WO2011144953A1

WO2011144953A1 - Procedure for producing recombinant human tissue factor

Info

Publication number: WO2011144953A1
Application number: PCT/HU2010/000055
Authority: WO
Inventors: Zsuzsanna Takatsy; Beata Nagy
Original assignee: Diagon Kft.
Priority date: 2010-05-18
Filing date: 2010-05-18
Publication date: 2011-11-24

Abstract

The invention relates to a procedure for producing recombinant human tissue factor TF in prokaryotic host cells, favourably in E. coli. The procedure is based on that pBAD promoter is used as a system for gene expression to enter the DNA sequence coding for human tissue factor into prokaryotic host cells. Bacteria are propagated under fermentation circumstances, and optimum protein production is ensured by using monosaccharide as inducing agent. Following yield via cell disruption a recombinant human tissue factor TF is created.

Description

PROCEDURE FOR PRODUCING

RECOMBINANT HUMAN TISSUE FACTOR

The invention relates to a procedure for producing recombinant human tissue factor, in the course of which using a prokaryotic expression system, under favourably reproducible and controllable fermentation circumstances, functionally stable human recombinant tissue factor product is created.

When the vascular bed is injured, the living organism attempts to prevent blood loss by coagulation, the rapid formation of a clot in response to the injury. In the chain reaction of coagulation (blood coagulation cascade) there are serine protease enzymes -coagulation factors- activated in sequence together with additional, so-called co-factors. Tissue factor TF (coagulation factor III, thromboplastin) is involved in the initiation of the physiologically more significant, so-called extrinsic (external) pathway of coagulation [Mackman N. et al.(2007): Arterioscler.Thromb.Vasc.Biol. 27: 1687-1693.]. Tissue factor TF is a member of the cytokine receptor superfamily [Bazan J.F.(1990): PNAS 87, 6934- 6938.], an integral transmembrane glycoprotein overlapping the cell membrane and providing high affinity receptor and cofactor for plasma coagulation factor VH/VIIa. Tissue factor TF participates in the process of thrombosis, atherosclerosis, intracellular cell signalling and blood vessel development [Carmeliet P. et al. (1996): Nature 383: 73-75.]. Although the detectable level of tissue factor TF in the circulation of healthy individuals has been disputed [Butenas S., Mann K.G.(2004): Nat.Med. 10: 1155-1156., Monroe D.M., Key N.S.(2007): J. Thromb. Haemost. 5: 1097-1105.], when the vascular bed is injured, tissue factor TF is released from the cells along the injury (such as adventitial fibroblasts, medial smooth muscle cells, endothelial cells, white blood cells), which initiates the blood coagulation cascade.

Below the extrinsic pathway of blood coagulation is described on the basis of figure 1. The released tissue factor TF binds and activates coagulation factor VII, which converts to activated coagulation factor Vila. In this way tissue factor TF is the cofactor and receptor of activated coagulation factor Vila. Tissue factor TF and activated coagulation factor Vila, as a receptor-ligand complex, facilitates the activation of coagulation factor X in the presence of Ca ions and membrane phospholipids, activated coagulation factor Xa is formed. The transformation of prothrombin to thrombin is triggered by the prothrombinase complex, which is created by activated coagulation factor Xa and activated coagulation factor Va, the latter as a regulating protein. The created thrombin activates fibrin stabilising factor XIII, activated fibrin stabilising factor XHIa is formed, which is a ligase enzyme within the enzyme family of transglutaminases. The stable fibrin network, the fibrin polymer is created from fibrin monomers by covalent cross-linking under the influence of activated fibrin stabilising factor Xllla [Edgington T.S. et al. (1991): Thrombosis and Haemostasis, 66: 67- 79., Triplett D.A. (2000): Clinical Chemistry 46: 1260-1269.].

