DK175109B1 - Activating recombinant non-glycosylated tissue plasminogen activator - Google Patents

Abstract

Method for activating non-glycosylated tissue plasminogen activator (t-PA) after its expression in prokaryotic cells comprises cell lysis; solubilisation under denaturing and reducing conditions, and reactivation under oxidising conditions in presence of reduced and oxidised glutathione (G5H, G55G). The new feature is that in the last stage is at pH 9-12 (pref. 9.5-11) with G5H and G55G concns. 0.1-20, pref. 0.2-10, mM and 0.01-3, pref. 0.5-1, mM, respectively, and with a non-denaturing concn. of the denaturing agent. Esp. the method is applied to t-PA expressed in E.coli and P. putida. The denaturing agent is pref. arginine, guanidine hydrochloride (both at 0.1-1, esp. 0.25-0.75, mM) or urea, at 0.5-4 (esp. 1-3.5) M in the last stage.

Description

DK 175109 B1 i

The invention relates to a method for activating gene-technologically prepared disulfide bridges containing eukaryotic proteins after expression in prokaryotic cells.

e

In the expression of heterologous proteins in prokaryotic cells, these proteins in the host cells often form inactive, highly soluble aggregates (so-called "refractile bodies") which are additionally contaminated with the proteins of the host cells. It is believed that the formation of such "refractile bodies" is a consequence of the high protein concentration caused by expression in the cell. It is known that the formation of large amounts of enzymes in the cell leads to the clumping of the enzymes into insoluble, high molecular weight, mostly inactive particles. Before such proteins can be used for e.g. for therapeutic purposes, these must therefore be purified and transferred to their active form.

According to known methods, reactivation of such proteins as aggregates of present proteins can be carried out in several steps (compare, e.g., R. Jaenicke, FEBS Federation of European Biochemical Societies, Vol. 52 (1979) 187 to 198; R. Rudolph et al. Biochemistry 18 (1979) 5572 to 5575); In the first step, solubilization is achieved by the addition of potent denaturants, for example guanidine hydrochloride or high concentration urea or by the addition of highly acidic agents, for example glycine / phosphoric acid mixtures. As additional adjuvants, reducing SH reagents (e.g., dithioerythritol, DTE) and 'EDTA have been found to be suitable, e.g. by renaturating LDH. If the protein is contaminated with proteins from the host cell, the next step is purification by itself, known and common methods, e.g. gel or ion exchange chromatography. Then vigorously dilute to reduce the denaturant concentration. Using guanidine hydrochloride is thereby diluted to concentrations below 0.5 mol / l. In enzymes with free SH groups, it was found to be advantageous to add SH group protecting agents (compare, e.g., R. Jaenicke, Journal Polymer Science, Part C 16 (1967) 2143 to 2160).

2 DK 175109 B1 European patent application EP-A-0114506 discloses methods for isolating, purifying and reactivating some heterologous expression products from bacterial cultures; for the reactivation, solutions of "refractile bodies" in a powerful denaturing agent are transferred a) directly to a solution of a weaker denaturing agent, which is then subjected to oxidative conditions to restore disulfide bridges; b) the protein is sulfonated, then transferred to a solution in a weaker denaturing agent, and the S-sulfonate groups are converted by treatment with a sulfhydryl reagent in its reduced and oxidized form, e.g. with GSH / GSSG, for -S-S groups; or c) the solution is treated in a weak denaturant directly with the sulfhydryl reagent, e.g. with 10 GSH / GSSG. A typical example whereby the above problems occur is t-PA.

The major component of the protein matrix in coagulated blood is polymeric fibrin. This protein matrix is dissolved by plasmin which is formed from plasminogen via activation through the so-called plasminogen activators, e.g. through t-PA (tissue plasminogen activator, tissue type plasminogen activator). The enzymatic activity of natural or from high-karyotic cells genetically recovered t-PA (catalytic activation of plasminogen to plasmin) is extremely low in the absence of fibrin or fibrin cleavage products (FSP), but can be significantly increased in the presence of these stimulators (more than factor 10). . This so-called activity stimulability is a crucial advantage of t-PA 20 over other known plasminogen activators such as urokinase or streptokinase (compare, e.g., M. Hoylaerts et al., J. Biol. Chem. 257 (1982) 2912 to 2919; Nieuwenhiuzenetal., Biochemica et Biophysica Acta 755 (1983) 531 to 533). Therefore, the stimulus-effectiveness factor with BrCN cleavage products is stated differently in the literature and numbers up to 35. 1

