AU624869B2 - Production of human prourokinase - Google Patents

Description

1 I

AUSTRALIA

PATENTS ACT 1952 COMPLETE SPECIFICATION Form

(ORIGINAL)

FOR OFFICE USE Short Title: Int. Cl:

I

c Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: Priority: Related Art: TO BE COMPLETED BY APPLICANT 4 4 4 I~r 4' Name of Applicant: FAR1ITALIA CARLO ERBA s.r.l.

Address of Applicant: VIA CARLO IMBONATI, 24 20159 MILN

ITALY

Actual Inventor: Address for Service: GRIFFITH HACK CO., 601 St. Kilda Road, Melbourne, Victoria 3004, Australia.

Complete Specification for the invention entitled: PRODUCTION OF HUMAN YOUROKINASE.

The following statement is a full description of this invention including the best method of performing it known to me:- -fP "FC 409" PRODUCTION OF HUMAN PRDUROKINASE The present invention relates to a recombinant DNA method of producing non-glycosilated single-chain prourokinase (hereinafter referred to as proUK). More particularly, it relates to a method of producing non-glycosilated proUK which comprises recovering mRNA from an established cell line, preparing cDNA based on said mRNA, inserting the cDNA into a vector, introducing the resulting plasmid into a bacterial cell to thereby produce a trasformant and recovering 0 non-glycosilated proUK from said bacterial cell.

o o The invention concerns also certain expression plasmids employed in the above method.

S0 0 Co Introduction The increasing knowledge of the molecular interactions that regulate physiological fibrinolysis has lead to I important implications in the understanding of the S' mechanisms that dissolve blood clots, and in the development of new thrombolytic agents.

In the human fibrinolytic system a proenzyme, St plasminogen, can be activated to the active enzyme, plasmin, by several types of plasminogen activators 4, (Collen, D. and Ljjnen, CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, Blood, 67, n. 6,p. 1529, 1986).

Plasmin is the major protease responsible for the degradation of the fibrin component of a blood clot (Rakoczi, Wiman, B. and Collen, Biochim.

Biophys. Acta, 540, p. 295, 1978; Robbins, K.C.

S

i i C 2 Summaria, Hsieh, and Shah, J. Biol. Chem.

242, p. 2333, 1967; Wiman, Eur. J. Biochem, 76, p.

129, 1977).

However, plasmin can also exert its proteolytic effect on several plasma proteins among which the components of the coagulation pathway fibrinogen, factor V and VIII (Collen, D. and Lijnen, CRC Critical Reviews in oncology/hematology, 4, n. 3,p. 249, 1986; Verstraete, M. and Collen, Blood, 67, n. 6,p. 1529, 1986; Wiman, Lijnen, H.R, and Collen, Biochim, Biophys. Acta, 579, p. 1--12, 1979).

Activation of plasminogen may occur at the systemic level, leading to circulating plasmin that is rapidly i neutralized by alfa2-antiplasmin and thus not available for fibrinolysis (Collen, D. and Lijnen, CRC SCritical Reviews in oncology/hematology, 4, n. 3,p.

249, 1986; Verstraete, M. and Collen, Blood, 67, n.

(€it 6,p. 1529, 1986).

When the alfa2-antiplasmin level is markedly reduced, plasmin is less rapidly neutralized and can exert its proteolytic effects not only on fibrin, but also on the blood coagulation proteins as described previously.

S< Excessive lowering in the plasma concentrations of fibrinogen, factor V and VIII, together with the t t inhibitory effects exerted by some of the fibrinogen degradation products on the hemostatic process, on platelet aggregation and on fibrin polymerisation lead C' •to hemostatic deficiency and subsequently to high bleeding risk (Latallo, Z.S. and Lopaciuk, S.; Thrombos. Diath. Haemouh., 56, p. 253, 1973; Totty, Gilula, Mc. Clennman, Ahmed, and Sherman, L. Radiology, 143, p. 59, 1982). On the other hand, activation of plasminogen may occur at the fibrin level (fibrin-bound plasminogen activation) leading to i-i 3 fibrin-bound plasmin (Collen, D. and Lijnen, CRC Critical Reviews in oncology/hematology, 4, n. 3,p.

249, 1986; Verstraete, M. and Collen, Blood, 67, n.

6,p. 1529, 1986) which is, instead, not affected by alfa2-antiplasmin and cannot induce systemic fibrinogenolysis.

Urokinase and streptokinase, the most commonly used plasminogen activators in conventional thrombolytic therapy in man, have no specific activity for fibrin.

Both compounds activate relatively indiscriminately either circulating or fibrin-bound plasminogen (Zamarron, Lijnen, Van Hoef, and Collen, Thromb. Haemostas. 52, p. 19, 1984; Samama, and Kher, A. Sem. Hop. Paris. 61, n. 20. p. 1423, 1985).

Therefore, the systemic haemostatic breakdown often encountered during treatment with streptokinase and S€ urokinase and, consequently, the elevated bleeding risk have often hampered the widespread clinical use of these thrombolytic agents, despite their demonstrated clinical efficacy (Samama, and Kher, A. Sem. Hop.

Paris, 61, n. 20. p. 1423, 1985; Maizel, and Bookstein, Cardiovasc. Intervent. Radiol., 9, p.

236, 1986; Bell, W.R. Thromb. Haemostas., 35, p. 57, 1976; Acar, Vahanian, Michel, Slama, M., t Cormier, B. and Roger, Seminars in Thromb. and SHaemost., 13, n. 2, p. 186, 1987; Gruppo Italiano per lo studio della Streptochinase nell'infarto miocardico J (GISSI); Lancet, 1, p. 397, 1986).

On the contrary, tissue-type plasminogen activator (t-PA) (Hoylaerts, Ryken, Lijnen, H.R. and Collen, D.J. Biol. Chem., 257, n. 6, p. 2912, 1982), and more recently prourokinase (pro-UK) (Husain, S.S., and Gurewich, Arch. Biochem. Biophys. 220 p. 31, 1983), both natural proteins, were shown to be weak L I__ 4 activators of the circulating plasminogen and, conversely, strong activators of the fibrin-bound plasminogen, without either systemic haemostatic breakdown or consumption of alfa2-antiplasmin and plasminogen, thus their clinical use may cause lesser bleeding risk.