The first purified tissue factor TF was produced from bovine brain in 1980 [Bach R. et al. (1981): J. Biol. Chem. 256: 8324-8331.]. The sequence of purified human tissue factor TF was analysed in 1985 [Broze G.J. et al. (1985): J. Biol. Chem. 260: 10917-10920.]. Three domains are distinguished in the structure of tissue factor TF: the extracellular, the transmembrane and the intracellular domains.

Integral membrane proteins like tissue factor TF are characterised by polypeptide chains containing domains rich in hydrophobic amino acids, which occur in the chain alternately with domains consisting of hydrophilic amino acids. The hydrophobic domain of tissue factor TF consisting of 25-30 amino acids is embedded in the double layer of membrane lipids. The purified tissue factor TF polypeptide chain consists of 295 amino acids, having the N-terminal extracellular and receptor domain of 219 amino acids. The transmembrane domain is 23 amino acids long, and the cytoplasmic domain consisting of 21 amino acids leads to the C-terminal end of the molecule. As it was described above, the extracellular part of the molecule binds activated coagulation factor Vila, and the intracellular cytoplasmic domain provides the signal transmitting function of tissue factor TF [Jiirgen Jaenecke, Antikoagulanzien-und Fibrinolysetherapie, Thieme 5. Auflage (1996)].

The term thromboplastin reagent in our description is an assembly of phospholipids and tissue factor TF (see above), a component initiating the extrinsic pathway of coagulation, consequently an essential element of in vitro blood coagulation diagnostic tests.

One of them, the Prothrombin time (PT) test reports the time in seconds until fibrin polymer formes, the blood clot in the endpoint method. The Prothrombin time test, with great significance in everyday medical practice, provides important information in determining the blood coagulation status of the examined person, in monitoring patients receiving anticoagulant therapy. The first PT test reported [Quick A.J.(1935): J.Biol.Chem.109: 73-74.] was based on thromboplastin reagent derived from rabbit brain to prove bleeding abnormalities in patients with obstructive jaundice. Later on, other animal and human tissue (e.g. bovine lung, heart, or human placenta, etc) were extracted to prepare thromboplastin reagents rich in tissue factor TF for the PT test.

Regarding tissue factor TF procoagulant properties and the use of thromboplastin reagent to reflect alterations in clotting factor activities of the extrinsic pathway of the blood coagulation cascade, diagnostic reagents of animal origin have the disadvantage of great fluctuation in the functionality of individual batches produced. This fluctuation may be due, among others, to differences in species, age, nutrition and sex of the organism. According to our experience, fluctuation in the functionality of individual batches can be followed by the variations in the sensitivity of individual reagent Lots used in laboratory PT tests. Seasonal fluctuations in the individual reagent Lots have also been observed, and different - unrecognised - diseases of the animal stock may result in differences in the reproducibility of the diagnostic reagent [Paborsky, L.R. el al.(1989): Biochemistry 28: 8072-8077.].

An attempt to improve Lot-to-Lot consistency in the sensitivity of thromboplastin reagents, a WHO primary standard reference preparation was given (WHO/LAB/98.3) to introduce the International Sensitivity Index (ISI). This internationally accepted primary standard reference preparation having ISI value of 1.0 serves as a blueprint reagent for calibrating local reference (in-house reference) reagent preparations at manufacturers. The local reference preparations work by comparing each batch of reagent produced to them [WHO/LAB/98.3]. The other format introduced to help standardization of highly variable thromboplastin reagents was the International Normalized Ratio (INR). The INR is a mathematical calculation incorporating ISI as an exponent in the formula of prothrombin time ratio [Poller L.J.(2004): 2: 849-860.]. The Prothrombin time ratio is the patient prothrombin time in seconds divided by the mean normal prothrombin time (MNPT) in seconds, the latter calculated at local laboratories. The purpose of introducing INR was to create the uniform monitoring of patients receiving anticoagulant therapy [Jackson CM. et al.(2003): Pathophysiol.Haemost.Thromb. 33: 43-51.], even if INR has also been reported in the interpretation of other disorders with secondary coagulation deficiencies (Bellest L. et al.(2007): Hepatology 46: 528-534.]. The incorporation of ISI into the format of INR emphasizes the importance of Lot-to-Lot consistency of thromboplastin reagents issued. Regarding the inverse relationship between ISI and the sensitivity of thromboplastin reagent, an ISI value below 1,2 is favourably accepted in medical practice. To keep ISI values in the required range, thromboplastin reagents should be of standard quality without fluctuation in their functionalities.