A t-PA-like, non-glycosylated product is also formed in genetically engineered prokaryotic cells (after cDNA entry); however, such a product does not stimulate the activity of a t-PA from eukaryotic cells. This may be due to the fact that the reaction conditions in the prokaryotic cell are in such a way different from that in the eukaryotic cell from which the gene originates, that from the beginning no active product is formed, which could be due, for example, to the numerous SS bridges containing the naturally active molecule are incorrectly linked or not formed at all. However, in the therapeutic use of t-PA, not only is the enzymatic activity necessary, but also its stimulability. The fact that the prokaryotic cell probably does not create the right conditions to provide the activity of eukaryotic proteins properly is referred to in the context of other substances in EMBO Journal 4, No. 3 (1985) 775 to 780.

According to European patent application EP-A-0093639, the 10 cell pellets obtained from E. coli for the reactivation of t-PA are suspended in 6 mol / l guanidine hydrochloride, treated with ultrasound, incubated and then dialyzed for a solution of Tris -HCl (pH = 8.0), sodium chloride, EDTA and Tween 80. After dialysis, centrifuge the plasminogen activator activity in the supernatant. The t-PA thus renaturated is proteolytically active, but does not show any measurable stimulability through fibrin BrCN cleavage products (BrCN-FSP) according to that of J. H. Verheijen, Thromb. Haemostas., 48, (3), 260-269 (1982).

For the reactivation of denatured proteins, there is no generally applicable method in the prior art; this is especially true for t-PA, since the natural protein has a highly complex structure; it contains a free diol group and 17 SS bridges, which theoretically can be linked in 2.2 x 1020 different ways, and only one structure corresponds to the natural state. Prior art methods for reactivating t-PA well lead to a proteolytically active t-PA, but do not show any measurable stimulability.

An activation method leading to stimulable t-PA is not known. The object of the present invention, therefore, is to provide a method for the complete activation of genetically engineered heterologous disulfide bridges containing eukaryotic proteins after expression in prokaryotic cells.

DK 175109 B1 4

The invention relates to a method for activating gene-technologically prepared heterologous disulfide bridges containing eukaryotic proteins after expression in prokaryotic cells, which is obtained in claim 1 by means of cell pickup, solubilization under denaturing and reducing conditions, and activation (renaturation) under oxidizing conditions. in the presence of GSH / GSSG. The peculiarity of the process is that in the activation step one works at a pH of 9 to 12, a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol per liter and with a non-denaturing concentration of the denaturing agent.

Preferred embodiments of the method are set out in the subclaims.

As a denaturant, a denaturant or arginine usually used for the activation under oxidizing conditions can usually be used; of the known denaturing agents, guanidine hydrochloride or urea or derivatives thereof are preferred.

Furthermore, arginine has proven to be suitable. Furthermore, mixtures of these denaturing agents can be used. Preferably, this activation step is also performed in the presence of a foreign protein; as such, as a rule, any foreign protein is suitable as long as it does not appear proteolytic; preferably, bovine serum albumin (BSA) is used, e.g. in an amount of 1 to 3 mg / ml. The BSA addition provides slightly elevated yield and stabilization of the protein (this is likely due to protection against surface denaturation and / or proteolytic degradation). 1 2 3 4 5 6

The other process conditions may correspond to the usual and usual conditions known for the reactivation steps of the prior art 2. Activation duration (incubation) is preferably 20 to 48 hours at room temperature. The half-life of activation is 4 in the presence of 0.5 mmol / l reduced (GSH) and oxidized (GSSG) glutathione at 5 ca. 10 to 15 hours at 20 ° C. As a rule, for longer incubation (48 hours) under reoxidation conditions, the stimulatability through CNBr-FSP decreases. The activation step is preferably carried out in the presence of EDTA, where the most suitable concentration is approx. 1 mmol / 1 EDTA.

DK 175109 B1 5

The prior and subsequent process steps of the activation step (reoxidation / activation), such as cell unlocking, solubilization (solubilization / reduction) and optionally one or more of the preceding and / or subsequent purification operations, may be known in the art, e.g. EP-A-0114506 and EP-A-0093619, 5 are carried out by methods known and known in the art; however, for optimum yield and activation results, it may be useful to carry out some or all of the steps in accordance with one or more of the embodiments set forth herein. In particular, it is possible to carry out the steps of activation according to the invention in the mixture obtained after the opening without prior denaturation and / or reduction, however with lower yield. Expression is carried out in prokaryotic cells, preferably in P. putida, and especially in E. coli. However, the method of the invention is equally suitable when the expression occurs in other prokaryotic cells (e.g., Bacilli).