The fibrin-specific thrombolytic activity of t-PA has been explained by its ability to bind fibrin through specific lysine binding sites, located in the triple-disulfide-bonded "kringle domains" of the molecule.

Consequently, fibrin-bound plasminogen could be activated without significant haemostatic breakdown (Collen, and Lijnen, Haemostasis; 16, n. 3, p. 25, 1986). On the other hand, proUK (also denominated single chain urokinase type plasminogen activator, scu-PA) does not bind to fibrin, however it displays fibrin-specific thrombolytic activity without systemic hemostatic consumption (Pannell, R. and Gurewich, Blood, 67, p. 1215, 1986; Gurewich V., and Pannell, Seminars in Thromb. and Haemost., 13, n. 2, p. 146, 1987; Lijnen, Zamarron, Blaber, Winbler, and Collen, J. Biol. Chem. 261.

p. 1253, 1986).

C Recombinant t-PA was submitted to multicenter clinical trials in patients with acute myocardial infarctions and was shown to be significantly more effective than streptokinase in the recanalization of obstructed coronary arteries (The European Cooperative Study Group for Recombinant Tissue-type Plasminogen Activator; Lancet, 1 p. 842, 1985; Sheehan, F.H. Braunwald, E., Canner, Doodge, Gore, Van Natta P., Passamani, E.R. Williams, Zaret, Circulation, 4,p. 817, 1987).

Prourokinase is at present in advanced clinical trials and is thought to be, at least, as effective as t-PA in terms of thrombolytic activity and safety (Van de Werf, Nobuhara, and Collen, Annals of Internal Medecine, 104, p. 345, 1986; Van de Werf, F., Vanhaecke, De Geest, Verstraete, and Collen, Circulation, 74, n. 5, p. 1066, 1986)

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(4 40 4 1 4 (444 4 4I 4 44 tI S 4r 4 I (4r 4 BacKgrouna or tne invention Urokinase-type plasminogen activators (u-PAs) are found in at least three different forms in human urine, plasma and conditioned culture medium from a variety of cell lines. The first form to be characterized as u-PA consisted of a fibrinolytically active polypeptide of 410 amino acids, with an apparent moelcular weight of 54000 daltons, containing two disulfide-linked chains (Gunzler, Steffens, Oetting, Kim, SM.A.

Frankus, and Flohd, Hoppe Seyler's Z.

Physiol.Chem. 363, p. 1155, 1982).

The A-chain or light chain contains 157 amino acids and one triple disulfide-bonded "kringle" structure. This chain also contains a receptor binding domain for normal and neoplastic cells (monocytes, monocyte-like cells and A 431 epidermoid cells). The B chain or heavy chain (30000 daltons) consists of 253 amino acids and contains the catalytic domain.

This molecular form of u-PA is generally termed urokinase two. chain urokinase (TC-UK) or high molecular weight urokinase (HMW-UK)(Gunzler, W.A., Steffens, Oetting, Buze, and Floh6, L.: Hoppe-Seyler's Z. Phisiol. Chem. 363, p. 133, 1982).

The second form of u-PA has a molecular weight of 33000 daltons and results from proteolytic degradation of the HMW form by either plasmin or trypsin. It is called low ~vi; 6 I 0 00 00 00 00 0000 t00 molecular weight urokinase (LMW-UK). Protein sequence determinations have revealed that LMW-UK is identical to HMW-UK except for the absence of the NH2-terminal 135 amino acids that are specifically removed by the action of plasmin or trypsin (Steffens, Gunzler, W.A. Oetting, Frankus, and Floh6, L., Hoppe-Seyler's Z. Phisiol. Chem., 363, p. 1043, 1982).

Native prourokinase (proUK) is a single chain (54000 daltons) form of urokinase and is also termed single chain urokinase type plasminogen activator (scu-PA). As stated before, proUK displays a fibrin-specific thrombolytic activity and is therefore a better thrombolytic agent compared to the presently used high or low molecular weight urokinases.

In order to produce prourokinase, the authors of the present invention have developed a recombinant DNA procedure which allows the preparation of large amounts of the proUK polypeptidic chain.

Several methods have been described in the scientific and patent literature for the production of proUK (Holmes, Pennica, Blaber, Rey, M.W., Gunzler, Steffens, G.J. and Heynecker, H.L.; Biotechnology, 3 p. 923, 1985; European Patent Application 0092182). However, the method described within the text of the present int Tntion exploits parameters known to be important for the expression of heterologous proteins in E. coli, but whose combination has never been applied before to the production of recombinant proUK.

The main parameters, whose combination contributes to the establishment of a recombinant strain of E. coli, able to produce proUK, and which represents the object of the present invention, are the E. coli promoter Ptrp, the Shine-Dalgarno sequene MS-2 from the phage ii u i; i:i ii i: i ii -i i! i" r _1

I

t 7 MS-2, and E. coli strains of the type B as hosts for the expression of the human proUK gene (see below).

Such combination is crucial. Substitution of one of these parameters with an alternative expression signal may not yield as much proUK.

Accordingly, object of the present invention is a method for the preparation of non glycosilated pro-UK, characterized in that non-glycosilated pro-UK is expressed under the control of the E. coli promoter Ptrp and the Shine-Dalgarno sequence MS-2 by E. coli B.

I Description of the production procedure The present invention relates to the construction, by genetic engineering techniques, of strains of E. coli able to express the human proUK gene at high levels.

*synthesize large amouncs of the proUK polypeptidic Ichain.

In order to isolate said recombinant strains of the bacterium E. coli, it is necessary to go through a number of steps including: the isolation of the human cDNA gene coding for he desired proUK I the insertion of said gene in an appropriate expression plasmid the transformation of selected strain of E. coli I with the engineered plasmid and the cultivation of the transforrmants in appropriate conditions.