The experience mentioned above together with the international attempts for standardization of thromboplastin reagents resulted in the demand for producing recombinant tissue factor TF, which eliminates sources of animal origin and ensures the functional stability of the reagent, thus enabling PT test to report reliable results.

The known procedures for producing recombinant tissue factor TF employ recombinant DNA technology based on the principle of DNA cloning [Kopper L. et al.: Molekularis medicina, Medicina Konyvkiadol997; William Wu et al.: Methods in Gene Biotechnology, CRC Press LLC 1997]. During DNA cloning, with the help of a vector (expression vector, clone, recombinant plasmid) the selected genetic information is transferred artificially from a living organism (animal, plant, microorganism) into another, where under controlled circumstances it is multiplied and its expression (protein synthesis) is induced. The method aimed at the production of recombinant tissue factor TF consists of the following steps:

1. isolating the DNA containing the target gene to be cloned

2. combining of the vector and the target gene to be cloned (recombinant plasmid created through cleavage and ligation, with the insertion sequence inside it)

3. entering the recombinant plasmid into the host cell (transformation)

4. propagating the transformed (host) cells

5. activating of target gene: promoter induction followed by gene expression (heterologous protein production)

Eukaryotic Pichia pastoris, Saccharomyces cerevisiae host cell system, and insect and mammalian cells are also used for producing recombinant tissue factor TF.

Cheryl Brucato et al.(Instrumentation Laboratory S.p.A, Italy) produced rabbit recombinant tissue factor TF in Pichia pastoris yeast host cells [Brucato C. et al.(2002): Protein Expression and Purification 26: 386-393.]. Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2, which have a strong promoter effect. Methanol is the inducing agent of protein production, which is continuously added during fermentation. The alcohol oxidase enzyme of Pichia pastoris transforms the absorbed methanol into formaldehyde and hydrogen peroxide. Heterologous protein secretion takes place in the penplasmic space of the host cell, which may make subsequent purification of the target protein significantly easier. The disadvantage of the system is that the period of cell propagation and induction is long, 96 hours, as a result of which the entire fermentation process becomes difficult. Furthermore, according to our experience in practice, the yield of recombinant tissue factor TF produced using the Pichia pastoris system is much lower in quantity.

A possible further solution is the use of Baculovirus vector in insect and mammalian cells (American Diagnostica). Its disadvantage is that its use on industrial scale is rather costly.

Escherichia coli (E. coli) is a prokaryotic system studied for decades, in gene expression procedure it has proved to be the most suitable solution. As compared to eukaryotic cells it has several great advantages, one of which is its very short lifecycle; its generation time is about 20 minutes. Its further advantages as compared to the other system include much shorter induction time and efficient protein production. The system is easy to handle, and the media demand of cell propagation in the volume-increased culture can also be satisfied cheaply [Baneyx F. et al.(1991): Appl. Microbiol. Biotechnol. 36: 14-20.]. However, it is important to consider that specific protease activity of E. coli may influence the stability of heterologous protein expressed by the target gene. For this reason it is necessary to use protease inhibitor during host cell disruption aimed at yielding the produced protein [Maurizi M.R. et al.(1992): Experientia 48: 178-201.].

