SK278317B6 - Activation method of by genetic technology gained heterologous proteins with content of disulfidic bridges of eukaryotic origin after expression in prokaryotic organisms - Google Patents

Abstract

This patent describes a procedure dealing with an activation of the heterologous proteins, gained with the use of the genetic technology, containing the disulphide bridges of the eukariotic origin, after it has been expressed prokaryotic organisms, when a breakage of cells with the use of the solubilization is completed and the subsequent denaturation and reduction is provided, while the oxidation conditions are kept and the substance called CSH/GSSG is present, however this activation is provided in the environment with a pH value of 9 up to 12 and the concentration of the GSH substance is 0,1 up to 20 mmol/l, the concentration of the CSSG is 0,01 up to 3 mmol/l and the concentration of the denaturation means is of the such level, that no protein denaturation is observed.

Description

Technical field

The invention relates to a method of activation by genetically engineered heterologous proteins containing disulphide bridges of eukaryotic origin after expression in prokaryotic organisms.

BACKGROUND OF THE INVENTION

When expressing heterologous proteins in procarvotic organisms, these proteins often form inactive, sparingly soluble aggregates in the target cells. refractile bodics, which are, in addition, contaminated with proteins, inherent in the target cells. It is likely that the formation of these bodies is due to the high protein concentration in the cell when expressed. It is known that when large amounts of enzymes are formed in a cell, this condition in the cell results in the aggregation of the enzymes into insoluble, high molecular weight, and largely inactive particles. Before these proteins can be used, for example, for treatment, they need to be purified and converted from an inactive form to an active form.

According to known procedures, activation can be achieved in different ways in several stages, as described, for example, in B. R. Jaenicke, FEBS Federation of European Biochemical Societies, Vol. 52, (1979), 187-198; R. Rudolph, Biochemistry 18, (1979), 5572-5575.

In the first step, the solubility of the proteins is achieved by adding a strong denatured agent, for example guanidine hydrochloride or urea in high concentration, or by adding strongly acidic agents, for example a mixture of glycine and phosphoric acid. Possible auxiliaries are reagents which contain SH- groups, for example dithioerythritol or EDTA, in particular in the renaturation of LDH. Once the protein is contaminated with the target cell proteins, it is necessary to purify in the next step by conventional means, for example by gel or ion exchange chromatography. Thereafter, the material obtained is strongly diluted to reduce the concentration of the denaturing agent. When using guanidine hydrochloride, the sample should be diluted to less than 0.5 mol / l. In the case of enzymes having free SH-groups, it is expedient to add an agent protecting these groups, as described, for example, in B. R. Jaenicke, Joumal Polymer Science, Vol. C 16 (1967), 2143-2160.

In European patent application no. 114,506 describes a procedure for isolating, purifying, and activating a heterologous expression product from bacterial cultures. For activation, solutions of refractive bodies in a strong denaturing agent are used which are a) directly converted into solutions in a weaker denaturing agent and then oxidized to ensure disulfide bridging, b) the protein is subjected to sulfonation, then transferred to a a solution in a weak denaturing agent and an S-sulfonate group is converted to a reduced and oxidized form, for example by treatment with 60 GSH / GSSG to form -SS- groups, or c) the solution in a weak denaturing agent is directly treated with a sulfhydryl-type reagent such as GSH / GSSG. A typical example of such problems is t-PA. 65

The major component of the blood protein matrix is fibrin polymers. This protein is dissolved by the action of a plasmid, which is formed from plasminogen by activation of so-called. plasminogen activators, for example by treatment with t-PA (tissue-type plasminogen activator). In the absence of fibrin or fibrin cleavage products (PSF), the enzymatic efficacy of natural or genetic technology from a eukaryotic organism obtained by t-PA is very low, but it can be increased by more than 10-fold by adding said stimulators. This so-called. potency stimulation is a great advantage of t-PA over other known plasminogen activators, such as urokinase or streptokinase, as described, for example, in N. Hoylaerts et al., J. Biol. Chem. 257, (1982), 2912-2919, Nieuwenhiuzen et al., Biochemica et Biophysica Acta 755, (1983), 531-533. The BrCN-type cleavage stimulation factor is reported in the literature and has a value of up to 35.

