DK175091B1 - Method for activating gene-technologically prepared, heterologous, disulfide-bridged eukaryotic proteins after expression in prokaryotic cells - 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 175091 B1

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

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 host cells' proteins. It is believed that the formation of such "refractile bodies" is a consequence of 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 example, for therapeutic purposes. therefore, 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 proven useful, e.g. by renaturating LDH. If the protein is contaminated with proteins from the host cell, the next step follows a purification by methods known per se and in the art, 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. For enzymes with free SH groups, it was found advantageous to add 25 SH group protecting agents (compare, e.g., R. Jaenicke, Journal Polymer Science, Part C 16 (1967) 2143 to 2160).

UK Ί / 5Uyi bl 2 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 key 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; Nieuwenhiuzen et al., Biochemica et Biophysica Acta 755 (1983) 531 to 533). Therefore, the stimulus potency factor with BrCN cleavage products is stated differently in the literature and numbers up to 35 are mentioned.

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 so 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, that 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 exhibit any measurable stimulability through fibrin BrCN cleavage products (BrCN-FSP) according to that of J. H. Verheijen, Thromb. Haemostas., 48, (3), 260-269 (1982).

Orsini and Goldberg (J. Biol. Chem. 253 (1978), 3453-3458) describe the renaturation of reduced chymotrypsinogen A by transferring the denatured proteins into a purification buffer containing GSH, GSSG and a non-denaturing concentration of 20 · Guanidine HCl or urea.

4

From Proc. NaCl. Acad. Sci. United States, Vol. 81: pages 3273-3277 (1984) discloses a process for producing a recombinant protein in E. coli. The celiac extract with the dissolved, denatured and reduced antibody is centrifuged and dialyzed and the dialysate reactivated under a series of dialysis at pH 10.8 with 1 mM GSH and 0.1 mM GSSG and 25 urea, the latter dialyzed to a final concentration of 1M. During the reconstitution of antibody, among other things, the denatured, disulfide-free chains are converted to protein S-sulfonate by means of sodium sulfide and sodium tetrathionate. However, in the process of the invention, a number of intermediate steps are avoided which, according to the above article, must be carried out after disulfide formation and prior to purification.

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 thiol 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-bridged eukaryotic proteins after expression in prokaryotic cells.

The invention relates to a method for activating gene-technologically prepared, heterologous, disulfide-bridged eukaryotic proteins after expression in prokaryotic cells, which is obtained in claim 1 by 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 8 to 12, a 20 GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol per liter and having a non-denaturing concentration of the denaturing agent and using in the reactivation step as denaturing agent arginine and / or at least one compound of the general formula R2-CO-NRR, (I) wherein R and R are H or alkyl of 1 to 4 C atoms and R 2 means H or NHR, or alkyl of 1 to 3 C atoms, with the proviso that R and R are not simultaneously H.

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

DK 175091 B1 5

As the denaturing agent, arginine or urea derivatives of formula (I) are used.

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).

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

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 activation step (s) preceding and / or subsequent purification operations, may be known, e.g. EP-A-0114506 and EP-A-0093619 are carried out by methods known and customary 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 25 for 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 process vii (vvs u i 6 of the invention is equally suitable when expression occurs in other prokaryotic cells (e.g., bacilli).

Cell harvesting can be accomplished by conventional methods, e.g. by ultrasound, high pressure dispersion or lysozyme; The digestion preferably takes place in a buffer solution suitable for setting 5 of a neutral to slightly acidic pH value, such as suspension medium, e.g. in 0.1 mol / l Tris-HCl. After cell uptake, the insoluble components ("refractile bodies") are separated in any way, preferably by centrifugation at higher g-numbers and longer centrifugation times or by filtration. After washing with agents that do not interfere with t-PA, but which, as far as possible, dissolve 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 mercaptan group and 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. Conveniently, the concentration of guanidine hydrochloride is 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 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. In addition, to prevent the oxidation of the reducing agent 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, since a large proportion of the t-PA immunologically non-cross-reacting material (foreign protein) does not dissolve.

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 ion exchange (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 10 DTE, this must be separated in a purification step. The purification may e.g. performed at SEC 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.

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.

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

A further eukaryotic protein that can be activated according to the invention after expression 25 in prokaryotic cells is B-interferon.

8 DK 175091 Bl

The invention is illustrated by the following examples, but not limited thereto. Unless otherwise indicated, percentages indicate% by weight and temperature indications to ° C.

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, it was homogenized again and cooled to 3 ° C. Cell uptake was achieved by high pressure dispersion (550 10 kg / cm 2). Thereafter, 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, 4 ° C), the pellet was taken up in 1.3 l 0.1 mol / l Tris / HCl (pH 6.5), 20 mmol / l EDTA and 2.5% Triton-x 100 and homogenized.

After re-centrifugation (30 minutes at 27,000 g, 4 ° C), the pellet was taken up in 1,310.1 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 / 1 Tris / HCl (pH 6.5) and 20 mmol / 1 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 20 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 / 1 DTE and 1 mmol / 1 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 4 ° C.