According to the experience of others, the disadvantage of the E. coli system is that the produced protein - depending on its nature - may become trapped in inclusion bodies in the cytoplasm of the bacteria [Meyenburg, K. et al. (1984): EMBO Journal, 3: 1791-1797.]. When checking the possibility during our procedure, we did not find bacterial inclusion bodies according to our electronic microscopic tests.

Due to the above advantages E. coli is a widely used host cell system for producing recombinant proteins including recombinant tissue factor TF. The production of recombinant tissue factor in E. coli is described in several patent descriptions relating to blood coagulation diagnostic tests (mostly PT). US Patent no. 5625036 describe a reagent suitable for use in measuring prothrombin time, which contains full length or truncated recombinant human tissue factor TF and natural or synthetic phospholipids. Recombinant human tissue factor TF is expressed in E. coli host cells. US Patent no. 5858724 relates to a truncated recombinant tissue factor TF of rabbit origin, which is produced in bacterial host cells. The solubility of the product is increased by deleting the transmembrane domain. Purification of the protein is facilitated by that it is expressed as a fusion protein with thioredoxin in thioredoxin deficient E. coli host cells. The protein is lipidated and used as a reagent suitable for measuring blood coagulation.

There are several vectors for entering the target gene into bacteria. We ourselves performed experiments using the pMAL™ expression system, which enables purification of the produced target protein using maltose affinity chromatography, by fusing sequences coding for maltose-binding protein domain to the N-terminal domain of the tissue factor (pMAL™ Protein Fusion and Purification System, Instruction Manual, New England BioLabs Inc. Version 5.3.). The maltose-binding domain may also facilitate keeping the fusion protein in solution and maintaining its stability. In our practice the fusion protein with its large molecule weight (about 70 kDa) obtained by this method, made the biochemical identification of the product difficult.

The aim of the invention was to elaborate a procedure for producing recombinant human tissue factor TF, with which the unfavourable features of the known solutions can be eliminated.

The idea of the procedure according to the invention is based on our observation that the solubility of tissue factor TF is satisfactory even without the maltose-binding domain, and the subsequent and expensive purification of the product by affinity chromatography is unnecessary. We recognised that it is cheaper and simpler to use pBAD promoter instead of pMAL™ expression system. A further advantage of the former system is that the phenomenon described in the case of pMAL™ can be avoided, that is the trapping of recombinant fusion protein often toxic to the host cell in hard-to-handle bacterial inclusion bodies.

In our present procedure the amount of the induced, produced protein is modulated by changing the monosaccharide, favourably the arabinose and glucose concentration. A further great advantage of our system is that practically there is no mutation in the target gene, the phenotype is stable. We recognised that in order to produce a reproducible and functionally stable product, in the case according to the invention, there is no need for complicated purification procedures. The subject of the invention is a procedure for producing recombinant human tissue factor TF in prokaryotic host cells, favourably in E. coli. The procedure is based on that pBAD promoter is used as a system for gene expression to enter the DNA sequence coding for human tissue factor TF into the prokaryotic host cells. Bacteria are propagated under fermentation circumstances, and optimum protein production is ensured by using a monosaccharide as inducing agent. Following yield via cell disruption a recombinant human tissue factor TF is created.

In the case of a favourable realisation of the procedure according to the invention plasmid containing pBAD promoter is used as a gene expression system.

In the case of a possible solution the medium of the fermentation process is made of a blend of tryptone favourably 2.0 %, yeast extract favourably 0.5 %, sodium-chloride favourably 10 mM, in appropriate proportions. As a carbon source favourably glucose, maltose, glycerine is used, at a concentration of 0.1-1.0 %.

During the realisation of the procedure according to the invention protein production is induced favourably by adding 0.1-0.4 % arabinose or 0.1-0.4 % arabinose and 0.1-1.0 M isopropyl-P-D-l-thiogalactopyranoside (IPTG) as a monosaccharide.

