 ° C, 0.35 ml of S2251 (1.0 mM solution in the above buffer) was added and the reaction continued for 30 minutes at 37 ° C. Glacial acetic acid (25 μΥ) was added to complete the reaction. Samples were centrifuged and absorbance measured at 405 nm. Quantified amounts of activity are obtained by comparison with standard urokinase solution. The test conditions were modified to detect full-length plasminogen activator tissue by adding fibrinogen (0.2 mg) to the solution. Fibrinogen leads to stimulation of the observed activity of the plasminogen activator tissue, and therefore a slightly increased level of activity is generated. Activity was recorded in Plow Units, with 90,000 Plow Units equivalent to activity expressed by 1 mg of purified plasminogen activator.

2. Indirect Plasma Formation Testing

a. The theory

A sensitive test for the activity of plasminogen activator of tissue has been developed (87). The assay is based on the determination of plasmin formation by measuring the extent of plasmin digestion of fibrin on an agar plate containing fibrin and plasminogen. Plasmin produces a clear cleavage zone in the fibrin plate. The surface of the cleavage zone can be cholerated by the amount of plasminogen activator tissue in the sample.

b. Process

Following the procedure from Granelli-Piperno and Reich (87), the plates were incubated for 3.5 hours at 37 ° C and the cleavage zones were measured. Quantification is achieved by comparison with standard urokinase solution.

E.l.L. Plasminogen Tissue Activator Activity Detection

1. Bacterial growth and sample preparation

A plasmid-containing E. coli colony (pdeltaRIPA ⁰ ) was inoculated into a test tube containing 5 ml of LB growth medium containing 20 µg / ml ampicillin. Cells were cultured overnight at 37 ° C. An aliquot of this culture was diluted 1: 100 in 300 ml M9 substrates containing 20p, g / ml ampicillin. Cells were grown in a shaker at 37 ° C for 4 h, resulting in an absorbance at 550 nm of 0.419. The tryptophan analog of indolacrylic acid was added to a concentration of 30 µg / ml. The cells were incubated for 90 minutes, resulting in an absorbance at 550 nm of 0.628. Cells were harvested by centrifugation and resuspended in 0.8 ml of 0.001 M Tris, pH 8.0 containing 0.01 M EDTA. The resulting suspension was stirred rapidly at room temperature for 18 hours. The sample was centrifuged and the supernatant tested for plasminogen activator activity.

For the expression of pt-PAtrpl2, see the exact description in the legend for Figure 10.

2. Activity detection

Tables 1 and 2 show the results of plasminogen activation using the corresponding E. coli extracts during the course of the assay. Plasminogen-dependent activity was generated (Table 1). This activity is unaffected by the rabbit preimmune serum and is significantly inhibited by an antiserum prepared against purified melanoma cells producing plasminogen activator (88), Tables 1 and 2. This indicates that E. coli extracts produce plasminogen activating activity inhibited by antibodies against plasminogen activator.

Figure 7 shows the result of a fibrin test on a plate for fibrinolytic activity. A standard amount of urokinase was added to the central row in concentrations, from left to right, of 0.24, 0.14, 0.10, 0.05, and 0.02 Ploug units. The lower row are samples of a natural plasminogen activator tissue, with the same amount of enzyme in each well. The wells contain from left to right, plasminogen activator, antiplasminogen activator plus pre-immunological serum and plasminogen activator plus plasminogen activator antibody. All wells in the top row contain 8 μΐ of E. coli plasminogen activator activator extract. The first well is an extract only, the second well has preimmune serum added, and the third well has plasminogen activator activator antibodies added. It is clear that the pre-immunological serum does not affect the natural or recombinant plasminogen activator of the tissue and that plasminogen activator activator antibodies inhibit the activity of both the natural product and the E. coli extract. Based on the urokinase standards, the extracts contain only slightly less than 2.5 Plow units per ml. This can be compared favorably with the value obtained in Table 1, namely 1.3 Plow units per ml. Tables 1 and 2 show the results of tests performed as described above in E.l.K.l.b .;

TABLE 1: Plasminogen activation by E. coli extract of RIPA-containing cultures

The pattern A— Extract (no plasminogen) 0,043 Extract 0,451 th most common Extract plus preimmune serum 0,477 th most common Extract plus anti t-PA antibodies 0,079

Percentage Calculated activities - Plow units / ml (θ) (100) 1.3

106

The percentage of activity was calculated by subtracting the control (0.043) from the values obtained and dividing by the obtained value from the extract