In principle, it is possible to produce a non-glycosylated t-PA product also in prokaryotic organisms by genetic manipulation using c-DNA. However, when using this product, there is no possibility of stimulating efficacy as with t-PA from eukaryotic organisms. This is probably due to the fact that different conditions, especially reduction and oxidation, differ so much in the prokaryotic cell and in the eukaryotic cell from which the gene originates that from the outset the ineffective product is formed, probably due to the poor formation of SS bridges contained in the natural molecule or these bridges are not formed at all or are bound in an improper manner. However, the therapeutic use of t-PA requires not only the enzymatic activity of t-PA, but also the separate enzymatic stimulatability of the product. The fact that the cells of prokaryotic organisms usually do not produce active proteins of eukaryotic origin is also noted, for example, in The Joumal 4, no. 3 (1985), 755-780.

In European patent application no. 93 639, upon activation of t-PA cell pellets obtained from E. coli, were suspended in guanidine hydrochloride at a concentration of 6 mol / l, disrupted by ultrasound, the resulting material was incubated, and then dialyzed for 4 hours against a pH solution of tris-HCl 8.0 in admixture with sodium chloride, ethylenediaminetetraacetic acid and Tween 80. After dialysis, the material is centrifuged to detect plasminogen activator in the supernatant. The t-PA obtained in this way is proteolytically active, and cannot be stimulated by cleavage products after treatment with bromine cyanide on fibrin (BrCN-PSP) as described by JH Verheijen, Thromb Haemostas, 48, (3), 260-269, (1982).

No generally applicable procedure is described in the literature for the activation of denatured proteins. This is especially true for t-PA, since natural protein has a very complex structure. It contains a free thiol group and 17 SS bridges, which are theoretically linked by 2.2 x 10 ²⁰ methods, only one of which structures corresponds to the natural state. Methods for activating t-PA, which are known in the literature, lead to obtaining a protcolytically active t-PA which, however, has no measurable stimulatability. No activation procedure is known to result in a stimulatable t-PA.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for the complete activation of heterologous proteins obtained by genetic technology containing disulfide bridges of eukaryotic origin after expression in prokaryotic organisms.

Accordingly, the present invention provides a method of activating genetically engineered heterologous proteins containing disulphide bridges of eukaryotic origin after expression in prokaryotic organisms by cell disruption, solubilization by denaturation and reducing conditions, and activation (renaturation) under oxidative conditions in the presence of GSH / GSSG. The process according to claim 1, wherein the activation is carried out at a pH of 9-12, a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol / l, and in the presence of a denaturing agent in a concentration at which no denaturation occurs.

Preferred embodiments of the process of the invention will be described below.

Among the denaturing agents, agents commonly used for activation under oxidative conditions or arginine may be used in the process of the invention. A preferred denaturing agent is guanidine hydrochloride, urea or derivatives thereof. Another suitable agent is arginine. It is also possible to use mixtures of the denaturing agents mentioned. Preferably, the activation step is carried out in the presence of a foreign protein, which is preferably any protein that is not proteolytically active, preferably bovine serum albumin (BSA), for example in an amount of 1 to 3 mg / ml. The addition of BSA results in a small increase in yield and at the same time protein stabilization (presumably protects against surface denaturation and / or proteolytic degradation).

Further conditions of the process according to the invention correspond to the reactivation conditions known from the literature. Activation (incubation) preferably takes 20 to 48 hours at room temperature. The activation half-life in the presence of 0.5 mmol / l reduced GSH and oxidized GSSG glutathione is in the range of about 10-15 hours at 20 ° C. Upon further incubation, for example 48 hours under reoxidation conditions, the stimulant usually decreases due to CNBr-FSP. The activation step is preferably carried out in the presence of ethylenediaminetetraacetic acid, preferably at a concentration of 1 mmol / l.

Traces of the activation process of reoxidation and activation, or previous and subsequent steps, for example cell disruption, dissolution and optionally one or more steps of activation and / or subsequent purification, may be carried out according to the prior art as described, for example, in European patent applications no. 114 586 and 93 619. However, in view of the high yields and good activation, it is expedient to carry out individual steps or all steps with respect to the described process conditions. In particular, it is possible to carry out the steps of the process according to the invention consisting in activating the mixture obtained by disrupting the cells without prior denaturation and / or reduction, the yields being rather low. Prokaryotic organisms, preferably P. putida and especially E. coli, are used for expression, but the method of the invention may also be carried out in other prokaryotic organisms, such as Bacilli, in which expression of the desired protein is also achievable.