It is very favourable to yield protein during cell disruption by treating the host cells with buffer containing non-ionic detergent, favourably 0.1-1.0 % Triton-X-100 or 0.1-1.0 % n-octyl glucopyranoside in 50 mM Tris buffer. In the case of a further possible solution the recombinant product yielded using detergent-based cell disruption can be stored in liquid state.

The recombinant human tissue factor TF prepared in this way is suitable for further in vitro application in blood coagulation diagnostic reagent.

Short description of the f igures attached. Figure 1 Simplified diagram of the extrinsic pathway of blood coagulation (explained above in detail).

Figure 2 Correlation between cell density of bacteria and their absorbance measured at a wavelength of 600 nm. In figure 2 the correlation between the number of bacterial cells - axis "y" - and the absorbance measured at 600 nm - axis "x" - can be seen. During our procedure we monitor the increase in cell number by measuring absorbance continuously, in order to make sure that induction is performed at the optimal time, at the end of the log phase of bacterial multiplication, before reaching the stationary phase. The cell culture is induced at an absorbance value of 0.55-0.60 OD.

Figure 3 MNPT and ISI values (explained above in detail) of PT tests obtained by two individual Lots of thromboplastin reagent having our recombinant human tissue factor TF product in them. STA Compact represents the mechanical, while Sysmex CA represents the optical type of coagulometers. Compared to our local reference (in-house reference) preparation having been adjusted to the WHO primary standard reference preparation (explained above in detail), the sensitivity of individual Lots has shown Lot-to-Lot consistency. The ISI values are below the limit of 1 ,2 acceptable in medical practice and are close to that of the international standard reference preparation.

The procedure according to the invention is described below in detail.

During the procedure E. coli cells are transformed with pBAD vector containing DNA sequence (insert) coding for human tissue factor TF. The bacterial suspension is spread on a solid culture medium containing antibiotics. Due to the selective effect of the antibiotics, on the culture medium only the colonies of transformed cells will grow, after a certain period of incubation. The so-called inoculum is produced by injecting the colony selected from the grown colonies into a liquid culture medium The liquid culture medium in the shake culture or in the fermenter is infected with the inoculum appropriately multiplied in the culture medium. During fermentation the temperature, pH and oxygen saturation is monitored continuously. Bacterial multiplication is monitored by performing photometric absorbance measurement at given time intervals. The bacteria reach the optimal cell number at the end of the log phase, before the stationary phase. At this point the inducing agent is added, which initiates the protein production of the cells. After a given induction period the medium is centrifuged, then the bacteria are sedimented and the pellet is treated with lysis buffer on the basis of wet weight determination. The solution containing the product protein is obtained after repeated centrifugations. After conventional lipidation with synthetic phospholipids of the product protein, the functionality of recombinant human tissue factor TF is checked in PT test as recommended by WHO/LAB/98.3.

Examples of realisation

Example 1: 1 -litre shake culture fermentation : yielding protein from E. coli cytoplasmic space

With pBAD24 plasmid containing insert DNA expression competent E. coli K12 TBI strains are transformed. During transformation the plasmid is diluted on the basis of its DNA concentration, about 10-16 ng of plasmid DNA per 50 μΐ of bacteria.

The cell suspension produced in this way is kept on ice for 30 minutes, then it is dipped in 42°C water bath for 30 seconds, and then it is placed on ice again for 1 minute.

The temperature shock increases membrane fluidity and enables the plasmid to enter the cells.

250 μΐ of SOC medium (2.0 % tryptone, 0.5% yeast extract, 10 mM sodium chloride, 2.5 mM potassium chloride, 10 mM magnesium chloride, 10 mM magnesium sulphate, 20 mM glucose) preheated to 37 °C is measured out for the bacteria and then incubated at 37 °C for 1-2.5 hours with shaking.

50 μΐ of suspension is spread on LB agar containing ampicillin of a concentration of 0.1 mg/ml.

It is incubated at 37 °C overnight.