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 / 1 GSH, 0.2 mmol / 1 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 (pH 8.6), 6 mol / 1 guanidine hydrochloride, 0.2 mol / 1 DTE and 1 mmol / 1 EDTA for 2 hours at room temperature, then immediately reoxidation was started at a 1: 100 dilution in 0.1 mol / 1 Tris / HCl (pH 10.5), 1 mmol / 1 EDTA, 1 mg / ml BSA, 0.3 mol / 1 L-arginine and the amounts of GSSG listed in the table, and in the activation portion, a residual concentration of 0.06 mol / 1 guanidine / hydrochloride and 2 mmol was found. / 1 DTE.

10 υιν i / ου »i di

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

GSSG Yield 'Stimulability (mmol / 1) __ (Factor) _ 5 0.2 0 1 0.13 4.0 5 1.49 7.4 6 1.28 5.4 7 1.04 5.8 10 9 0 , 98 5.2 10 1.77 10.0 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 according to the invention on the activation and stimulability of t-PA. In this reduction study, according to Example 2, solubilized reduced protein was not further purified.

The reduced protein (in 0.01 mol / l HCl) was activated by dilution to from 1:10 to 1: 500 in "reoxidation buffer". Activation was determined after 22 to 48 hours incubation at room temperature. The activity of the reoxidized protein was based on a "standard reoxidation" (= 100%) in: 0.1 mol / l Tris / HCl (pH = 10.5) + 1 mmol / l EDT A 10 + 0.5 mol / 1 L-arginine

+ 1 mg / ml BSA

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

Stimulability is calculated from ΔΕ + CNBrFSP / AE-CNBrFSP (compare W

15 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 EDT A + 1 mg / ml BSA

w m · www 12

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

a) L-arginine L-arginine Activity Stimulability 5 (mol / l) (%) (factor) 0 4 Ϊ3 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) (%) 0 11 0.25 22 20 0.5 53 0.75 58 1.0 12 DK 175091 B1 13 2. The dependence of the activation yield on the addition of urea and urea derivatives.

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

0.2 mmol / 1 GSSG

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

Methylurea Activity (mol / l) (%) 0.5 22 20 1 174 1.5 313 2 375 2.5 332 3 215 25 4 12 5 0 L ^ IX I f I U1 14 c)

Ethylurea Activity (mol / l) (%) 0.5 46 5 1 212 1.5 323 2 300 2.5 107 3 19 10 4 0 i 5 0 d) Dimethyl urea

Dimethylurea Activity Stimulability (mol / l) (%) (Factor) 15 0.5 167 8.8 1 256 8.9 1.5 283 9.4 2 177 7.7 2.5 78 8.9 20 3 23 9.9 4 4 8.6 5 2 3.5 3. Dependence of activation yield on addition of fatty acid amides: DK 175091 B1 15

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) Formamide

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

Methylformamide Activity (mol / l) (%) 0.5 100 1 135 20 1.5 304 2 389 2.5 466 3 452 4 425 25 5 121 DK 175091 B1 16 c) Acetamide

Acetamide Activity (mol / l) (%) 0.5 72 5 1 134 1.5 207 2 261 2.5 204 3 237 10 4 198 5 141 d) Propionamide

Propionamide Activity (mol / l) (%) 15 0.5 95 1 99 1.5 197 2 150 2.5 101 20 3 39 4 2 5 0 DK 175091 B1 17 e) Butyramide

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

Reoxidation in 0.1 mol / 1 Tris / HCl + 1 mmol / 1 EDTA 10 + 0.5 mol / 1 L-arginine

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

pH Activity Stimulability 15 (%) (Factor) 7 1 8 22 3.0 9 89 13.6 10 105 20.3 20 11 95 21.3

1 ^ 1¾ I VVV I W I

5. Dependence of activation yield on GSH / GSSG concentration 18

Reoxidation in 0.1 mol / l Tris / HCl, pH 10.5,

+ 1 mmol / 1 EDTA + 0.5 mol / 1 L-arginine 5 + 1 mg / ml BSA

a) + 1 mmol / 1 GSH

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

b) + 0.2 mmol / l GSSG

DK 175091 B1 19 (GSH) Activity Stimulability (mmol / l) (%) (Factor) 0.05 15 2.2 5 0.1 40 3.8 0.2 112 6.8 0.5 142 7.4 1 273 6.8 5 260 7.9 10 10 143 6.3 / 20 55 5.1 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)

15 + 1 mmol / l EDT A

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

Dilution Activity Stimulability (%) (Factor) 1:10 29 15.3 1:20 45 25.4 1:50 69 37.9 25 1: 100 100 37.9 1: 200 79 52.7 1: 500 ___ 29__28, 7 -__

Ul \ I «IV? I Part 7. Dependency of Activation Yield on BSA Addition 20

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

+ 1 mmol / 1 EDT A + 0.5 mol / 1 L-arginine + 0.5 mmol / 1 GSH

+ 0.5 mmol / 1 GSSG

BSA Activity (mg / ml) (%) 0 47 10 0.5 83 1 100 3 102 5 52

Figures 1 and 2 show the activity with and without CNBr-FSP in the standard study 15 after 17 hours reoxidation at room temperature in 0.1 mol / 1 Tris / HCl (pH = 10.5) + 1 mmol / 1 EDTA + 0.5 mol / 1 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, curve (B) to activity without CNBr-FSP.