Cell harvesting can be accomplished by conventional methods, e.g. using 15 ultrasound, high pressure dispersion or lysozyme; The absorption is preferably carried out in a buffer solution suitable for neutralizing a weakly acidic pH value as a suspending medium, such as e.g. in 0.1 mol / l Tris-HCl. After cell opening, the insoluble components ("refractile bodies") are separated in any way, preferably by centrifugation in grouting at higher g-numbers and longer centrifugation times or by filtration. After washing with 20 agents that do not interfere with t-PA, but which dissolve as far as possible foreign cell proteins, e.g. water, phosphate buffer solution, is optionally subjected to the precipitate (pellet) with the addition of mild detergents such as Triton, the solubilization (solubilization / reduction). The solubilization is preferably in alkaline pH range, in particular at pH = 8.6 ± 0.4 and in the presence of a reducing agent of the mercaptan group and I.

25 a denaturing agent.

As denaturing agents, they can be used for the solubilization of the prior art, e.g. from European patent application EP-A-0114506, known and customary denaturing agents are used, in particular guanidine hydrochloride or urea. The concentration of DK 175109 B1 6 of guanidine hydrochloride suitably amounts to approx. 6 mol / l and for urea approx. 8 mol / l. Also, compounds of the general formula I may be used.

As a reducing agent of the mercaptan group, e.g. reduced glutathione (GSH) or 2-mercaptoethanol, e.g. at a concentration of approx. 50 to 400 mmol per liter 5 and / or in particular DTE (dithioerythritol) or DTT (dithiothreitol), e.g. at a concentration of approx. 80 to 400 mmol / l. Suitably, the solubilization occurs at room temperature over one or more hours (incubation), preferably over two hours. Additionally, to prevent the oxidant oxidation through the oxygen of the air, it may be useful to add EDTA. In addition to solubilization / reduction, the solubilization step also has a purification effect, with a large proportion of the t-PA immunologically non-cross-reacting material (foreign protein) not dissolving.

After the solubilization and before the activation step, the known and common purification steps can be pushed in; as cleaning methods come e.g. steric exclusion chromatography (SEC) (in the presence of guanidine hydrochloride or urea) or 15 ion exchangers (eg in the presence of urea or derivatives thereof) under consideration; a nonspecific reoxidation can be prevented by the addition of a reducing agent (e.g., 2-mercaptoethanol) or at pH 4.5 (compare, e.g., R. Rudolph, Biochem.

Soc. Transactions 13 (1985) 308 to 311). If used in the previous solubilization step DTE, this must be separated in a purification step. The purification may e.g. is carried out at SEC 20 over Sephadex G 100 in the presence of guanidine hydrochloride and a reducing agent, e.g. GSH at a pH of 1 to 4 (in this step a large amount of foreign protein can be separated); or via separation of the denaturant / reducing agent by desalting over Sephadex G 25 in 0.01 mol / l HCl or 0.1 mol / l acetic acid. Alternatively, the separation of the denaturing / reducing agent can be achieved by dialysis against the same solutions. 1

A further purification step may follow the reactivation step; such purification is usually done by dialysis, or also by subsequent isolation of the activated tPA, for example by affinity chromatography, e.g. over Lyssepharose. Another embodiment of the invention depends on the formation of mixed disulfides from genetically engineered heterologous disulfide bridges containing eukaryotic proteins and glutathione (hereinafter abbreviated t-PASSG). This can facilitate both the separation of foreign proteins in the denatured state and the further purification of the natural proteins. A purification after modification of the thiol groups has the advantage that the protein is protected against air oxidation and thereby stable a larger pH range, and that a change in the net charge facilitates the purification. In particular, a separation from the unmodified protein can advantageously be effected by ion exchange treatment.

For the formation of the mixed disulfides, the dialyzed, reduced, purified protein from the denaturing and reducing agent was incubated with diluted, e.g. 0.2 mol / l GSSG solutions containing a denaturing agent. The activation occurred after the separation of the denaturing and oxidizing agent at a pH of 7 to 10.5, a GSH concentration of 0.5 to 5 mmol / l, and at a non-denaturing concentration of the denaturing agent.

In all other reaction steps, the activation of the protein via the formation of the mixed disulfides with GSSG is similar to the embodiments of the activation of the aforementioned part of the invention. In this embodiment, the pH optimum is at 8.5, the yield is approx. twice as high, and the activated protein is stable over a longer period in the naturalization buffer.