1) Cloning of the human cDNA gene coding for proUK To obtain the cDNA clone coding for human prourokinase, Lj 8 the authors have utilized the proteins sequence data published in the literature (Gunzler, Steffens, Oetting, Kim, SM.A., Frankus. and Floh6, Hoppe Seyler's Z. Physiol.Chem., 363, p. 1155, 1982; Gunzler, Steffens, Oetting, Buze, and Floh6, Hoppe-Seyler's Z. Phisiol. Chem.

363, p. 133, 1982; Steffens, Gunzler, W.A., Oetting, Frankus, and Floh6, Hoppe-Seyler's Z. Phisiol. Chem., 363, p. 1043, 1982).

Accordingly, specific probes have been prepared and an appropriate cDNA library has been screened.

Oligonucleotides coding for selected peptides of single-chain urokinase-type plasminogen activator (pro-UK) were chemically synthesized (Caruthers, M.H., 0 Gassen, H.G. and Lang, J.A. (eds) Verlag-chimie, Weinheim, Deefield Beach, Basel, p. 71, 1982) to serve as specific probes to monitor enrichment of proUK mRNA o e and to select for clones containing prourokinase cDNA from an enriched cDNA library. The oligomers were 14 to 17 mer in lenght, and each one was synthetized either as unique sequence (named p7) or in pools containing two (named pl, p2, p3) or 16 (named p6) o 0' oligonucleotides as indicated in Fig. 1. The oligomers were tested for specificity to proUK by northern *4 4 t* hybridization. For this analysis polyA-containing RNA was extracted from the HEp-3 epidermoid carcinoma (Miskin, Haemostasis (Switzerland), 11, No. suppl aj 1, p. 63, 1982). For each oligomer the temperature of the washing following the hybridization reaction was adjusted so as to be 2 to 5 degree C below the minimal melting temperature, as calculated according to Suggs et al. for hybridization to southern blots (Suggs, Hirose, Miyake, Kawashima, Johnson Itakura, K. and Wallach, Developmental 9 Biology Using Purified Genes; Brown D.D. and Fox, C.F.

(eds) Academic Press, New York, p. 638, 1981). In this text the five proUK probes, shown in Fig. 1, reacted with one common major carcinoma mRNA band of about 2.3 kb, which is the size expected for proUK mRNA.

Cloning took place using enriched mRNA fractions from the HEp-3 epidermoid carcinL.Ta. RNA preparations were extracted and enriched about 3 fold on two successive sucrose gradients. cDNA was synthesized according to published procedures (Efstratiadis, Kafatos, F.C., Maxam, A.M. and Maniatis, T. Cell, 7, p. 279, 1976; Buell, Wickens, Payvar, F. and Schimke, SR.T. J. Biol. Chem. 253, p. 2471, 1978) using oligo-dT 0 as a primer. Longer molecules were isolated using 00o polyacrilamide gel electrophoresis followed by e? ec.tro-elution of the appropriate gel fractions. The cDNA was then extracted using standard 0i< phenol/chloroform extraction followed by ethanol precipitation.

These cDNA molecules were first ligated to EcoRI linkers and then cloned into the phage Agtl0 vector according to a modification of the technique of Davis (Maniatis, Fritsch, Sambrook, Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY, 1982). By doing so, a library containing 2x10 5 pfu (plaque forming units) was constructed.

Half of the library was screened on duplicate filters, one filter with 3 2 P-labelled probe pl, and the counterpart filter with a mixture of probes p3 and p6.

A total of 36 positive clones were obtained, seven of which were positive in the duplicate filters, thus indicating cDNA inserts corresponding to a large portion of the proUK coding sequence.

Recombinant phages that hybridized with the 3 probes were plaques purified using probe pl, and further characterized by restriction mapping with EcoRI and by DNA sequencing. The fraction of the positive clones in the total cDNA library indicated that the frequency of prourokinase mRNA in the HEp-3 carcinoma is approximately 0.01%.

Sequence analysis of four proUK cDNA clones revealed that three of the clones had deletions or sequences not consistent with the amino acid sequence of the enzyme.

Only one clone, A Ucl7, had a sequence with complete concordance with the known amino acid sequence.

However,AUcl7 did not include the entire 3' non-coding end of the mRNA and was missing 30 nucleotides of the coding sequence. A full lenght pre-prourokinase cDNA clone was constructed by ligation of a 1325 bp AUcl7 SmaI-BamHI fragment, containing the 5' non-coding S° region and the majority of the coding sequence, with a BamHI-EcoRI fragment containing the remaining missing 2' 3' region from another cloneAUc6 (Fig. 2).

This construct was ligated into the Smal-EcoRI site of the plasmid vector pUN121 (Nilsson Uhlen M., Josephson Gatenbeck S. and Philipson Nucleic Acid Research 11, p. 8019, 1983), eliminating, thus, most of the cI gene, and given the name pcUK176 (Fig.

3).

j The DNA sequence of the complete cDNA clone is depicted in Fig. 4. It consists of 2296 nucleotides, including 69 non-coding nucleotides at the 5' end, 1296 coding nucleotides, and 931 non-coding nucleotides at the 3' end, followed by a poly(A) tail of more than residues.

The coding sequence starts with 60 bp coding for amino acids comprising the "pre-prourokinase" ;i i 11 (Heyneker, Holmes, W.E. and Vehar, G.A. (1983).

SEuropean Patent Application Publ. No. 0092182), and is followed by the sequence coding for the entire prourokinase wterl, which is in complete concordance with the amino acid sequence.

The complet'. fragment has been checked by sequence and restriction analysis. The sequence coding for mature prourokinase has been inserted into the expression vector used for production.

2) Construction of the proUK expression plasmid The original full lenght cDNA, present in pcUK176, was used to construct a prourokinase expression plasmid, named pFC44, in which the proUK gene is under the transcrptional and the translational control of the promoter Ptrp and of the "Shine-Dalgarno" sequence S' MS-2, respectively. The plasmid pFC44 is shown in fig.

