SI8611796A - Method for activating gentechnological prepared of heterolog,eukaryotic proteins, which contain disulphide's bridges, after their expression in the procaryotes. - 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

BOEHRINGER MANNHEIM GMBH

Method for activating gentechnologically prepared, heterologous, eukaryotic proteins having disulfide bridges after expression in prokaryotes

The present invention relates to a method for activating genetically engineered, eukaryotic proteins containing disulfide bridges upon expression in prokaryotes.

In the expression of heterologous proteins in prokaryotes, these proteins are often inactive hard-soluble aggregates (so-called refractile bodies) in host cells, which are further contaminated with host cell proteins. The formation of such refractile bodies is thought to be due to the expression of the resulting high concentration of proteins in the cell. It is known that when large amounts of enzymes are formed in a cell, the enzyme aggregates into insoluble, high molecular weight, mostly inactive particles. However, before such proteins can be used, e.g. however, for therapeutic purposes, they must be purified and translated into active form.

According to known methods, the activation of such aggregate proteins can be effected in several steps (e.g., eg BR Jaenicke, FEBS Federation of European Biochemical Societies, Vol. 52 (1979) 187 to 198; R. Rudolph, Biochemistry J8 (1979 ) 5572 to 5575):

In the first stage, they achieve solubilization by the addition of strong denaturing agents, e.g. guanidine hydrochloride or urea in high concentration, or with the addition of strongly acidic agents, e.g. glycine / phosphoric acid mixtures. Reducing SH reagents (eg dithioerythritol, DTE) and EDTA, e.g. when renaturating LDH. To the extent that the protein is contaminated with the proteins of the host cell, then the next step is purification by methods known per se and conventional, e.g. by gel or ion exchange chromatography. It is then diluted sharply to reduce the concentration of the denaturing agent. When using guanidine hydrochloride, it is diluted to below 0.5 mol / l. For enzymes with free SH groups, it appears to be a suitable adjunct to agents protecting SH groups (see, e.g., B. R. Jaenicke, Journal Polymer Science, Part C 16 (1967) 2143 to 2160).

EP-A-O1145O6 describes methods for isolating, purifying and activating certain heterologous expression products from bacterial cultures; to activate, transfer solutions of refractile bodies in a strong denaturing agent a) directly to a solution in a weaker denaturing agent, which is then subjected to oxidizing conditions to re-form disulfide bridges; b) the protein is sulphonated to then be converted into a solution in a weak denaturing agent and the S-sulphonate groups by treatment with the sulfhydryl reagent in its reduced and oxidized form, e.g. with GSH / GSSG, translated into -S-S-groups; or c) treat the solution in a weak denaturing agent directly with a sulfhydryl reagent, e.g. with GSH / GSSG. A typical example of the problems presented above is t-PA.

The major component of the blood clotting protein matrix is the fibrin polymer. This protein matrix is dissolved by plasmin formed from plasminogen via activation by e.g. plasminogen activators, e.g. with t-PA (tissue-plasminogen activator). The enzymatic activity of natural or eukaryotes of gentechnologically derived t-PA (catalytic activation of plasminogen to plasmin) is very low in the absence of fibrin or fibrin cleavage products (FSP), but is significantly enhanced (by more than a factor of 10) in the presence of these stimulators. This t.i. stimulation of efficacy is a crucial advantage of t-PA compared to other known plasminogen activators such as urokinase or streptokinase (cf. e.g. M. Hoylaerts et al, J. Biol. Chem. 257 (1982)

2912 to 2019; Nieuwenhiuzen et al, Biochimica et Biophysica Acta 755 (1983) 531 to 533). The stimulus factor with BrCN vaccine products is therefore variously cited and numbered up to 35 in the literature.