One of the colonies grown selectively on the agar is injected into 10 ml sterile LB Broth medium containing 0.1-1.0 % glucose and 0.1 mg/ml ampicillin.

It is incubated for 16 hours at 37 °C with shaking.

With the inoculum obtained in this way 1 1 of sterile liquid medium is injected in a proportion of 1:100. The medium is modified LB medium, which favourably contains: 10 g/1 tryptone, 5 g/1 yeast extract, 5 g/1 sodium chloride, 1.0 M magnesium sulphate, 0.1 mg/ml ampicillin, as carbon source 0.1-1.0 % glucose, mannose, maltose, galactose, glycerine, glyceric aldehyde can be used.

The shake flask is incubated at 37 °C with continuous shaking under air. A sample is taken every hour, and absorbance is measured using a photometer (600 nm wavelength). At a value of OD= 0.55-0.60 the cell culture is induced with 0.1-0.4 % arabinose, 0.1-1.0 M isopropyl-P-D-l-thiogalactopyranoside (IPTG) (Figure 2). Until the end of the log phase - under the circumstances determined herein - bacterial multiplication takes an average of 4.5-5 hours.

Induction can be performed at 37 °C and/or at room temperature for 2-4 hours. After the end of the induction period the nutrient solution is centrifuged at 10,000g for 10 minutes at 4 °C. Before centrifugation the centrifuge tubes are measured so that after removing the supernatant the wet weight of the pellet can be determined. The bacterial mass is carefully suspended with lysis buffer of an amount suiting the weight of the wet mass.

As lysis buffer BugBuster, favourably 0.1-1.0 % Tryptone XI 00 or TRIS or TBS buffer containing 0.1-1.0 % n-octyl glucopyranoside (OGP) can be used. In each case phenylmethanesulfonyl fluoride (PMSF) of a 1 :100 dilution must be added to the lysis buffer as protease inhibitor. Besides chemical cell lysis, ultrasound-mediated disruption can also be used. After carefully suspending the cell pellet, it is gently shaken at room temperature for 20 minutes, then the cell mass is centrifuged at 16,000g at 4 °C for 20 minutes. The supernatant containing the yielded protein recombinant human tissue factor TF is stored in a sterile vessel. The sensitivity of lipidated recombinant human tissue factor TF is checked in the PT test as recommended by WHO/LAB/98.3 .

Example 2: 1 -litre shake culture fermentation : yielding protein from E. coli periplasmic space

The process of transformation and fermentation is the same as the process described in detail in the previous example.

At the end of fermentation, after the induction period the medium is centrifuged at 10,000g for 10 minutes at 4 °C. The precipitation is re-suspended in an ice-cold solution containing 20 mM Tris, 2.5 mM EDTA 20 % sucrose (pH=8.0). The amount of solution in which the pellet is absorbed must be chosen so that in tenfold dilution the OD measured at 550 nm must be OD=0.550. Then it is placed on ice for 10 minutes. After centrifuging it at 15,000g for 30 minutes, the pellet is suspended in an ice-cold solution containing 20 mM Tris, 2.5 mM EDTA 20% sucrose (pH=8.0). It is kept on ice for 10 minutes, then it is centrifuged at 15,000g for 10 minutes. The yielded protein is contained in the supernatant. The sensitivity of lipidated recombinant human tissue factor TF is checked in the PT test as recommended by WHO/LAB/98.3 . Example 3 : Fermentation in a 5 -litre fermenter

Transformation and spreading is performed as described in example 1. One of the colonies grown on LB agar is injected in 50 ml of sterile LB Broth medium favourably containing 0.1-1.0 % glucose and 0.1 mg/ml ampicillin.

It is incubated for 16 hours at 37 °C with shaking.