7 In order to obtain pFC44, several intermediate plasmids were constructed. Starting with pDS20 (Fig. Helfard, R.M. and Holmes, Cell 30, p. 855, 1982), we have first replaced the EcoRI-HindIII fragment coding for the galactose operon promoter Pgal with the EcoRI-HindIII polylinker sequence from the M13 mp8 vector (Vieira, J. and Messing, I. Gene, 19, p.

259, 1982), obtaining a new plasmid, named pAB1 (Fig.

The promoter Ptrp has been obtained from the plasmid pDR720 (bought from Pharmacia) as an EcoRI-SalI restriction fragment. This fragment has been inserted in the polylinker region of pABI between the EcoRI and the SalI site. By doing so, we have obtained a new plasmid, named pFC10 (Fig. can be considred as the base vector into which we 12 have inserted the proUK gene as well a the "Shine-Dalgarno" sequence from the phage MS-2.

To achieve expression of mature prourokinase, it is necessary to fuse the proUK coding sequence, from the first codon of the mature protein to the initiator triplet ATG. This fusion must then be preceded by the "Shine-Dalgarno" sequence.

The ribosome binding site (RBS) from the bacterial phage MS-2 was known and its nucleotide sequence had already been published (Fiers, Contreras, R., Duerinck, Haegeman, Iserentant, Merregaert, Min Jou, Molemans, Raeymaekers, Van den Berghe, Volckaert, G. and Ysebaert, M. Nature, 260, .o p. 500, 1976).

It is thought to be a strong signal for an efficient 0 6 0 translation of the mRNA. Therefore, we have chosen this regicn as translation signal for the production of proUK. In order to obtain the correct nucleotide fusion I with the proUK gene, we have synthesized a double strand DNA region of the MS-2 RBS directly joined to the beginning of the proUK gene. A Taq:' site is present on the 25th nucleotide of the mature proUK sequence. We have taken advantage of this site and isolated, by chemical synthesis, the following DNA sequence: HindIII MetSer 3'-AATTATCTGCGGCCGGTAAGTTTGTACTCCTAATGGGTACTCGT S' TaqI ATGAACTTCATCAAGTTCCAT-3' which is flanked upstream by an HindIII site and down 13 stream by a TaqI site. The initiator codon ATG is shown in bold face. The sequence coding for the beginning of the mature pr"'K sequence is underlined.

The synthetic fragment has been used in a ligation reaction with the two following restriction fragments: -the TaqI-BglII fragment from pcUK176 (Fig. which carries the proUK sequence from nucleotide 155 to nucleotide 392 (see Fig. 4); -the large BamHI-HindIII fragment from pFC10 (Fig. which carries the antibiotic resistance to ampicillin as well as the promoter Ptrp.

Through this construction, we have isolated a new plasmid, named pAB8, whose schematic map is shown in fig. 6. In this plasmid, the promoter Ptrp and the MS-2 BS are fused to the first 260 nucleotides of the 1 mature proUK gene (corresponding to nucleotides 131-391 in Fig. In addition, pAB8 has a unique NcoI site into which we have inserted the rest of the proUK sequence through a NcoI-NcoI restriction fragment from pcUK176. This ligation has caused the duplication of an NcoI-BglII fragment downstream of the proUK gene in the non-coding region. However, this duplication does not affect plasmid stability. Through this construction signals can now dilct the synthesis of the complete proUK sequence (see Fig. 6).

All the plasmids, described so far, were selected in the E. coli K-12 host strain C-600 galK (ATCC 33955), on the basis of ampicillin resistance. Indeed, they carry the gene coding for B-lactamase, the enzyme responsible for the degradation of the antibiotic ampicillin in the culture medium. Early experiments have shown that pFC16 could be successfully inserted in E. coli type B strains and cause high level production of recombinant proUK.

14 However, to comply with international guidelines for the production of recombinant DNA-derived products, we have modified pFC16 to create a new tetracycline-resistant plasmid able to express the proUK gene at high levels. In particular, from the well-known plasmid pBR322 (Maniatis, Fritsch, E.F.

Sambrook, Molecular cloning. A laboratory manual.

Cold Spring Harbour Laboratory. Cold Spring Harbour, NY. 1982)(Fig. we have isolated a EcoRI-Aval fragment where the sticky ends were filled in using the klenow fragment of DNA polymerase I (Perbal,B., A Wiley-Interscience Publication John Wiley and Sons, p.

231, 1984)_ This fragment was ligated to the larger AatII-PvuI fragment from pFC16, whose ends were made blunt by DNA polymerase I (Perbal, A 0: Wiley-Interscience Publication John Wiley And Sons, p.

231, 1984). By doing so, we have replaced the amino terminal portion of the 8-lactamase 'gene and its controlling sequence with the tetracycline-resistance gene. Following ligation the tetracycline resistance gene is in the same orientation as the proUK gene.

Moreover, a new EcoRI site has been created at the junction between the PvuI and EcoRI sites, previously Sfilled in. The new plasmid, pFC44 (see fig. is the final construction that has been used for the production of recombinant prourokinase.

Plasmid pFC44 (tetracycline-resistant) and pFC16 I ,(ampicillin resistant) are one of the objects of the present invention. The expression signals, present in these two plasmids, namely the promoter Ptrp and the Shine-Dalgarno sequence have already been described in the literature for the expression of heterologous proteins (Remaut Stranssens P. and Fiers W. Nucl. Acid. Res. 11, p. 4677, 1983), However, their combination has never been applied before to the expression of the proUK gene.

iI 3) Transformation of E.coli type B strains The second main object of the present invention is the use of E. coli strains of the type B for the expression and production of prourokinase. Indeed, the authors of the present invention have found that insertion of plasmids pFC16 or pFC44 in type B strain of the bacterium E. coli brings to high level productions of the prc-TT polypeptidic chain. Interestingly, insertion of plaFmids pFC16 or pFC44 in other strains of E. coli (type K-12, type C, type W, etc) does not yield as much proUK. Consequently, the host strain type seems to be crucial for the successful production of proUK.