- 4 t-PA-nast, non-glycosylated product is also formed in genetically manipulated prokaryotes (after c-DNA insertion); such product does not, however, belong to the stimulatory efficacy of t-PA from eukaryotes. This may be due to the fact that the redox conditions in the prokaryotic cell are so different from the eukaryotic cell from which the gene originates that an inactive product is initially formed, which can be led e.g. to the point that many SS bridges contained in the natural active molecule are either incorrectly bonded or not formed at all. However, therapeutic use of t-PA requires not only enzymatic activity per se, but also its stimulation. Also, the fact that a prokaryotic cell is unlikely to create the right conditions for the eukaryotic protein activity to be expressed in the correct manner is indicated for other substances in The EMBO Journal 4, Nr. 3 (1985) 775 to 780.

EP-A-0093639 suspends E. coli cell pellets in 6 mol / l guanidine hydrochloride to activate t-PA, treated with ultra-sound, incubated and then dialyzed for 4 hours against Tris-HCl solution (pH = 8, 0), sodium chloride, EDTA and Tween 80. After dialysis, they were centrifuged, finding the plasminogen activation efficiency in the top layer (supernatant). Natured t-PA thus produced is proteolytically active, but it does not show measurable stimulation with BrCN-cleavage products (BrCN-FSP) of fibrin in the sense of the procedure described in J. H. Verheijen, Thromb. Haemostas.,

48, (3), 260-279 (1982).

- 5 No generally applicable method is known in the art for the activation of denatured proteins; this is especially true for t-PA because the native protein has a very complex structure; contains a free thiol group and 17 SS bridges that can theoretically be connected in 2.2 x 10 different ways, with only one structure corresponding to the native state. The prior art methods for activating t-PA do lead to proteolytically active t-PA, but do not show measurable stimulation; however, the activation process leading to the stimulatory t-PA is not yet known.

It is an object of the present invention to provide us with a process for the complete activation of genetically engineered, heterologous, eukaryotic proteins containing disulfide bridges upon expression in prokaryotes; this task is solved by the object of the present invention.

The subject of the invention is a method for activating gene-technologically prepared, heterologous, eukaryotic proteins containing disulfide bridges after expression in prokaryotes, upon cell cleavage, solubilization under denaturing and reducing conditions and activation (renaturation) under oxidizing conditions of GS, in the presence of GS characterized in that, at the activation stage, we operate 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 / l, and a non-denaturing concentration of the denaturing agent.

Preferred embodiments of the present process are the subject of claims.

- 6 As a rule, a denaturing agent or arginine can generally be used as a denaturing agent to activate under oxidizing conditions; guanidine hydrochloride or urea or derivatives thereof are preferably used among known denaturing agents. On top of this, arginine also proved to be suitable. Furthermore, mixtures of these denaturing agents may be used. Preferably, this level of activation is also carried out in the presence of a foreign protein; as such, any foreign protein is generally suitable as long as it is not proteolytically effective; preferably bovine serum albumin (BSA) is used, e.g. in an amount of 1 to 3 mg / ml. BSA supplementation causes a slight increase in yield and stabilization of the protein (probably by protection against surface denaturation and / or proteolytic degradation).

Other conditions of the process may correspond to conditions known and common to the prior art reactivation steps. The activation time (incubation) is preferably 20 to 48 hours at room temperature. The activation time is about 10 to 15 hours at 20 ° C in the presence of 0.5 mmol / l reduced (GSH) and oxidized (GSSG) glutathione. On prolonged incubation (48 hours) under reoxidation conditions, stimulation with CNBr-FSP generally declines. The activation rate is preferably carried out in the presence of EDTA, with the most appropriate concentration being about 1 mmol / l EDTA.

The stages of the process performed before or after the activation step (reoxidation / activation), such as cell digestion, solubilization (solubilization / reduction), and optionally one or more purification operations performed before and / or after the activation step, are given performed according to a technique known or known. common in such procedures, e.g. from EP-A01 14506, EP-A-0093619; however, in order to achieve the optimum result in terms of gain and activation, it may be advisable to carry out individually or all stages of the process, taking into account one or more of the embodiments explained in the description provided. Thus, it is particularly possible to carry out the activation step of the invention in the mixture obtained after digestion without prior denaturation and / or reduction, and in any case in low yield. Expression is carried out in prokaryotes, preferably in P.putida and in particular E.coli. The process of the invention is also suitable when expressed in other prokaryotes (e.g., in Bacilla).