According to the invention, it is possible to activate t-PA derived from prokaryotic cells so that not only an activation of the normal biological activity is obtained, but also a stimulus, as defined above, which greatly exceeds the stimulability of the natural t-PA and which may be greater than factor 10, even factor 50. 1

A further eukaryotic protein that can be activated according to the invention after expression in prokaryotic cells is B-interferon.

DK 175109 B1 8

The invention is illustrated by the following examples, but not limited thereto. Unless otherwise stated, percentages indicate wt% and temperature indications to aC.

EXAMPLE 1 a) Preparation of "refractile bodies" 100 g of E. coli moist cell mass, taken up in 1.5 L, 0.1 mol / l Tris / HCl (pH 6.5) and 20 mmol / l EDTA were homogenized (Ultra - Turrax, 10 sec) and 0.25 mg / ml lysozyme was added. After 30 minutes of incubation at room temperature, the mixture was homogenized again and cooled to 3 ° C. Cell uptake was achieved by high pressure dispersion (550 10 kg / cm 2). Then, rinsed with 300 ml of 0.1 mol / l Tris / HCl (pH 6.5) and 20 mmol / l EDTA. After centrifugation (2 hours at 27,000 g, 4CC), the pellet was taken up in 1.3 I 0.1 mol / l Tris / HCl (pH 6.5), 20 mmol / l EDTA and 2.5% Triton-x-100 and homogenised.

After centrifugation again (30 minutes at 27,000 g, 4 ° C), the pellet was taken up in 1.3 10.1 mol / l Tris / HCl (pH 6.5), 20 mmol / l EDTA and 0.5% Triton-x 100 and homogenized.

Alternately centrifugation (30 minutes at 27,000 g, 4 ° C) and homogenization of the pellet in 1 1 0.1 mol / l Tris / HCl (pH 6.5) and 20 mmol / l EDTA were performed three more times.

The t-PA content of "refractile bodies" preparations was quantified by SDS-PAGE, identification of the t-PA bands by "Western blotting" and by densiometric analysis. The "refractile bodies" exhibited, by SDS-PAGE and "Western blotting, a strong t-PA band having a molecular weight of approx. 60 kDa. The t-PA proportion of the total protein content in "refractile bodies" is approx. 21%.

b) Solubilization / reduction of "refractile bodies" "Refractile bodies" were incubated at a protein concentration of 1 to 5 mg / ml in 0.1 mol / 1 Tris / HCl (pH 8.6), 5 mol / 1 guanidine hydrochloride, 0.15 to 0.4 mol / L DTE and 1 mmol / l EDTA for 2 to 3 hours at room temperature. Subsequently, insoluble material (cell wall fragments, etc.) was centrifuged, e.g. 30 minutes at 35,000 to 50,000 g, 4 ° C. The pH of the supernatant was adjusted with the wife. HCl to pH 3. The denaturant and reducing agent were then separated by dialysis against 0.01 mol / l HCl at 4CC.

C) Reoxidation / activation

Reoxidation / activation was performed at a 1:50 to 1: 200 dilution in 0.1 mol / l Tris / HCl (pH 10.5), 1 mmol / l EDTA, 1 mg / ml BSA, 0.5 mol / l L -arginine, 2 mmol / l GSH, 0.2 mmol / l GSSG. After 17 to 24 hours of activation at approx. 20 ° C, the activity was determined and the yield compared with the activity of natural glycosylated t-PA derived from eukaryotic cells.

Yield calculated on the total protein content of "refractile bodies": 2.5 ± 0.5% stimulability: 10 ± 5

Yield calculated on the t-PA proportion of 15 "refractile bodies": approx. 12% d) Reoxidation / activation without separation of the denaturing / reducing agent "Refractile bodies" was incubated at a protein concentration of 1.25 mg / ml in 0.1 mol / 1 Tris / HCl (9H 8.6), 6 mol / 1 guanidine hydrochloride, 0.2 mol / 1 DTE and 1 mmol / 1 EDTA for 2 hours at room temperature. Thereafter, reoxidation was immediately initiated at a 1: 100 dilution in 0.1 mol / L Tris / HCl (pH 10.5), 1 mmol / L EDTA, 1 mg / ml BSA, 0.3 mol / L L-arginine, and the amounts of GSSG listed in the table. In addition, in the activation portion, a residual concentration of 0.06 mol / l guanidine / hydrochloride and 2 mmol / 1 DTE was found.

Dependence of activation yield on GSSG concentration upon activation without separation of the denaturing / reducing agent.