Several type B strains of E. coli are available and can be used for successful expression of the proUK gene.

S0 Prefered strains are: ATCC 12407, ATCC 11303, NCTC 10537. Below is an example of transformation of strain S NCTC 10537 with plasmid pFC44 and subsequent cultivation of the transormnant.

Competent cells of strain NCTC 10537 were prepared using the calcium chloride procedure of Mandel and Higa (Mandel, M. and HigF, J. Mol. Biol. 53, p. 154, 1970). Approximately 200 fl of a preparation of these cells at 1 x 109 cells per milliliter were transformed with 2.l of plasmid DNA (approximate concentration Ag/ml). Transformants were selected on plates of L-agar containing 12.5 Ag/ml tetracycline. Two small colonies were streaked with wooden tooth picks (each as three streaks about 1 cm long) onto L-agar containing the same antibiotic. After 12 hours incubation at 37 0

C,

E portions of the streaks were tested for human prourokinase production by inoculation onto 10 ml of LB medium (containing tetracyc ine at a concentration of U g/ml) and incubated overnight at 37°C. The following day the cultures were diluted 1:100 in M9 medium, containing the same concentration of tetracycline, and incubated for 6 hours at 37 0 C. Total i 'i 16 cell proteins from 250 Al aliquots of culture 5 o= 1-1.5) were analysed by sodium dodecylsulfate polyacrylamide gel electrophoresis as described by Laemmli (Laemmli, U.K. Nature, 227, p. 680, 1970). A major protein band having a molecular weight corresponding to that of non-glycosilated human prourokinase (45000 daltons) was observed for the two independent samples (Fig. 8).

The set of streaks corresponding to colony no. 2 (clone 2) was chosen arbitrarily for further characterization and then selected as a proUK producing strain.

Materials and Methcus Growth Media: The media used were prepared using recipes as described by Maniatis et al. (Maniatis, T., Fritsch, E.F. Sambrook, Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY 1982). LB Medium, LB agar and MacConkey agar were prepared using Difcobacto Products.

M9 medium is comprised of the following components: Na 2 HPO,, 6 grams/l; KH 2

PO

4 3 g/1: NaCl, 0.5 g/l; NHCl, 1 g/1. After sterilization of the above components by autoclaving (1 atm.; 120 0 C for 20 min.), 1 ml of 1M MgSO4, 0.1 ml of 1M CaCl 2 16 ml of Glucose, 20 ml of 0.5 mg/ml Thiamine (SIGMA) and 20 ml of 20% Casamino Acids (DIFCO) were added per litre. The above solution is sterilized by filtration.

Use of Restriction Endonucleases and Other Enzymes: L Restriction endonucleases, T4 DNA Ligase and DNA Polymerase I (Klenow Fragment) were obtained from New England Biolabs and from Boehringer Mannheim and were used in conditions recomended by the manufacturer.

Preparation of Plasmid DNA: Plasmid DNA was prepared byI I 17 a method involving dye-buoyant density centrifugation based on the procedure of Birnboim and Doly (Birnooim, H.C. and Doly. Nucleic Acid Res. 7, p. 1513, 1979).

DNA sequence analysis: Sequence data were obtained using the Amersham M13 sequencing kit according to the instruction of the manufacturer. Briefly, this technique, based on the Sanger method (Sanger, F., Science, 214, p. 1205, 1981) consists of subcloning various restriction fragments in convenient M13 vectors (the "mp family") which can be obtained in their single strand configuration. After annealing the single strand forms with "universal primers", it is possible to copy the DNA template with DNA polymerase I (klenow fragment). By copying the template in the presence of 9 the four dideoxynucleotides it is possible to cause random terminations of the chain elongation. The truncated fragments are then separated on denaturing polyacrylamide gels and the electrophoretic profile is evidenced by autoradiography.

Oligonucleotides: Synthetic oliognucleotides, utilized in plasmid constructions and as primers in DNA sequencing, were synthesized using the Applied Biosystem (ABI) DNA synthesizer 380B according to the ABI manual.

Other procedures: Procedures for agarose and polyacrilamide gel electrophoresis of nucleic acids \were as described by Maniatis et al. (Maniatis, T., Fritsch, E.F. Sambrook, Molecular cloning. A laboratory manual. Cold Spring Harbour Laboratory. Cold Spring Harbour, NY, 1982). Proteins were separated by polyacrylamide gel electrophoresis as described by Laemmli (Laemmli, U.K. Nature, 227, p. 680, 1970). DNA 18 was extracted from agarose gels by electrophoresis into dialysis bags and was concentrated by ethanol precipitation.

Legends to Figures Fig. 1: The nucleotide sequence of probes P1, P2, P3, P6 and P7, the complementary mRNA sequence and the proUK peptides coded by the probes are depicted.

Fig. 2: The size and location of restriction endonuclease cleavage products were estimated by electrophoresis and confirmed by DNA sequence analysis.

The filled region indicates the coding sequence of the mature proUK protein, the cross-hatched region oo represents the "pre-pro" peptide coding sequence and Oo.* the open regions indicate the 5' and the 3' onon-translated sequences. The 5' end of the mRNA is to the left. The lines below the restriction map indicate the contribution of the two partial clones AUcl7 and AUc6.

Fig. 3: The EcoRI-Smal fragment carrying the cTNA

I

clone has been inserted in pUN121 replacing most of the ScI gene. Plasmid pcUK176 is still tetracycline and anpicillin resistant. *cI represents an inactivated cI protein..

Fig. 4: The complete cDNA sequence of clone pcUK176 is depicted with the corresponding translated amino acid sequence. Restriction sites which have been used for plasmid constructions are underlined. Two polyadenilation sites at position 2264 and 2277 and the Serine residue at position 1 of the mature proUK ii t 1 19 sequence are a:so underlined.