Cellular digestion can then be performed by conventional methods, e.g. using ultra-sound, high pressure dispersion or lysozyme; it is preferably carried out in a buffer solution suitable for adjusting the neutral to weakly acidic pH, such as a suspension medium, e.g. in 0.1 mol / 1 Tris-HCl. After cell digestion, the refractile bodies are chosen in any way, preferably by centrifugation at higher g-numbers and longer spin time, or by filtration. After washing with t-PA-free agents, foreign cellular proteins are not preferably dissolved, e.g. in water, phosphate buffer solution, optionally subjected to solubilization (solubilization / reduction) with the addition of soap detergents as tritone. Solubilization takes place preferably in the alkaline pH range, especially at pH 8.6 + 0.4 and in the presence

- 8 reducing agent from mercaptan group and denaturing agent.

As denaturing agents, they can be used to solubilize the prior art, e.g. from EP-A-0114506, known and conventional denaturing agents, in particular guanidine hydrochloride or urea. The guanidine hydrochloride concentration is preferably about 6 moles / l and urea is about 8 moles / l. Compounds of general formula I may also be used.

As reducing agents from the mercaptans group, e.g. reduced glutathione (GSH) or 2-raercaptoethanol, e.g. at a concentration of about 50 to 400 mmol / l dithiothreitol and / or in particular DTE (dithioerythritol) or. DTT (dithiothreitol), e.g. at a concentration of about 80 to 400 mmol / l. Solubilization is preferably carried out at room temperature for a period of (incubation) for 1 to several hours, preferably 2 hours. It is advisable to use EDTA to prevent oxidation of the reducing agent with air oxygen. In addition to solubilizing / reducing, the solubilization rate also has a cleansing effect, since most of the material that does not react immunologically with t-PA (foreign proteins) does not pass into solution.

After solubilizing and before the activation step, known and conventional purification steps can be restored; as cleaning methods, for example, steric digestion chromatography (SEC) (in the presence of guanidine hydrochloride or urea) or ion exchangers (in the presence of urea or its derivatives); nonspecific reoxidation can be prevented by the addition of a reducing agent (eg 2-mercaptoethanol) or by pH values

- £ 9. 4,5 (cf. e.g. R. Rudolph, Biochem. Soc.

Transactions 13 (1985) 3θθ to 311). If a DTE is used in the previous solubilization step, it must be removed in the purification step, for example, cleaning may take place. with SEC via Sephade * G 100 in the presence of guanidine hydrochloride and a reducing agent, e.g. GSH at pH 1 to 4 (a large amount of foreign protein can be decided at this stage); or by decontamination of a denaturing-reducing agent by desalination via Sephadex G 25 in 0.01 mol / l HCl or. 0.1 mol / l acetic acid. Alternatively, the denaturing-reducing agent can be made by dialysis against the same solutions.

A further purification step may be attached to the reactivation step; such purification is generally carried out by dialysis, or by subsequent isolation of activated t-PA, e.g. by affinity chromatography, e.g. over Lys-Sepharose

A further embodiment of the invention is based on the formation of mixed disulfides of genetically engineered, heterologous, eukaryotic proteins containing disulfide bridges and glutathione (hereinafter abbreviated as t-PASSG). This can facilitate both the decision-making of foreign proteins in the denatured state as well as the further purification of the native protein. Purification after modification of thiol groups has the advantage that the protein is protected from oxidation from air and thus stable over a larger pH range, and a change in net charge facilitates purification. The ion exchange treatment makes it particularly convenient to perform the unmodified protein decision.

To form mixed disulfides, we incubate a dialysed, reduced protein from which the denaturing and reducing agents have been purified, with diluted e.g. 0.2 M GSSG solution containing denaturing agent. Activation takes place after the denaturing and oxidizing agent is selected at a pH of 7 to 10.5, a GSH concentration of 0.5 to 5 mmol / l, and a non-denaturing concentration of the denaturing agent.