DK 175109 B1 10 GSSG Yield 'Stimulability (mmol / 1) __ (Factor) 0.2 0 1 0.13 4.0 5 5 1.49 7.4 6 1.28 5.4 7 1.04 5.8 9 0.98 5.2 10 1.77 10.0 10 15 0 20 0 '= Yield of active t-PA calculated on the total protein content of "refractile bodies".

EXAMPLE 2 A RB ("refractile bodies") preparation (about 5 mg) was incubated in 1 ml of 0.1 mol / l Tris / HCl (pH = 8.6), 6 mol / l guanidine hydrochloride and 0 15 - 0.2 mol / l in DTE for 2 to 3 hours at room temperature. Insoluble material (cell wall fragments) was then separated by centrifugation (20 minutes at 17,000 g). The denaturing and reducing agent was removed by gel filtration through Sephadex 25 (superfine) in 0.01 mol / l HCl. Thereby, the sample was diluted ca. by factor 5 to 10. The reduced material was stored in 0.01 mol / l HCl at -20 ° C.

EXAMPLE 3 The following tables show the influence of various parameters of the invention on the activation and stimulability of t-PA. In this reduction study the solubilized reduced protein of Example 2 was not further purified.

DK 175109 B1 11

The reduced protein (in 0.01 mol / l HCl) was activated by dilution to ffa 1:10 to 1: 500 in "reoxidation buffer". Activation was determined after 22 to 48 hours of incubation at room temperature. The activity of the reoxidized protein was based on a "standard reoxidation" (= 100%) in:

5 0.1 mol / l Tris / HCl (pH = 10.5) + 1 mmol / l EDTA

+ 0.5 mol / l L-arginine + 1 mg / ml BSA

+ 0.5 mmol / 1 GSH (reduced glutathione) + 0.5 mmol / 1 GSSG (glutathione disulfide).

Stimulability is calculated from AE + CNBrFSP / AE-CNBrFSP (cf. W. Nieuwenhuizen et al., Biochimica et Biophysica Acta 755 (1983) 531 to 533). Activity (as a percentage) and stimulability (factor) were determined according to J. H. Verheijen Thromb. Haemostas. 48 (3), 266-269, (1982).

The following results were obtained: 1. Dependence of activation yield on L-arginine or guanidine hydrochloride addition.

Reoxidation in 0.1 mol / l Tris / HCl (pH 10.5)

+ 1 mmol / 1 EDTA + 1 mg / ml BSA + 0.5 mmol / 1 GSH 20 + 0.5 mmol / 1 GSSG

DK 175109 B1 12 a) L-arginine L-arginine Activity Stimulability (mol / l) (%) (Factor) 5 _ 0 4 2.5 0.25 98 7.5 0.5 100 21.9 0.75 27 16.3 10 1.0 23 3.5

It should be noted that in this study, t-PA is inhibited by L-arginine. Decrease in the activation yield at higher L-arginine concentrations must therefore be corrected for the inhibition.

b) Guanidine hydrochloride (Gdn / HCl) (Gdn / HCl) Activity (mol / l) (%) 20 _ 0 11 0.25 22 0.5 53 0.75 58 25 1.0 12 DK 175109 B1 13 2 The dependence of the activation yield on the addition of urea and urea derivatives.

Reoxidation in 0.1 mol / 1 Tris (pH 10.5), 1 mmol / 1 EDTA, 1 mg / ml BSA, 5 mmol / 1 GSH, 0.2 mmol / 1 GSSG

a) Urea 5 _

Urea Activity (mol / l) (%) 0 1 10 0.5 20 1 59 1.5 126 2 162 2.5 141 15 3 72 4 12 5 0 DK 175109 B1 14 b) Methylurea

Methylurea Activity (mol / l) (%) 5 ___ 0.5 22 1 174 1.5 313 2 375 10 2.5 332 3 215 4 12 5 0 15 c) Ethylurea

Ethylurea Activity (mol / l) (%) 1 2 3 4 5 6 0.5 46 2 1 212 3 1.5 323 4 2 300 5 2.5 107 6 3 19 4 0 5 0 DK 175109 B1 15 d) Dimethyl urea

Dimethylurea Activity Stimulability (mol / l) (%) (Factor) 5 _ 0.5 167 8.8 1 256 8.9 1.5 283 9.4 2 177 7.7 10 2.5 78 8.9 3 23 9.9 4 4 8.6 5 2 3.5 1 in 3. The activation yield's dependence on the addition of fatty acid amides:

Reoxidation in 0.1 mol / 1 Tris (pH 10.5), 1 mmol / 1 EDTA, 1 mg / ml BSA, 5 mmol / 1 GSH, 0.2 mmol / 1 GSSG.