With the inoculum obtained in this way the medium in the 5-1 sterile fermenter is injected in a proportion of 1 :100. The medium is modified LB medium: lOg/1 tryptone, 5 g/1 yeast extract, 5 g/1 sodium chloride, 1.0 M magnesium sulphate, 0.1 mg/ml ampicillin, as a carbon source 0.1-1.0 % glucose, mannose, maltose, galactose, glycerine, glyceric aldehyde can be used. 0.5 ml of foam inhibitor containing silicon oil is added to the medium.

The E. coli multiplied in the fermenter is incubated at 37°C, with continuous stirring at 250 rpm, 0₂ saturation (80-100%), pH (7.0-7.4), permanently controlling the liquid level. A sample is taken every hour, and absorbance is measured (Figure 2) using a photometer (600 nm wavelength). At a value of OD= 0.55-0.60 the cell culture is induced with 0.1-0.4 % arabinose, 0.1-1.0 M isopropyl-p-D-l-thiogalactopyranoside (IPTG).

Induction is performed at 37 °C and/or at room temperature for 2-4 hours. The protein is yielded from the fermented solution using the methods described in examples 1 and 2. The sensitivity of lipidated recombinant human tissue factor TF is checked in the PT test as recommended by WHO/LAB/98.3 .

In PT tests the sensitivity of our lipidated recombinant human tissue factor TF obtained by any of the procedures detailed in the examples was compared to our local reference reagent preparation previously adjusted to the WHO primary standard reference preparation (see above text). With each reagent Lot produced the PT tests were performed on five series of fresh plasmas, altogether minimum 80 samples (normal : patient = 20 : 60) for each reagent Lot. PT tests were run on mechanical (STA Compact) and optical (Sysmex CA) coagulometers. Results were read in secundum, the ISI value and the mean normal prothrombin time (MNPT) were calculated as recommended by WHO/LAB/98.3. The sensitivity of our recombinant human tissue factor TF reagent proved Lot-to-Lot consistency as required. This is illustrated by the MNPT and ISI values shown in Figure 3. Recombinant human tissue factor TF produced using our procedure, is very efficient and sensitive in the in vitro functional test. These features make it a suitable component for in vitro diagnostic reagent (thromboplastin reagent in blood coagulation PT tests). With our recombinant protein produced under controlled fermentation circumstances, sources of animal origin are eliminated, so the diagnostic product can be favourably reproduced in the individual reagent Lots. Our procedure makes subsequent complicated, costly and time consuming modification steps unnecessary, and the procedure is applicable for continuous and large scale production.

Claims

1. Procedure for producing recombinant human tissue factor TF in prokaryotic host cells, favourably in E. coli, characterised by that pBAD promoter is used as a system for gene expression to enter the DNA sequence coding for human tissue factor TF into the prokaryotic host cells, bacteria are propagated under fermentation circumstances, and optimum protein production is ensured by using monosaccharide as inducing agent, then following yield via cell disruption a recombinant human tissue factor TF product is created.

2. Procedure as in claim 1, characterised by that plasmid containing pBAD promoter is used as a gene expression system.

3. Procedure as in claim 1 or 2, characterised by that the fermentation medium is made of a blend of tryptone favourably 2.0 % , yeast extract favourably 0.5% , sodium- chloride favourably 10 mM.

4. Procedure as in claim 3, characterised by that as a carbon source favourably glucose, maltose, glycerine is used in the fermentation medium, at a concentration of

0.1-1.0%.

5. Procedure as in any of claims 1-4, characterised by that protein production is induced favourably by adding 0.1-0.4% arabinose or 0.1-0.4% arabinose and 0.1-1 M isopropyl-P-D- 1 -thiogalactopyranoside (IPTG) as monosaccharide.

6. Procedure as in claim 5, characterised by that in order to yield the recombinant human tissue factor product cell disruption is performed in 50 mM Tris buffer containing a non-ionic detergent, favourably 0.1-1% Triton-X-100 or n-octyl glucopyranoside.

7. Procedure as in claim 6, characterised by that following yielding by cell disruption the recombinant human tissue factor TF product is stored in liquid state.