Table 2: Plasminogen activation by E. coli culture extract

pt-PAtrpl2 The pattern —405 Percentage of activities Extract 0.657 (100) Extract plus preimmune serum 0.665 101 Extract plus anti t-PA antibodies 0,059 9

Figure 10 presents the results of a fibrin assay on a plate derived from extracts from 10 l of E. coli fermentation cultures containing a plasmid for expression of plasminogen activator. Fibrinolytic activity of the extract containing plasminogen tissue activator is presented in Fig. 10 by well A. This fibrinolytic activity is inhibited by anti t-PAIgG (well C) but not pre-immunological IgG (well B) or urokinase IgG (well D) see activities from an extract prepared from cells containing the leukocyte interferon plasmid pLeIPAtrpl03 (well H) as a control.

E.2 Production of tPA using DHFR protein with low binding affinity for ΜΤΧ

E.2.A. Vector construction

The sequence encoding a plasminogen activator of tissues (t-PA) is inserted into an expression plasmid containing the low affinity mutant DHFR for binding to ΜΤΧ described in U.S. Pat. report serial no. No. 459,151, filed Jan. 19, 1983, which is pending at the same time and which corresponds to European patent application no. 117.060, incorporated herein by reference, by the following procedure (see Figure 11).

Three plasmids from overlapping t-PA plasmids, pPA25E10 and pPA17 and pt-PAtrpl2 (above) were prepared as follows: plasmid pPA17 was digested with Dde I, supplemented using Klenow DNA polymerase 1, and trimmed with Pst I. Thus, the corresponding plasmid was generated and isolated. 200 bp fragment containing a 5 'terminal t-PA sequence. The second t-PA fragment is obtained by cleaving pt-PAtrpl2 with Pst I and Nar I and isolating the fragment at about 310 bp. A third t-PA fragment is obtained by digesting pPA25E10 with Narl and Bgl II and isolating the fragment at about 1645 bp, which contains, in addition to a large portion of the t-PA coding region, some 3 'non-translated sequences.

Plasmid pE342 expressing the HBV surface antigen (also called pHBs348-E) is described in Levinson et al, patent application no. 326,980 that was filed

December 3, 1981 and corresponding to European patent application no. 73,656, which is incorporated herein by reference. (Briefly, we isolate the SV40 virus source by digesting SV40 DNA with Hind III and converting Hind III ends into EcoRI ends by adding AGCTGAATTC converters). This DNA was cut with PvuII and RI binding agents were added. After digestion with EcoRI, a 348 base pair-encapsulated fragment was isolated by electrophoresis on a polyacrylamide gel and electroeluted and cloned into pBR322. We construct a plasmid for expression of pHBs348-E by cloning a 1986 base-pair fragment derived from EcoRI and Bgl II by digesting HBV (Animal Virus Genetics, (Ch. 5) Acad. Press, Ν.Υ. (1980) (embracing gene encoding for HBsAg) in the pML plasmid (Lusky et al. Nature, 293: 79 (1981) at EcoRI and BamHI sites. (pML is a pBR322 derivative that has missing elimination sequences that inhibit plasmid replication in monkey cells). The resulting plasmid (pRI-Bgl) is then linearized with EcoRI and the 348 base pair fragment representing the SV40 region of the origin is introduced into the EcoRI site of pRI-Bgl, the source fragment can be inserted in any orientation, since this fragment encodes in addition to the replication source as early as also later SV40 promoters, HBV genes can be expressed under the control of any promoter, depending on this orientation (pHBS348-E represents HBs expressed under the control of the early promoter) .PE342 is modified by partial digestion with EcoRI, by cleavage on mes here using Klenow DNA polymerase and total plasmid ligation by removing the EcoRI site that is pre-SV40 in origin in pE342. The resulting plasmid, labeled pE342deltaR1, was digested with EcoRI, supplemented using Klenow DNA polymerase I, and trimmed with BamHI. After electrophoresis on an acrylamide gel, the fragment was electroelected at about 3500 bp, extracted with phenol-chloroform and precipitated with ethanol as above.

The thus prepared p342E 3500 bp vector and the above described 2160 bp t-PA fragments were ligated together using standard techniques. We isolate a plasmid containing three t-PA fragments for encoding in the correct orientation, characterize it, and it is designated as pE342-t-PA. This plasmid was digested with Sac II and treated with bak37-terial alkaline phosphatase (BRL). To provide the DHFR sequence (together with control sequences for its expression), a fragment of about 1700 bp was generated by Sac II digestion of pEHER. (pEHER is a plasmid expressing the mutant DHFR described in U.S. Serial No. 459,151 (above). This fragment is ligated into the pE342-t-PA plasmid to create a pETPAER400 plasmid analogous to pEHER, except that the HBsAg coding region is replaced by cDNA sequences from t-PA.