EXAMPLE 4 Activation of genetically engineered Interferon-β.

"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, DK 175091 B1 21 1 mmol / l EDTA and 0.2 mol / l DTE and after 30 minutes centrifugation at 4 ° C and 48,000 g the pH of the supernatant was adjusted to approx. 3 with wife. HC1. Subsequently, 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 / 1 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 / 1 EDTA, 5 mmol / l GSH, 9.5 mmol / l GSSG and incubated for 20 hours at 0 ° C .

L-arginine dependence of activation L-arginine (mol / l) Activity (%) 0 8 0.25 8 15 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.

I «WWW I w I

Urea dependence on activation 22

Urea (mol / I) Activity (%) 0 13 0.5 100 5 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. Formamide dependence on activation 10 Formamide (mol / I) Activity 0 13 1 13 2 13 3 0 15 4 0 d) Dependence of activation yield on redox buffer

The eluate was diluted 1:50 in 0.1 mol / L Tris / HCl, pH 8.5, 1 mmol / L 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 DK 175091 B1 23 GSH (mmol / l) GSSG (mmol / l) Activity (%) 1 0.5 6 5 0.5 13 10 0.5 25 5 20 0.5 25 5 0 , 1 13 5 0.5 13 5 1.0 13 5 5 6 10 e) Dependency of activation yield on 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.

BSA dependence on activation 15 BSA (mg / ml) Activity (%) 0 13 1 13 2 25 5 13 DK 175091 B1 24

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 mmol / GSSG and 0.25 mol / L L-arginine and tested after 17 hours of activation at 0 ° C.

pH Dependence on Activation 5 pH Activity (%) 6.5 0 7.5 6 8.5 13 9.5 50 10 10.5 100

Claims (20)

A method for activating gene-technologically prepared, heterologous disulfide-bridged eukaryotic proteins after expression in prokaryotic cells by cell uptake, solubilization under denaturing and reducing conditions, and reactivation under oxidizing conditions in the presence of GSH / GSSG, characterized in that by the reactivation step is worked at a pH of 8 to 12, a GSH concentration of 0.1 to 20 mtnol / 1, a GSSG concentration of 0.01 to 3 mmol / 1 and at a non-denaturing concentration of the denaturant and at wherein in the reaction step, as the denaturing agent, arginine and / or at least one compound of the general formula R2-CO-NR-R, (I) wherein R and R are H or alkyl of 1 to 4 C atoms are used. and R 2 is H or NHR, or alkyl of 1 to 3 C atoms, with the proviso that R and R are not simultaneously H. Process according to claim 1, characterized in that the pH of the reaction step is 9.5 to 11. Process according to claim 1 or 2, characterized in that the GSH concentration is 0.2 to 10 mmol / 1 and / or the GSSG concentration 0.05 to 1 mmol / 1 in the reactivation step. Process according to any one of the preceding claims, characterized in that a purification step is carried out after solubilization and before reactivation. Process according to any one of claims 1 to 3, characterized in that the reactivation is carried out without prior separation of the denaturing / reducing agents, wherein the reaction solution is diluted after denaturation / reduction with reactivation buffer and that GSSG concentration upon subsequent reactivation is exceeded. the residual residual concentration of DTE increases. "" "^ mmmtm DK 175091 B1 26 Process according to any one of claims 1 to 6, characterized in that the expression is carried out in E. coli or P. putida. Process according to any one of claims 1-6, characterized in that the concentration of arginine is 0.1 to 1.0 mol / l, in particular 0.25 to 0.8 mol / l. Process according to any one of claims 1-6, characterized in that the concentration of compounds 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 means of ultrasound, high pressure dispersion or lysozyme. Process according to claim 10, 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. DK 175091 B1 27 Process according to claim 13, characterized in that it is used in the presence of compounds of the general formula I as a denaturing agent. Process according to claim 14, characterized in that the concentration of compounds of the general formula I is 8 mol / l. Process according to any one of claims 13 to 15, characterized in that it operates in the presence of DTE, β-mercaptoethanol, cysteine or GSH. Process according to any one of the preceding claims, characterized in that purification and separation of reducing, oxidizing 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 18, characterized in that t-PA is used as a genetically-engineered eukaryotic protein. Method according to any one of claims 1 to 19, characterized in that Interferon-β is used as a genetically-engineered eukaryotic protein.