For all other reaction steps, activation of the protein via the formation of mixed disulfides with GSSG embodiments for the activation of the previous part of the invention is appropriate. In this embodiment, the pH optimization is at 8.5, the yield is about twice that, and the activated protein is stable for a longer time in the renaturation buffer.

According to the invention, it is fortunate for t-PA to be activated from prokaryotes such that not only activation of normal biological activity is achieved, but also stimulation in the above defined sense, which far exceeds the stimulation of native t-PA and is greater than factor 10, it even exceeds the factor of 50.

A further eukaryotic protein, which according to the invention can be activated after expression in a prokaryote, is N - interferon.

The following examples further illustrate the invention without limiting it. Unless otherwise stated, percentages refer to mass percentages and temperature data to degrees Celsius.

EXAMPLE 1

a) Preparation of refractile bodies

100 g of a wet cell mass of E. coli in 1.5 l,

0.1 mol / l Tris / HCl (pH 6.5) and 20 mrool / l EDTA were homogenized (Ultra-Turrax, 10 sec) and 0.25 mg / ml lysozyme was added After 30 minutes of incubation at room temperature, homogenized and cooled to 3 ° C. Cell digestion was achieved by high pressure dispersion (550 kg / cm ² ). It was further rinsed with 300 ml of 0.1 mol / 1 Tris / HCl (pH 6.5) and 20 mmol / 1 EDTA. After centrifugation (Sorvall GSA, 2 hours at 13000 rpm,

21000 g, 4 ° C) the pellet was taken up in 1.2 1 0.1 mol / 1 Tris / HCl (pH 6.5), 20 mmol / 1 EDTA and 2.5% Triton-x-100 and homogenized. After re-centrifugation (Sorvall GSA, 30 min. At 13000 U / min, 27000 g, 4 ° C), the pellet was taken up in 1.3 1 0.1 mol / 1 Tris / HCl (pH 6.5), 20 mmol. / 1 EDTA and 0.5% Triton-x and homogenized. Alternative centrifugation was performed three more times (Sorvall GSA, 30 min at 13000 rpm, 27000 g 4 ° C) and homogenized pellets in 1 1 0.1 mol / 1 Tris / HCl (pH 6.5) and 20 mmol / 1 EDTA.

The content of t-PA in refractile bodies was quantified by SDS-PAGE, identification of t-PA bands by Western blotting and densitometric analysis. Refractile bodies show a strong t-PA band with a molecular weight of about 60 kDa in SDS-PAGE and Western blotting. The proportion of t-PA in the total protein content of refractile bodies is about 21%.

1.2

b) Solubilization / reduction of refractile bodies ”

Refractile bodies with a protein concentration of 1 to 5 mg / ml were incubated in 0.1 mol / 1 Tris / HCl (pH 8.6), 6 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. We then centrifuged the insoluble material (cell wall fragments, etc.) (e.g.

Sorvall SS 34, 30 minutes at 15000 to 20000 rpm, 35000 to 50000 g, 4 ° C). The pH of the top layer (supernatant) was adjusted to pH 3 with concentrated hydrochloric acid. · The denaturing and reducing agent was then dialyzed against 0.01 mol / l HCl at 4 ° C.

c) Reoxidation / activation

Reoxidation / activation expired from 1:50 to 1: 200 dilution in 0.1 mol / 1 Tris / HCl (pH 10.5), 1 mmol / 1 EDTA, mg / ml BSA, 0.5 mol / 1 L- arginine, 2 mmol / 1 GSH, 0.2 mmol / 1 GSSG. 1 each? up to 24 hours of activation at about 20 ° C, activity was determined and yield was determined relative to that of native glycosylated t-PA from eukaryotes.

Yield based on total protein content in refractile bodies ”: 2.5 +/- 0.5% stimulation: 10 +/- 5

Yield with respect to the proportion of t-PA in refractile bodies:

Ca. 12%.

d) Reoxidation / activation without decision of denaturing / reducing agent

Refractile bodies were 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. Subsequently, reoxidation was immediately initiated with a 1: 100 dilution in 0.1 mol / 1 Tris / HCl (pH 10.5), 1 rnmol / 1 EDTA, 1 mg / ml BSA, 0.3 mol / 1 L-arginine, and the quantities of GSSGs listed in the table. In addition, a residual concentration of 0.06 mol / 1 guanidine hydrochloride and 2 mmol / 1 DTE was located in the activation extension.