E.2.B. The expression and amplification of the t-PA sequence of pETPAER400 (pETPER) were transfected in both dhfr 'CHO-DUX Bil cells and DHFR ⁺ CHO-K1 (ATCC CCL61) cells by the procedure from Graham and Van der Eb (above). Transformed dhfr 'cells are selected by growth in a glycine, hypoxanthine and thymidine deficient substrate. Transformed DHFR ⁺ cells are selected based on growth in> 100 nM ΜΤΧ. Colonies growing on an appropriately selected basis are isolated using cloning rings and propagated in the same manner, on the same basis for several generations.

For amplification, cells from the colonies are divided into substrates containing 5x10 ⁴ , 10 ⁵ , 2.5x10 ⁵ , 5x10 ⁵ and ΙΟ ⁶ nM ΜΤΧ and allowed to pass several times. Cells were applied in very low (10 ² -10 ³ cells / plate) cell density in 10 cm cups and the resulting colonies isolated.

E.2.C. Testing procedures

The expression of t-PA in transfected amplified colonies can be appropriately evaluated by procedures similar to those shown in E.l.K.l.b (above).

The co-amplification of DHFR and t-PA sequences was tested by isolating DNA from the infused layers of (mono) amplified colonies as follows: The confluent monolayers in 150 mm plates were washed with 50 ml of sterile PBS and split with the addition of 5 ml of 0.1% SDS, 0.4 M CaCl ₂ , 0.1 M EDTA, pH 8. After 5-10 minutes, the mixture was separated, extracted with phenol, extracted with chloroform and precipitated with ethanol. The DNA was resuspended in 1 ml (150 mm plate) of 10 mM Tris-HCl pH 8, 1 mM EDTA (TE), RNase was added to 0.1 mg / ml and the solution was incubated for 30 minutes at 37 ° C. SDS is then added up to 0.1 percent and find (Sigma) up to 0.5 mg / ml is added. After 3-16 hours of incubation at 37 ° C, the solution was re-extracted with phenol, extracted with chloroform and precipitated with ethanol. The DNA granule was resuspended in 0.5 ml of water and digested with restriction enzymes. About 5-10 / xg of digested DNA was subjected to agarose gel electrophoresis / 1 percent agarose in Tris-acetate buffer (40 mM Tris, 1 mM EDTA supplemented to pH 8.2 with acetic acid); (Crouse, et al, J. Biol. Chem. 257: 7887 (1982)). When the bromophenol blue color migrates through the gel up to 2/3, the gel is separated and stained with ethidium bromide. After visualization of the DNA by ultraviolet light, DNA is transferred from the gel to nitrocellulose filters according to the procedure from Southern (J. Mol. Biol. 98: 503. (1975)). The filters were then hybridized with a translated probe prepared from a 1700 bp Sac II fragment of pEHER (prepared and hybridized as described above) or from a Bgl II fragment of pETPER of about 1970 bp.

E.3. Production of t-PA together with wild-type DHFR protein

E.3.A. Vector construction

In a manner analogous to that used in the pETPER construct, we construct a plasmid pETPFR containing a DNA sequence encoding for wild-type DHFR. The construction proceeded as in the case of E.2.A., except that the plasmid pE342.HBV.E400 was used instead of the plasmid pEHER, as a solution of the gene sequence for the DHFR protein. D22 is described in a pending application, Genentech Docket no. 100/92. In the US ser. no. 459.152 amended on 19 January 1983 corresponding to European patent application no. 117,058, which is incorporated herein by reference. Plasmid pE342.HBV.E400.D22 is the same as pEHER except for the difference in one base pair between wild-type DHVR and mutant. The plasmid pETPFR thus obtained is analogously analogous to pETPER, except that the DNA sequence encoding the wild-type DHFR is replaced by the mutant sequence.

E.3.B. Expression of the t-PA sequence of pETPFR was used for transfection of DHFR-deficient CHO cells (Urlaub and Chasin (above)) using a calcium phosphate staining procedure from Graham and Van der Eb. Twenty-one colonies grown on a selective basis (-HGT) were evaluated by detection of plasmin formation as assessed by fibrin digestion on an agar plate containing fibrin and plasminogen described in Granelli-Piperno, et al. J. Exp. Med., 148: 223 (1978).