Fig. 5: In this figure four intermediate constructions are depicted. The starting plasmid, carries the general background which, through the different intermediate plasmids, looses the promoter Pgal, the galK gene and the B-lactamase gene.

Fig. 6: Three additional intermediate constructions including plasmid pFC16 which expresses high level of proUK. For details, see the text.

Fig. 7: In pFC44 the mature proUK coding sequence is 1 under control of the promoter Ptrp and of the "ribosome binding site" from the phage MS-2. The tetracycline gene has been inserted at the place of the B-lactamase gene. pFC44 is therefore ampicillin sensitive.

Fig. 8: The samples were prepared and analysed as described in the' text and loaded on a 12.5% SDS-polyacrylamide gel (acryl to bisacrylamide ratio 40:1). Lanes 1 and 2 contain material derived from two cultures of strain NCTC 10537 transformed'with pFC44.

The position of the recombinant prourokinase protein is indicated by the arrow. Lane 3 shows material derived from the control host strain NCTC 10537. A molecular weight standard is shown in lane 4.

Discussion and conclusions The present invention relates to a recombinant DNA method for the production cf non-glycosilated prourokinase. This method is based on the insertion of the human gene coding for proUK in bacterial strains of E. coli and the subsequent cultivation of said

L

1 transformed strains.

The production of heterologous proteins in E. coli is a well studied field of modern biotechnology (Harris T.J.R. and Emtage J.S. Microbiological Sciences, 3, p.

28-31, 1986). Today the molecular biologists dispose of several expression signals such as promoters, Shine-Dalgarno sequences, terminators, etc. that can be used for the protein of choice. The promoter is responsible for the synthesis of messenger RNA while the Shine-Dalgarno sequence should guarantee an efficient translation of the mRNA in polypeptidic chain.

The combination, however, of these parameters is an important feature in the heterologous gene expression.

r For example, fusion of an efficient Shine-Dalgarno sequence to different promoter regions can lead to different expression levels. In addition, the lenght of the restriction fragments carrying the expression signals often affects the levels of production (McCarthy Sebald Gross G. and Lammers R.; Gene, 41, p. 201, 1986).

The choice of the host strain is also a critical step in the development of an efficient method of production. It is, in fact, known that insertion of the same expression plasmid in different strains can lead to ery different expression efficiencies (Harris T.J.R. and Emtage J.S. Microbiological Sciences, 3, p.

28-31, 1986).

While the expression signals described in the present invention were already known in the scientific literature, their combination had never been exploited before for the specific expression of human prourokinase. More particularly, plasmids pFC16 and pFC44 carry the proUK gene under control of the E. coli -I J i 21 promoter Ptrp and the phage Shine-Dalgarno sequence MS-2.

Consequently, the production method, disclosed within the text of the present invention, is based on expression plasmids essentially different from other expression plasmids previously described. These plasmids, pFC16 and pFC44, represent therefore one of the novelty aspects of the present invention and, as already said, are an object thereof.

In addition, the method here disclosed takes advantage of E. coli strains of the type B. The vast majority of the expression methods, described in the scientific literature, i- based on strains of E. coli of the type S' K-12. Thus, the production of proUK in E. coli strains °o of the type B represents another novelty aspect of the present invention.

This second aspect is extremely important. The choice S' of the host organism can, in fact, affect the global production process at several steps.

For instance, fermentations at high biomass may dramatically be influenced by the type of host. The present inventors as well as other groups of S' researchers have consistently found that E. coli S' strains of the type B can be grown more easily than, K-12 strains. Insertion of the same expression plasmids, pFC16 or pFC44, in K-12 strains such as C600 generates recombinant strains, which cannot grow, in fermentators, as efficiently as the recombinant B strains. In other words, yields of recombinant non-glycosilated pro-UK are higher from B strains, when using the same expression plasmids.

Another important feature related to the choice of the host strain is the different nature of the bacterial contaminants during the pro-UK production process.

C 4 Interestingly, in 1986, Winkler and Blaber (Winkler, Blaber, Biochemistry, 25, n. 14, p.

4041, 1986) have described a pro-UK production process based on the K-12 strain 294 (ATCC 31446).

In this process, the authors had to take several precautions to avoid proteolytic digestion of pro-UK.

According to the authors these proteolytic activity was due to bacterial proteases from the host strain.

In contrast the use of B strains according to the present invention yields cell extracts with much lower proteolytic activity. In particular, it has been found that pro-UK extracted from the K-12 strain C600 is contaminated by urokinase to a higher extent compared Sto pro-UK from B strains.

In conclusion, the authors of the present invention, believe that the higher yields of recombinant pro-UK observed with the here described procedure compared with the prior art, represent an unpredictable result and an improvement over the known procedures.

Sp j c

Claims (5)

1. A method for the preparation of non-glycosilated pro-UK, characterized in that non-glycosilated pro-UK is expressed under the control of the E. coli promoter Ptrp and the Shine-Dalgarno sequence MS-2 by E. coli B.

2. A method according to claim 1 wherein the non-glycosilated single chain prourokinase has a molecular weight of about 45000 daltons. 0 3. The method according to claim 1, characterized in o that the E. coli B expresses primarily the sequence of pro-UK.