Dependence of activation gain on GSSG concentration on activation without denaturing / reducing agent.

GSSG (mmol / l)

Yield Stimulation (%) (Factor)

0.2 0 1 0.13 5 1.49 6 1,28 7 1.04 9 0.98 10 1.77 15 0 20 0

4.0

1.4

5.4 5.8 5.2

10,0 '= yield of active t-PA with respect to the total protein content of the refractile bodies.

EXAMPLE 2

Preparation of RB (refractile bodies) (450 0D _ccn / ml) at u was incubated in 1 ml of 0.1 mol / 1 Tris / HCl (pH = 8.6), moles / 1 of guanidine hydrochloride and 0.15 to 0. 2 mol / 1 DTE for 2 to 3 hours at room temperature. The insoluble material (cell wall fragments, etc.) was then determined by centrifugation (20 minutes at 17,000 rpm). The denaturing and reducing agent was removed by gel filtration via Sephadex C 25 (superfin) in 0.01 mol / l HCl. The sample was diluted by a factor of about 5 to 10. The reduced material in 0.01 mol / l HCl was stored at -20 ° C.

EXAMPLE 3

The following tables summarize the effect of various parameters of the invention on the activation and stimulation of t-PA. For these reoxidation experiments, however, solubilized, reduced protein was not further purified by Example 1.

The reduced protein (in 0.01 mol HCI) was activated in reoxidation buffer by dilution to 1:10 to 1: 500. Activation was determined after 22 to 48 hours of incubation at room temperature. Reoxidated protein activity refers to standard reoxidation (= 100%) 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 (reduced glutathione) + 0.5 mmol / l GSSG (glutathione disulfide).

l '-. t

Stimulation is calculated from E + cugppsp ^ .cNBrFSP (cf. W. Nieuwenhuizen et al, Biochimica et Biophysica Acta 755 (1983) 531 to 533). Activity (percentage) and stimulation (factor) were determined according to J. H. Verheijen Thromb. Haemostas. 48 (3), 266-269, (1982).

We obtained the following results:

1. Activation efficiency dependence upon the addition of L-arginine, guanidine hydrochloride

Reoxidation in 0.1 mol / 1 Tris / HCl (pH 10.5) + 1 mmol / 1 EDTA + 1 mg / ml BSA + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG

a) L-arginine

L-arginine) Activity Stimulation (mol / 1) (%) (Factor)

0 4 2.5 0.25 98 7.5 0.5 100 21.9 0.75 27 16.3 1.0 23 3.5

In this experiment, it should be noted that t-PA is inhibited by L-arginine. Therefore, the decrease in activation gain should be corrected at higher concentrations of L-arginine with respect to inhibition.

b) Guanidine hydrochloride (Gdn.HCl)

(Gdn'HCl, (mol / 1) Activity (%) 0 11 0.25 22 0.5 53 0.75 58 1.0 12 Dependence of activation efficiency from the supplement urea and urea derivatives. Reoxidation in 0.1 mol / l Tris (pH 10.5), 1 mmol / l EDTA, 1 mg / ml BSA, 5 mmol / 1 GSH, 0.2 mmol / l GSSG a) Urea Urea Activity (mol / 1) (%) 0 1 0.5 20 1 59 1.5 126 2 162 2.5 141 3 72 4 12 5 0

b) Methylurea

Methylurea Activity (mol / 1) (%)

0.5 22 1 174 1.5 313 2 375 2.5 332 3 215 4 12 5 0 _c ) Ethylurea Ethylurea Activity (moi / 1) (%) 0.5 46 1 212 1.5 323 2 300 2.5 107 3 19 4 0 5 0

d) Dimethylurea Dimethylurea (mol / 1) Activity (%) Stimulation (Factor) 0.5 167 8.8 1 256 8.9 1.5 283 9,4 2 177 7.7 2.5 78 8.9 3 23 9.9 4 4 8.6 5 2 3.5