DK 175109 B1 16 a) Formamide

Formamide Activity (mol / l) (%) 5 0 42 0.5 59 1 175 1.5 245 2 325 10 2.5 423 3 444 4 416 5 341 15 b) Methylformamide

Methylformamide Activity (mol / l) (%) 0.5 100 20 1 135 1.5 304 2 389 2.5 466 3 452 25 4 425 5 121 DK 175109 B1 17 c) Acetamide

Acetamide Activity (mol / l) (%) 0.5 0.5 72 1 134 1.5 207 2 261 2.5 204 10 3 237 4 198 5 141 d) Propionamide Propionamide Activity (mol / l) (%) 0.5 95 1 99 20 1.5 197 2 150 2.5 101 3 39 4 2 25 5 0 DK 175109 B1 18 e) Butyramide

Butyramide Activity (mol / l) (%) 5 0.5 55 1 52 1.5 17 2 0 10 4. Dependence of activation yield on pH

Reoxidation in 0.1 mol / 1 Tris / HCl + 1 mmol / 1 EDTA + 0.5 mol / 1 L-arginine + 1 mg / ml BSA + 0.5 mmol / 1 GSH 15 + 0.5 mmol / 1 GSSG

pH Activity Stimulability (%) (Factor) 1 2 3 4 5 6 7 1 - 2 8 22 3.0 3 9 89 13.6 4 10 105 20.3 5 11 95 21.3 6 _ DK 175109 B1 19 5. Dependence of activation yield on the GSH / GSSG concentration

Reoxidation in 0.1 mol / 1 Tris / HCl, pH 10.5, + 1 mmol / 1 EDTA 5 + 0.5 mol / 1 L-arginine

+ 1 mg / ml BSA

a) + 1 mmol / 1 GSH

(GSSG) Activity Stimulability 10 (mmol / I) (%) (Factor) 0.1 239 14.9 0.2 273 15.3 0.5 193 13.3 15 1 198 12.5 5 17 2.1 10 0 20 0 DK 175109 B1 20

b) + 0.2 mmol / l GSSG

(GSH) Activity Stimulability (mmol / l) (%) (Factor) 5___ 0.05 15 2.2 0.1 40 3.8 0.2 112 6.8 0.5 142 7.4 10 1 273 6, 8 5 260 7.9 10 143 6.3 20 55 5.1 15 6. Dependence of activation yield on protein concentration upon reoxidation (dilution 1:20 - 1: 500)

Reoxidation in 0.1 mol / l Tris / HCl (pH 10.5)

+ 1 mmol / l EDTA + 0.5 mol / l L-arginine 20 + 1 mg / ml BSA

+ 0.5 mmol / l GSH + 0.5 mmol / l GSSG

DK 175109 B1 21

Dilution Activity Stimulability (%) (Factor) 5 1:10 29 15.3 1:20 45 25.4 1:50 69 37.9 1: 100 100 37.9 1: 200 79 52.7 10 1: 500 29 28.7 7. Dependence of Activation Yield on BSA Addition

Reoxidation in 0.1 mol / 1 Tris / HCl (pH 10.5) + 1 mmol / 1 EDTA 15 + 0.5 mol / 1 L-arginine

+ 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG

BSA Activity 20 (mg / ml) (%) 0 47 0.5 83 1 100 25 3 102 5 52 DK 175109 B1 22

Figures 1 and 2 show the activity with and without CNBr-FSP in the standard study after 17 hours reoxidation at room temperature in 0.1 mol / l Tris / HCl (pH = 10.5) + 1 mmol / 1 EDTA + 0.5 mol / l L-arginine + 1 mg / ml BSA + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG. In Figures 1 and 2, curve (A) refers to the activity in the presence of CNBr-FSP, the curve (B) to activity without CNBr-FSP.

Example 4 t-PA activation via the mixed disulfides of t-PA and glutathione. The "refractory bodies" used were obtained according to one of the aforementioned examples. The reduction of the "refractile bodies" was carried out by 2 hours incubation at room temperature in 0.1 mol / l Tris / HCl, pH 8.6, 1 mmol / 1 EDTA, 6 mol / l Gdn-HCl, 0.2 mol / l 1 DTE at a protein concentration of ca. 1 mg / ml.