4. The method according to claim 1, characterized in that the cDNA sequence for pro-UK is obtained from mRNA of HEp-3 epidermoid carcinoma cells. The method according to claim 1, characterized in that the promoter Ptrp constitutes of an EcoRI-SalI restriction fragment obtained from the plasmid pDR-720. The method according to claim 1, characterized in Sthat the sequence comprising the Shine-Dalgarno sequence MS-2, the ATG start codon and the beginning of the pro-UK gene, flanked upstream by a HindIII site and downstream by a TaqI site is as follows: t 7, .A fJO 24 HindIlI '-AGCTTAATAGACGCCGGCCATTCAAACATGAGGATTACCCATGAGC 3' -AATTATCTGCGGCCGGTAAGTITGTACTCCTAATGGGTACTCG TagI AATGAACTTCATCAAGTTCCAT- 3' TTACTTGAAGTAGTTCAAGGTAGC-

7. Expression plasmid FC-16 according to Fig. 6.

8. Expression plasmid FC-44 according to Fig. 7. DATED this 26th day of OCTOBER 1989. FARMITALIA CARLO ERBA s.r.l. By its Patent Attorneys: GRIFFITH HACK CO. Fellows Institute of Patent Attrorneys of Australia. 4 4-' .4 t 04 4 8 4 44 V V V N 4 C S iIG. 1 PROBE WN NA 3' GTG ACC ACG 5' CAC T-!G UGC u U TTG ACG G AAC UGC CC U U Asn Cys Pro 34 3' GTT CTT ACG TAC CA C 5' CAA GAA UGU AUG GU G G C Gin Glu Cys Met Val, 248 Pis Trp Cys I A Chv!ii PRO-UROKINASi B Chain 411 I- rflr% F 407 403 Gly AnGiu Glu Lys CGG UAA GAG AAG CAA A C A CCC ATT CTC TTC CTT 287 282 Pro Asp Asn Tyr Met 3' CC UAG UAA UAU 1GUTA C C c 5' CC GTC CTT ATA CAT 236 231 Val Glu Phe Lys Met Glu UG AAG UUU AAA GUA AAG G C G G AC TTC AAA TTT CAT TTC C G C C P6 rnRNA PROBE FIG. 2 ~L Ii~ "4 U4 E 4 44 Lambda Ucl7 Lambda IUc6 EZ~ZInon coding sequence E= sequence coding for the signal peptide mature proUK coding sequence FIG. 3 Sma I Sma I Eco R! \cDNA Eco RI pcUKl76 2 FIG. 4 ISmal fffQCTCCGGCTGCGGTCTCCTGCCGCAGCACCGAGCGCCGTCTAGCGCCCGACCTC 2.00 GCCAkCC ATG AGA GCC CTG CTG GCG CGC CTG CTT CTC TGC GTC CTG GTC 4 4 4 #4 4 C C tee. 'COO Met Arg Ala GTG A GC GAC TCC AAA Val Ser Asp Ser Lys TGT GAG TGT CTA AAT Cys As) Cys Leu Asn AAC ATT CAC TGG TGC Asn Ile His Trp Cys GAA ATA GAT AAG TCA Giu Ile Asp Lys Ser CGA GGA AAG GCC AGO Arg Gly Lys Ala Ser AAC TCT GOC ACT GTC Asn Ser Ala Thr Val. 400 GCT CTT GAG CTG GGC Ala Leu Gin Leu Gly 450 AAC CGG AGG OGA CCC Asn Arg Arg Arg Pro 500 GTC CAA GAG TGC ATG Val Gin Giu Cys Yel. 550 TCT COT CCA. GAA GAA Ser Pro Pro Giu Giu 600 CCC CGC TTT AAG ATT Pro Arg Phe Lys Ile 650 CCC TGG TTT GCG GCC Pro Trp The Ala Ala Gly GGA Gly AAC Asn AAA Lys ACT Thr CTT Leu CTG Leu TGG Trp GTG Val1 TTA Leu ATT Ile 3eL GGA Gly TGC Cys ACC Thr GAG Asp GAG Gin GGG Giy TGO Cys CAT Hi-Ls AAA Lys GGG Giy A-sn Giu Leii His Gin Val ACA TGT GTG TCG Thr Cys Val Ser GCA AAG AAA TTG Pro Lys Lys Phe TGO TAT GAG GG Cys Tyr Glu Giy NcoI ACCATG-(GC CGG Thr Met Gly Arg CPA AGG TAG CAT Gin Thr Tyr His AAA CAT AAT TAG Lys His Asn Tyr TAT GTG GAG GTG Tyr Vai Gin Val GAG TGG GCA. GAT Asp Cys Ala Asp TTT GAG TGT GGG Phe Gin Cys Gly GGA GAA TTO AGO Gly Giu Phe Thr AAC Asn GGA Gly AAT Asn CCC Pro GCC Al a TGC Gys GGG Gly GGA Gly OAA. Gin ACC Thr AAG Lys GGG Giy GGT Gly- TGC Cys CAG His AGG Arg OTA Leu MA Lys AAG Lys ATG Ile Pro Ser Asn 200 TAO TTC TGG Tyr Phe Ser 250 CAG GAO TGT Gin His Cys 300 GAO TTT TAG His Phe Tyr. 350 CTG CCC TGG Leu Pro Trp BgiII AGA TCT CAT Arg Ser Asp AAG OGA GAG Asn Pro Asp AAG CCG CTT Lys Pro Leu AAG CCC C. Lys Pro Ser ACT CTG AGG Thr Leu Arg GAG AAC GAG Giu A-sn Gin Leu Leu Ala Arg Leu Leu Leu Gys, Val .Leu Val 150 TaqI GGC AGO AAT GAA OTT CAT CAA GTT OGA ZIC--AC ATO TAG AGG AGG CAC CGG Ile Tyr Arg Arg His Arg GGG GGG TOT GTC ACC Gly Gly Ser Val Thr I FIG. 4 cont'd 700 TAG