Dependence of activation efficiency 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.

a) Formamide

Formamide Activity

(mol / 1) (%) 0 42 0.5 59 1 175 1.5 245 2 325 2.5 423 3 444 4 416 5 341

b) Methylformamide Methylformamide (mol / 1) Activity (%) 0.5 100 1 135 1.5 304 2 389 2.5 466 3 452 4 425 5 121

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

d) Propionamide

Propionamide (mol / 1) Activity (%) 0.5 95 1 99 1.5 197 2 150 2.5 101 3 39 4 2 5 0

e) Butyramide Butyramide (mol / 1) Activity (%) 0.5 55 1 52 1.5 17 2 0

Dependence of activation efficiency 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 + 0.5 mmol / 1 GSSG

_P H Activity (%) Stimulation (Factor) 7 1 8 22 3.0 9 89 13.6 10 105 20.3 11 95 21.3

5, Dependence of activation yield on GSH / GSSG concentration

Reoxidation in water, 1 mol / 1 Tris / HCl, pH 10,5, + 1 mmol / 1 EDTA + 0,5 mol / 1 L- ^ar S ^inine + 1 mg / ml BSA

a) + 1 mmol / 1 GSH

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

b) + 0.2 mmol / l GSSG

(GSH) (mmol / l) Activity (%) Stimulation (Factor) 0.05 15 2.2 0.1 40 3.8 0.2 112 6,8 0.5 142 7.4 1 273 6,8 5 260 7.9 10 143 6,3 20 55 5.1

Dependence of activation efficiency on protein concentration during reoxidation (dilution 1:20 - 1: 500)

Reoxidation in 0.1 mol / 1 Tris / HCl (pH 10.5) + 1 mmol / l EDTA + 0.5 mol / 1 L-arginine + 1 mg / ml BSA +0.5 mmol / l GSH + 0, 5 mmol / l GSSG

Dilution Activity (%) Stimulation (Factor) 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 1: 500 29 28.7

Dependence of activation yield on BSA supplementation

Reoxidation in 0.1 mol / 1 Tris / HCl (pH 10.5) + 1 mmol / 1 EDTA + 0.5 mol L + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG

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

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

EXAMPLE 4

Activation of t-PA via mixed t-PA and GSSG disulfides

The refractile bodies used were obtained from one of the preceding examples. Reduction

- 23 refractile bodies were performed after 2 hours of incubation at room temperature in 0.1 mol / 1 Tris / HCl, pH 8.6, 1 mmol / 1 EDTA, 6 mol / 1 Gdn / HCl, 0.2 mol / 1 DTE at a protein concentration of about 1 mg / ml.

The reduced protein dialyzed against 0.01 mol / 1 HCl was diluted 1: 1 with 0.1 mol / 1 Tris, pH 9.3, mol / 1 urea and 0.2 mol / 1 GSSG and incubated for 5 hours at room temperature.

Acidification with concentrated HCl at pH 3 was followed by dialysis against 0.1 mol / l HCl at 4 ° C. Following dialysis, the total protein concentration was 0.33 mg / ml. With t-PASSG prepared in this way, optimal reactivation conditions were determined.

a) pH-optimum activation of t-PASSG

In this case, as well as in the following optimization experiments, (1) GSSG was not used and (2) activation was determined after 17 hours of incubation at room temperature. Activation expired with a 1: 100 dilution in 0.1 mol / 1 Tris, mmol / 1 EDTA, 0.5 mol / 1 L-arginine, 1 mg / ml BSA and 2 mmol / 1 GSH at variation in pH.

pH Yield (%) Stimulation 6 0.04 3.3 6.5 0.37 9.5 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 9.5 8,32 11.7 10 6.15 12.5 10.5 3.07 11.2

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

b) Reproducibility of activation results from t-PASSG Under identical activation conditions different gains are observed for different measured species, which are conditioned, among other things, by the fluctuation of standard t-PA. To explain this error width, we have all the activation data at 1: 100 oz. 1: 200 dilutions were collected in 0.1 mol / 1 Tris / HCl, pH 8.5, mraol / 1 EDTA, 0.5 mol / 1 L-arginine, 1 mg / ml BSA and mmol / 1 GSH.