The reduced protein dialyzed against 0.01 mol / l HCl was diluted in a 1: 1 ratio with 0.1 mol / l Tris, pH 9.3, 9 mol / l urea and 0.2 mol / l GSSG and incubated for 5 hours at room temperature. 1 2 3 4 5 6

After acidification with wife. HCl to pH 3 followed dialysis against 0.01 mol / l HCl at 4SC. After the 2 dialysis, the total protein concentration was 0.33 mg / ml. The optimum reactivation conditions were determined using the t-PASSG prepared in this way.

4

a) pH optimum upon activation of t-PASSG

5

Here, as in the following optimization studies, (1) GSSG was not used and (2) 6 activation was determined after 17 hours of incubation at room temperature. Activation was carried out at a 1: 100 dilution in 0.1 mol / l Tris, 1 mmol / l EDTA, 0.5 mol / l L-arginine, 1 mg / ml BSA and 2 mmol / l GSH at various pH values. is.

DK 175109 B1 23 pH Yield (%) Stimulability 6 0.04 3.3 6.5 0.37 9.5 5 7 1.35 11.4 7.5 5.66 7.1 8 7.32 8.2 8.5 8.65 7.0 9 8.59 8.7 10 9.5 8.32 11.7 10 6.15 12.5 10.5 3.07 11.2

The yield was determined in% active t-PA based on the amount of protein used.

15 b) the reproducibility of the results in the activation of t-PASSG

At identical activation conditions, divergent yields were observed for different measurements, due in part to the fluctuations of standard t-PA. To clarify this error width, all activation data were summarized after dilution to 1: 100 or 1: 200 in 0.1 mol / 1 Tris / HCl, pH 8.5, 1 mmol / 1 EDTA, 0.5 mol / 1 L-arginine , 1 mg / ml 20 BSA and 2 mmol / 1 GSH.

DK 175109 B1 24

Experiment Yield (%) Stimulability 1 8.65 7.0 2 4.47 9.3 5 3 4.49 9.7 4 8.50 6.5 5 3.45 17.2 6 4.32 8.3 7 3.29 14.0 10 8 3.54 13.4 9 5.07 16.4

Mean 5.1 ± 1.9 11.3 ± 3.8 15 c) Stability of the activated protein

The activation is carried out in the above example by a 1: 200 dilution in 0.1 mol / l Tris / HCl, 1 mmol / l EDTA, 0.5 mol / l L-arginine, 1 mg / ml BSA and 2 mmol / l GSH .

Time pH 9.5 (h) Yield Stim.

20 (%) 1 0 - 6 0.89 15.5 23 2.43 23.1 25 47 2.83 23.6 71 2.62 21.5 DK 175109 B1 25 215 2.21 22.6 239 2, 28 14.3 EXAMPLE 5 5 Activation of genetically engineered Interferon-fi.

"Refractile bodies" were prepared according to the aforementioned methods. The reduction / solubilization of the "refractile bodies" was carried out as follows: The pellet was incubated for 3 hours at 25 ° C in 10 ml of 9.1 mol / l Tris / HCl, pH 8.6, 6 mol / l Gdn-HCl, 1 mmol / 1 EDTA and 0.2 mol / 1 DTE and after 30 minutes centrifugation at 4 ° C and 10 48,000 g, the pH of the supernatant was adjusted to about 3 with wife HCl, then a gel filtration over Sephadex G25 F was performed in 0.01 mol / l HCl.

The conductivity, protein concentration and reactivability of the eluate were investigated.

The activity of the reoxidized protein is based on a "standard activation" (= 100%) in 0.1 mol / l Tris / HCl, pH 10.5, 1 mmol / 1 EDTA, 5 mmol / 1 GSH, 0.5 mmol / 1 GSSG and 0.25 mol / l L-arginine.

a) Dependence of activation yield on L-arginine addition

The eluate was diluted 1:50 with 0.1 mol / l Tris / HCl, pH 8.5, 1 mmol / l EDTA, 5 mmol / l GSH, 9.5 mmol / l GSSG and incubated for 20 hours at 0 ° C.

L-arginine dependence of activation DK 175109 B1 26 L-arginine (mol / l) Activity (7c) O 8 0.25 8 5 0.5 15 0.75 15 b) Activation yield dependent on urea addition

The activation solution is similar to that of point a); however, activated for 17 hours at 0 ° C.

10 Urea dependence on activation Urea (mol / l) Activity (%) 0 13 0.5 100 15 1 200 1.5 100 c) Dependence of activation yield on formamide addition

The activation as in a); the samples were examined after 17 hours of activation at 0 ° C.