GTG TGT GGA GGG AGC CTC ATC AGC CCT TGC TGG GTG ATO AGG GCC Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Gys Trp VaJ. Ile Ser Ala ACA GAG Thr His GTG GGT Leu Gly GAG GTG ~Glu Val G ZT GAG ~Ala His 0t AGG TGT to&Axg Cys ATG TAT Met Tyr GGA AAA Gly Lys AGT GTT Thr Val TAG- GGC Tyr Gly TGG A-AA Trp Lys TGC CTC Ser Leu GGA TGT Gly Gys TGG Gys GG Arg GAA Glu CAC His GCG Ala AAC Asn GAG G iu GTG Val TCT Ser ACA Thr G.AA Gin CC Ala 750 TTC ATT GAT TAG CGA AAG AAG GAG GAG The Ile. Asp Tyr Pro Lys Lys Giu Asp 8 00 TCA AGG CTT AAC TCG AAG ACG CAA GGG Ser Arg Leu Asn Ser Asn Thr Gin Cly 850 AAC GTG ATC GTA GAG AAG GAG TAG AGG Asn Leu Ile Leu His Lys Asp Tyr Ser 900 AAC GAG ATT GGG TTG GTG AAG ATC GGT Asn Asp Ilie Ala Leu Leu Lys Ile Arg 950 GAG GGA TCC CGG ACT ATA GAG ACC ATC Gin Pro Ser Arg Thr Ile Gin Thr Ile 1000 GAT CCG GAG TTT GGC ACA AGG TGT GAG Asp Pro Gin Phe Gly Thr Ser Cys Giu 1050 PAT TCT ACC GAG TAT CTG TAT CCG GAG Asn Ser Thr Asp Tyr; Leu Tyr Pro Ciu 1100 AAG CTG ATT TCG GAC CGG GAG TGT GAG Lys Leu Ile Ser His Arg Giu Cys Gin 1150 GAA GTC ACC ACC AAA ATG CTA TGT GOT Giu Val Thr Thr Lys Met Leu Cys Ala i200 GAT TCG TGG CAd GCA GAG TGA GGG GGA Asp Ser Cys Gin Gly Asp Ser Giy diy: 125( GGG GGC ATG ACT TTG ACT GGA ATT GTG. Gly Arg Met Thr Leu Thr Giy Ile Vai CTG AAG GAGC AAG CCA GGC GC TAG AG Leu Lys Asp Lys Pro diy Vai Tyr Thr BamH I TAG Tyr GAG Giu GGT Al a TCG Ser TGC Gys ATG Ile GAG Gin GAG Gin GC T Ala. CCC Pro ATC Ile ATG Met GAG Asp AAG Lys CTG Leu ACT Thr CG Leu CCC Pro GAG Asp CTC Leu GTC 'Val AAG Ly s ACG Thr GAG diu CCC Pro GGG Gly AAA Lys CAG His CCA Pro GTC Val GGG Gly TCA Ser *TAd Tyr TTT Phe OTT Leu GGG Gly TOG Ser TTT Phe ATG Met TAG Tyr CAA Gin TGT Cys CdT Arg CAC His 0 AGG TGG Ser Trp 1300 AGA GTG krg Val 1350 TTG TTA CCC TC-Gr7 T( CGC AGT GAG AGO AAC GAA GAG AAT GGG CTG GOG Phe Leu Pro TrD Ile A-rg Ser His Thr Lys Giu Giu Asn Cly Leu Ala FIG. 4 cont'd 1400 CTC MG GGGTCCCCAGGGAGGAAACGGGCACCACCCGCTTTCTTGCTGGTTGTCATTTTT Leu end 1450 GCAG~fAGAGTCATCTCCATCAGCTGTAAGAAG--,CACTGC-GAAGATAGGCTCTGCACAGATGGA 1500 TTTGCCTGTGCCACCCACCAGGGTGAACGACAATAGCTTTACCCaCAGGCATAC GCCTGGGTG 1550 1600 CTGGCTGCCCAGACCCCTCTGGCCAGGATGGAGGGGTGGTCCTGACTCAACATGTTACTGACC 1650 AGCAACTTGTC rTTTTCTGGACTGAAGCCTGC-AGGAGTTAIAAAAGGGcAGGGCATCTCCTGTG 1700 NcoI I CATGGGTCAAGGGAGAGCCaGCTCCCCCGACGGTGGGCATTTGTGAGGC TTAGAAA 1750 TGAAT.AATTTCCCAATTAGAAGTGTA-ACAGCTGAGGTCTCTTGAGGGAGCTTAGCCAATGTG 1800 1850 GC-AGCAGCGGTTTGGGGGAGCAGAGA-CACTAACGACTTCAGGGCAGGGCTCTGATATTCCATG 1900 AATGTATCAGGAAATATATATGTGTGTGTATGTTTGCACACTTGTGTGTGGGCTGTGAGTGTA 1950 AGTGTGAGTAAGAGCTGGTGTCTGATTTTAAGTCTAATATTTCCTTAACTGTGTGGACTG 2000 2050 TGATGCCACACAGAGTGGTCTTTCTG.GAGAzGGTTATAGGTCACTCCTGGGGCCTCTTGGGTCC Ii (42100 CCCACGTC-ACAGTGCCTGCGAATGTATTATTCTGCAGCATGACCTGTC-ACCAGCACTGTCTCA 2150 GTTTCACTTTCACATAGATGTCCCTTTCTTGGCCAGTTATCCCTTCCTTTTAGCCTAGTTCATt 2200 CCAATCCTCACTGGGTGGGGTGAGGACCACTCCTTACACTCATATTTATATTTCACTATTTT 2250 EcoRI TATTTATATTTTTGTAATTTTAAA~kzAGTGATC-AkATGTGATTTTTCTG MIT. FIG. EcoRI ga EcoR! 1-p R R PP PDS20 Amp pAB I ~j ;.:~galK galK :*Pvu gene Pvulgene BamHI BamHI PP R R *Ap pDR72O m F1 galK gene BarnHl iii 8/fo r 1 3 .0 FIG. 6 Ptr Hind I TaqI .NcoI R Amp t 4 1 44 f 4 0 (B gill/B a nHI) H in dIll Taql NcoI B giIl Amp proUK P vU I' 4. 4 EcoRI (B g iI I/a2m HI) Tet Amp P v. u I Aval t 2. (7/1jo FIG. 7 C I 6~ I-. t I t 44 ~r ~I 0 I I. I CIII EcoRI HindIll TaqI .Ncol -B gill Tet proULK EcoRI/(Pyul) (BglII/BamHl) 4 .I t t I o/o FIG. 8 4 41 I II 0 41, II 44' 411' 0 44 C 414*41 1234 'A 4 4.41. Ii ii ii 444 4 4 1~ I 4 44 41 4 4 1 44 4441 41 41 44414 4144444 4 41