The four most potent positive clones were quantitatively evaluated for plasmin formation per cell, according to the procedure described in E.l.K.l.b.

After such quantification, we found that the four clones tested expressed the same or comparable t-PA secretion into the substrate determined as unit / cell / day. Subclones were prepared by transferring the inoculum from two clones to special plates containing -HGT substrate. Two resulting subclones, 18B and 1, were used for further analysis.

E.3.C Amplification and production levels of t-PA

The upper subclones were coated with 2x10 ⁵ cells per 100 mm plates in 50 nM ΜΤΧ to promote amplification. Those cells that survive when tested as described above are in each case administered about 10 times the non-amplified amount of plasminogen activator tissue activator activity. We selected two of these clones for further study and are named 1-15 and 18B-9.

Subclone 1-15 was further amplified by implanting 2x10 ⁵ cells into 100 mm plates containing 500 nM ΜΤΧ. Testing of cells thus amplified gave a further increase (about 3-fold) in the production of t-PA; when evaluated quantitatively by the ClC procedure, the levels were 7xl0 ' ⁴ units / cell / day in the interval. A portion of these amplified cells were then transferred and maintained in the presence of 10,000 nM ΜΤΧ. Subclones 1-15 and 18-B-9 were further tested after being maintained for about 1-2 months under the conditions specified in Table T.

TABLE 1 ' not t-PA / cell / day The cell line Start growing 1-1 ^ 500 500nMMTX 28.5 χ ΙΟ ' ³ Ι '- ^ οο 500nMMTX 26,0xl0 ' ³ 1'15 _5O o (-HGT lining, no ΜΤΧ) 8.3 χ ΙΟ ' ³ (-HGT lining, no ΜΤΧ) 18.0 χ 10 ' ³ 1-15 10,000 ΙΟμ, ΜΜΤΧ 29.3 χ ΙΟ ' ³ 1-15 10,000 ΙΟμΜΜΤΧ 49.0 χ 10 ' ³ 18B-9 50nMMTX 14.3 χ ΙΟ ³ 18B-9 50 nM ΜΤΧ 14.4 χ 10 ' ³ 18B-9 (-HGT lining, no ΜΤΧ) 14.3 χ 10 ' ³ 18B-9 (-HGT lining, no ΜΤΧ) 14.4 xl0 ' ³ 1 (-HGT lining, no ΜΤΧ) 1,0 xl0 ' ³ 1 (-HGT lining, no ΜΤΧ) 0.7 χ 10 ' ³

* t-PA in the culture medium was quantitatively evaluated in radioimmunoassay as follows: purified t-PA and purified iodinated tracer t-PA derived from melanoma cells were serially diluted to include concentrations of 12.5 to 400 ng / ml. in buffer containing phosphate-infused salt solution, pH 7.3, 0.5 percent albumen oxide serum, 0.01 percent Tween 80, and 0.02 percent NaN _y Add to the radiolabeled tracer proteins appropriate dilutions of the substrate samples provided by need to be tested. The antigens were allowed to incubate overnight at room temperature in the presence of a 1: 10,000 dilution of the IgG fraction of the rabbit antiserum anti-t-PA.

The antibody-antigen complex was precipitated by absorption on the IgG of anti-rabbit immunogen (BioRad) for 2 hours at room temperature. The grains were clarified by the addition of saline diluent and then centrifuged at 2000 x g at 4 ° C for 10 minutes. The supernatants are discarded and radioactivity is recorded in the precipitate. Concentrations are determined by comparison with a reference standard.

The cell lines are as follows: cell line 1 is a non-amplified clone from the original set of four. 1-15 _5θ0 is an amplified subclone of cell line 1, which is amplified initially at 50 nM ΜΤΧ, so that 1-15 is obtained and then transferred to further amplification at 500 nM ΜΤΧ. 1-15 _10θ0θ is a 1-15 _{5θθ subclone} further amplified in the presence of 10,000 nM ΜΤΧ. The 18B-9 cell line is a subclone of one of four detected initially amplified to 50 nM ΜΤΧ.

All the amplified cells showed increased levels of t-PA production above those expressed by the unamplified cell culture. Even non-amplified culture produces amounts of t-PA greater than 0.5 pg / cell / day; amplification leads to levels approaching 50 pg / cell / day.