20 Formamide dependence on activation DK 175109 B1 27

Formamide (mol / l) Activity 0 13 1 13 5 2 13 3 0 4 0 d) Dependence of activation yield on redox buffer 10 The eluate was diluted 1:50 in 0.1 mol / l Tris / HCl, pH 8.5, 1 mmol / 1 EDTA and 0.25 mol / l L-arginine, and the samples were tested after 17 hours of activation at 0 ° C.

GSH / GSSG Dependence on Activation GSH (mmol / 1) GSSG (mmol / I) Activity (%) 15 1 0.5 6 5 0.5 13 10 0.5 25 20 0.5 25 1 5 0.1 13 5 0.5 13 5 1.0 13 5 5 6 DK 175109 B1 28 e) Dependence of the activation yield on the BSA addition

The eluate was diluted 1:50 in 0.1 mol / l Tris / HCl, pH 8.5, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG and 0.25 mol / l L-arginine and tested after 17 hours of activation at 0 ° C.

5 BSA dependence on activation BSA (mg / ml) Activity (%) 0 13 1 13 10 2 25 5 13

f) Dependence of activation yield on pH

The eluate was diluted 1:50 in 0.1 mol / L Tris / HCl, 1 mmol / L EDTA, 5 mmol / L GSH, 0.5 15 mmol / GSSG and 0.25 mol / L L-arginine and tested after 17 hours. activation at 0 ° C.

pH dependence on activation pH Active (%) 6.5 0 20 7.5 6 8.5 13 9.5 50 10.5 100

Claims (18)

A method of activating heterologous, disulfide-bridged eukaryotic proteins made from gene technology by expression in prokaryotic cells by a) a) inclusion of the prokaryotic cells, b) dissolving the eukaryotic proteins under denaturing and reducing conditions, c) separating the reducing / d) reactivation under oxidizing conditions at 10 e) conversion of the thiol groups of the solubilized proteins to the mixed disulfides of protein and glutathione by addition of GSSG under denaturing conditions, f) formation of active protein from the mixed disulfides at a GSH concentration of 0.5 to 5 mmol / l, a pH of 7 to 10.5 and in the presence of a non-denaturing concentration of a denaturing agent. Method according to claim 1, characterized in that the expression is carried out in E. coli or P. putida. Process according to claim 1 or 2, characterized in that in the reactivation step, arginine, guanidine hydrochloride and / or at least one compound of the general formula R2-CO-NRRj (I) wherein R and R are H are used. or alkyl 20 having 1 to 4 C atoms, and R 1 is H or NHR, or alkyl having 1 to 3 C atoms. DK 175109 B1 Process according to claim 3, characterized in that the concentration of arginine and / or guanidine hydrochloride is 0.1 to 1.0 mol / l, in particular 0.25 to 0.8 mol / l. Process according to claim 3, characterized in that the concentration of the compound of the general formula I is 0.5 to 4 mol / l, in particular 1 to 3.5 mol / l. Process according to any one of the preceding claims, characterized in that during the reactivation step, work is carried out in the presence of a non-proteolytically active protein, in particular in the presence of bovine serum albumin. Method according to any one of the preceding claims, characterized in that the cell pickup is carried out by ultrasound, high pressure dispersion or lysozyme. Process according to claim 7, characterized in that the digestion is carried out in a dilute aqueous buffer solution, in particular in 0.1 mol / l Tris, at a neutral to slightly acidic pH. Process according to any one of the preceding claims, characterized in that after the cell opening, the insoluble components are separated. Process according to any one of the preceding claims, characterized in that the solubilization step is carried out at alkaline pH in the presence of a reducing agent from the mercapto group and in the presence of a denaturing agent. 1 Process according to Claim 10, characterized in that one works in the near room of guanidine hydrochloride and / or compounds of the general formula I as denaturing agent. DK 175109 B1 Process according to claim 11, characterized in that the concentration of guanide hydrochloride is 6 mol / l, the concentration of compounds of the general formula I is 8 mol / l. in· Process according to any one of claims 10 to 12, characterized in that 5 is operated in the presence of DTE, B-mercaptoethanol, cysteine or GSH. Process according to any one of the preceding claims, characterized in that purification and separation of reducing, oxidation or denaturing agents are carried out by steric exclusion chromatography or dialysis. Process according to any one of the preceding claims, characterized in that after the reactivation step a purification step is carried out by dialysis. Method according to any one of claims 1 to 15, characterized in that t-PA is used as a genetically-engineered eukaryotic protein. A stimulable, non-glycosylated tPA, obtained according to the method of any one of claims 1 to 16. 1 Process according to claim 6, characterized in that the mixed disulfide of protein and glutathione is separated from non-modified protein by means of ion exchange treatment.