Try it Profit (%) Stimulation 1 8.65 7.0 2 4,47 9.3 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 8 3.54 13,4 9 5.07 16.4 Medium value 5.1 +/- 1.9 11.3 +/- 3.8

c) Stability of activated protein

Activation expired in the above cases with a 1: 200 dilution in 0.1 mol / 1 Tris / HCl, 1 mmol / 1 EDTA,

0.5 mol / 1 L-arginine, 1 mg / ml BSA and 2 mmol / 1 GSH.

EXAMPLE 5

Activation of a genetically engineered interferon- ^.

Refractile bodies were manufactured using the above methods. The reduction / solubilization of the refractile bodies was performed as follows: the pellet was incubated for 3 hours at 25 ° C in 10 ml of 9.1 mol Tris / HCl, pH 8.6, 6 mol / 1 Gdn / HCl, mmol / 1 EDTA and 0 , 2 mol / 1 DTE, and after 3θ minutes of centrifugation at 4 ° C and 48000 g, the pH of the top layer (supernatant) was adjusted to about 3 with concentrated HCl. Subsequently, gel filtration was performed via Sephadex G 25 F in 0.01 mol / l

HCI.

The eluate was controlled by transmission (280 nm) for conductivity, protein concentration and reactivity.

Standard activation (100%) was performed 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 / 1 L-arginine.

a) Dependence on activation

The eluate was diluted 1:50 in 0.1 mol / 1 Tris / HCl, pH 8.5, .1.mol / 1 EDTA, 5 mmol / 1 GSH, 0.5 mmol / 1 GSSG and 0.25 mol / 1 L-arginine.

Activation time (h)

Activity

16 ° C

b) The dependence of the activation gain on the addition of L-arginine Eluate was diluted 1:50 with 0.1 mol / 1 Tris / HCl, pH 8.5, 1 mmol / 1 EDTA, 5 ramol / 1 GSH, 0.5 mmol / 1 GSSG and activated at 0 ° C for 20 hours.

Activation dependence on L-arginine

L-arginine (M) Activity (%)

8

0.25 8

0.5 15

0.75 15

c) Dependence of activation gain on urea supplementation

The activation solution corresponded to that of point b), but was activated for 17 hours at 0 ° C.

Activation dependence on urea

Urea (M) Activity (%)

0 13 0.5 100 1 200 1 »5 100

d) Dependence of activation gain on formamide supplementation Activation as in b); the samples were monitored after 17 hours of activation at 0 ° C.

Formamide activation dependence

Formamide (M)

Activity

e) Dependence of activation gain on redox buffer

The eluate was diluted 1:50 in 0.1 M Tris / HCl, pH 8.5 mM EDTA and 0.25 M L-arginine, and samples were controlled after 17 hours of activation at 0 ° C.

Activation dependence on GSH / GSSG

GSH (mM) GSSG (mM) Activity (%)

0.5 6

0.5 13

0.5 25

0.5 25

0.1 13

0.5 13

1.0 13

5 6

- 28 f) Dependence of activation gain on Eluate protein concentration was diluted 1:10 to 1: 100 in 0.1 M Tris / HCl, pH 8.5 1 mM EDTA, 5 mM GSH, 0.5 mM GSSG and 0.25 M L-arginine and counterbalanced after 17 hours of activation at 0 ° C. cp-activation-dependent cp (mg / ml) Activity (%)

0.018 13

0,036 13

0,072 13

0.108 8

0.180 10

g) Dependence of activation gain on BSA supplementation

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

BSA-dependent activation

BSA (mg / ml) Activity (%)

13

13

25

13

- 29 h) Dependence of activation gain on pH

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

pH-dependent activation of pH Activity (%)