F. Pharmaceutical preparations

The compounds of the present invention can be formulated according to known methods for the preparation of pharmaceutically useful preparations, wherein the subject product, a human tissue plasminogen activator, is combined into a mixture with a pharmaceutically acceptable carrier. Suitable carriers and their formulation, including other human proteins, e.g. human serum albumin are described, e.g. in Remington's Pharmaceutical Sciences, E.W. Martin, cited here as a reference. Such preparations will contain an effective amount of the protein in question, together with an appropriate amount of carrier, for the preparation of pharmaceutically acceptable preparations suitable for effective administration to the host.

For example, the subject plasminogen activator of human tissue may be administered parenterally to patients who have cardiovascular disease or conditions. Dosage and dosage rates may be parallel to those currently used in clinical trials of other cardiovascular, thrombolytic agents, e.g. about 440 IU / kg body weight as an intravenous primary dose, followed by continuous intravenous infusion of about 440 IU / kg / hour for 12 hours in patients with pulmonary embolism.

As one example of a suitable dosage form for a substantially homogeneous parental plasminogen activator in parenteral form useful herein is a viola containing 25000 IU plasminogen activator activity, 25 mg mannitol and 45 mg NaCl, which can be reconstituted with 5 ml sterile water for injection and mixed with a suitable volume of 0.9% sodium chloride for injection or with a 5% dextrose solution for intravenous administration.

G. Accurate description of recombinant human t-PA

The structure of a particular embodiment of human t-PA prepared in the examples herein has been studied in certain detail, both in terms of explaining the coding gene sequence and in terms of protein biochemistry techniques. The current understanding of protein structure is illustrated in Figure 12.

Collen and colleagues (88) have also previously demonstrated that human double-stranded t-PA is formed by proteolytic cleavage of single-stranded molecules of two polypeptides that are coupled by a disulfide bond. The work done here concludes that the heavy chain (30882 molecular weight) is derived from the NH ₂ terminal portion, and the light chain (28126 molecular weight) comprises COOH, the terminal region. N-terminal sequencing of the double-stranded molecule suggests that the double-stranded form is generated by cleavage of a single arginyl-isoleucine bond (Fig. 12; drawn arrow).

The primary structure of part of the human t-PA heavy chain region (Figure 12) reveals a high degree of sequence homology with the plasminogen kringle regions (89) and prototrombin (40, 41 kringle region refers to the characteristic triple disulfide structure initially discovered in the pro the prothrombin fragment, first described precisely by Magnuson et al (91, 92) The primary t-PA sequence shows the so-called 82 amino acid kringle regions, which have a high degree of homology to the 5 kringle plasminogen regions. amino acids have little homology to the conventional kringle region, however, it can be assumed that this region may have a structure containing multiple disulfide bonds, as 11 additional cysteine residues are located here.

The human t-PA light chain catalytic site, the so-called serine protease region, is most likely formed, as in other serine enzymes, by residues of histidine ₃₂₂ , aspartic acid _371, and serine _47g . Furthermore, the amino acid sequences surrounding these residues are very homologous with the corresponding portions of other serine proteases, such as trypsin, prothrombin, and plasminogen.

Without neglecting the fact that certain desired embodiments are indicated, it is clear that the present invention is not limited thereto, but limited only by the legal scope of the claims submitted.

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Claims (9)

PATENT APPLICATIONS A DNA isolate comprising a DNA sequence encoding a human tissue plasminogen activator. A DNA isolate according to claim 1, characterized in that the human tissue plasminogen activator has the amino acid sequence 1-527 given in Figures 5a, 5b and 5c. A recombinant expression vector comprising a DNA sequence encoding a human tissue plasminogen activator, characterized in that the vector is capable of expressing a human tissue plasminogen activator into a transformed microorganism or cell culture. Recombinant expression vector according to claim 3, characterized in that the human tissue plasminogen activator has the amino acid sequence 1-527 given in Figures 5a, 5b and 5c. The vector-transformed microorganism of claim 3, which is capable of expressing a human tissue plasminogen activator such as E. coli. A cell culture capable of expressing a human tissue plasminogen activator, characterized by being obtained by transformation of a mammalian cell line such as the vector of a Chinese hamster ovary vector according to claim 3. A method comprising expressing the DNA sequence of claim 1 in a recombinant host cell, such as an E. coli microorganism or a Chinese hamster ovary cell line. 8. A method for producing a recombinant plasminogen activator of human tissue, characterized in that it comprises: a) the growth of recombinant cells transformed with the expression vector, the vector of claim 3, and b) simultaneously expressing said DNA according to claim 1. A method for producing a recombinant plasminogen activator of human tissue, characterized in that it comprises: a) transforming the host cell of claim 7 with a replicator vector comprising the DNA sequence of claim 1, and b) expressing said DNA into said host cell.