6.5 0 7.5 6 8.5 13 9.5 50 10.5 100

Claims (23)

1. A method for activating gene-technologically prepared heterologous, eukaryotic proteins containing disulfide bridges after expression in prokaryotes by cell digestion, solubilizing under denaturing and reducing conditions, and activating under oxidizing conditions in the presence of GSH / GSSG, labeled with GSH / GSSG partly with 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 / l and a non-denaturing concentration of the denaturing agent. Method according to claim 1, characterized in that at the activation stage the pH is 9.5 to 11. Method according to one of Claims 1 and 2, characterized in that in the activation stage the GSH concentration is 0.2 to 10 mmol / l and / or the GSSG concentration is 0.05 to 1 mmol / l. Method according to one of the above claims, characterized in that a purification step is carried out after solubilization and before activation. Method according to one of Claims 1 to 3, characterized in that activation is carried out without prior determination of the denaturing / reducing agent, diluting the reaction solution after denaturing / reduction with activating buffer and exceeding the residual residual DTE concentration on subsequent activation. 6. Process variant for the activation of genetically engineered, heterologous, eukaryotic proteins exhibiting disulfide bridges after expression in prokaryotes with cellular digestion, solubilization under denaturing and reducing conditions, and activation under oxidizing conditions in the presence of GSH, characterized in that denaturing agent is decided, with the addition of GSSG under denaturing conditions, the thiol groups of proteins are converted into mixed disulfides of protein and glutathione, at the activation rate we operate at pH values of 7 to 10.5 and a GSH concentration of 0.5 to 5 mmol / l and at a non-denaturing concentration denaturing agent. A method according to any one of claims 1 to 6, characterized in that expression is carried out in E. coli or p. putida. Process according to one of Claims 1 to 7, characterized in that, in the activation step, arginine, guanidine hydrochloride and / or at least one compound of the general formula R ^CO-NRR ^ (I) is used as denaturing agent, in which R and R ₁ is hydrogen or alkyl of 1 to 4 carbon atoms, and R ₂ is hydrogen or NHR ₁ or alkyl of 1 to 3 carbon atoms. A process according to claim 8, 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. 32 A process according to claim 8, characterized in that the concentration of the compound of general formula I is 0.5 to 4 mol / l, in particular 1 to 3.5 mol / l. Method according to one of the preceding claims, characterized in that we operate at the rate of activation in the presence of a non-proteolytically effective protein, in particular in the presence of bovine serum albumin. Method according to one of the preceding claims, characterized in that the cell digestion is performed by means of ultra-sound, high-pressure dispersion or lysozyme. 13 The method of claim 12, characterized in that it is digested in dilute aqueous buffer solution, in particular in 0.1 mol / l Tris, at a neutral to slightly acidic pH. Process according to one of the preceding claims, characterized in that insoluble constituents are selected after cell digestion. Process according to one of the preceding claims, characterized in that, at the solubilization step, it is operated at an alkaline pH in the presence of a reducing agent from the mercapto group and in the presence of a denaturing agent. A process according to claim 15, characterized in that it is acted in the presence of guanidine hydrochloride and / or a compound of general formula I as a denaturing agent. - 33 Process according to claim 16, characterized in that the guanidine hydrochloride concentration is 6 mol / l and the concentration of the compound of general formula I is 8 mol / l. Process according to one of Claims 15 to 17, characterized in that it operates in the presence of DTE, N-mercaptoethanol or GSH. Method according to one of the preceding claims, characterized in that the purification and decontamination of reducing, oxidizing or denaturing agents is carried out by steric separation chromatography or dialysis. Method according to one of the preceding claims, characterized in that after the activation step, a cleaning step is carried out by dialysis. Process according to one of Claims 1 to 20, characterized in that t-PA is used as a genetically engineered eukaryotic protein. Method according to one of Claims 1 to 20, characterized in that the interferon - / - is used as a genetically engineered eukaryotic protein. Process according to claim 6, characterized in that the mixed disulfide of protein and glutathione is removed by ion-exchange treatment from unmodified protein.