Cell disruption can be accomplished by known methods, for example, by sonication, dispersion at elevated pressure, or lysozyme. The suspension medium preferably comprises a buffer solution which allows a neutral to slightly acidic pH, for example 0.1 mol / l tris-HCl, to be adjusted. After cell disruption, insoluble components of these cells, so-called. the refractive bodies are separated in any manner, preferably by vigorous centrifugation for a longer period of centrifugation or filtration. After washing with substances that do not destroy t-PA but, if possible, dissolve cellular proteins such as water or phosphate buffer, optionally containing mild surfactants such as tritone, the precipitate or pellet is solubilized under reducing conditions. The solubilization is carried out at an alkaline pH, in particular at a pH of 8.6 ± 0.4 in the presence of a mercaptans reducing agent and a denaturing agent.

As the denaturing agent, reagents known from the literature can be used, for example from European patent application no. 114 506 for solubilization, in particular guanidine hydrochloride or urea. Guanidine hydrochloride is preferably used at a concentration of 6 mol / l, urea at a concentration of 8 mol / l. Compounds of formula (I) may also be used equally well.

IVCO-NRR ¹ (I) wherein

R 1 and R ¹ are hydrogen or C 1 -C 4 alkyl

R ² represents a hydrogen atom, an NHR ¹ radical or an alkylated radical having 1 to 3 carbon atoms.

Reducing GSH or mercaptoethanol, for example at a concentration of 50 to 400 mmol / l of dithiothreitone and / or dithioerythritol (DTE) or dithiothreitol (DTT), for example at a concentration of 80 to 400 mmol / l, can be used as mercaptan reducing agents. The solubilization is preferably carried out at room temperature for one or more hours, preferably two hours. In order to prevent oxidant oxidation of the reducing agent, it is expedient to add EDTA to the reaction mixture. In addition to solubilization and reduction, this step also has a purifying effect, since most of the material that is not immunologically cross-linked with t-PA, especially foreign proteins, does not pass into solution.

After solubilization and prior to activation, conventional cleaning steps may be employed. Suitable purification processes are, for example, steric chromatography (SEC) in the presence of guanidine hydrochloride or urea, or an ion exchanger in the presence of urea or a derivative thereof. Non-specific reoxidation can also be used, which can be prevented, for example, by 2-mercaptanol or by adjusting the pH to 4.5 or less as described by R. Rudolph, Biochem, Soc. Transactions 13 (1985), 308-311. Once the solubilization step is complete, any DTE used may need to be removed in the purification step. The purification can be carried out on a Sephadex G 100 gel in the presence of guanidine hydrochloride and a reducing agent such as GSH glutathione at pH 1-4, in which way a large amount of foreign protein can be separated or a denaturing and reducing agent can be separated by removing salts on Sephadex G 25 gel. 0.01 mol / l hydrochloric acid or 0.1 mol / l acetic acid. The denaturing / reducing agent can also be separated by dialysis.

The next purification step may follow the reactivation step. This purification is generally carried out by dialysis or isolation of activated t-PA, for example by affinity chromatography, for example on Lys-Sepharex.

A further embodiment of the method according to the invention consists in the formation of mixed disulfides by genetically engineered, heterologous, disulfide bridges containing eukaryotic proteins and glutathione (hereinafter t-PASSG). This procedure can be carried out by separating the foreign protein in the denatured state or by further purification of the natural protein. The tanks after modification of the thiol groups have the advantage that the protein is protected against oxidation by the action of air and is therefore stable over a wide pH range and at the same time facilitates the entire purification due to the change in charge. The isolation of the unmodified protein is preferably carried out by treatment with an ion exchanger.

For the formation of mixed disulfides, dialyzed, reduced, denaturing and reducing agents purified protein is incubated with a solution containing a denaturing agent, for example 0.2 M GSSG solution. Activation can be carried out after separation of the denaturing and oxidizing agent at a pH of 7 to 10.5, at a GSH concentration of 0.5 to 5 mmoles and at a denaturing agent concentration which is not yet denatured.

For all other reaction steps, protein activation corresponds to the formation of mixed GSSG disulfides under the same conditions as in the above procedure. In this embodiment, the pH is optimally 8.5, the yield is about twice and the activated protein is stable in the renaturation buffer for a longer period of time.

Thus, by the method of the invention, it is possible to activate t-PA from prokaryotic organisms so that not only does activation for the usual biological activity be achieved, but additionally stimulates the above-mentioned stimulus in an amount far beyond that of t-PA at a factor greater than 10, sometimes 50.

Another protein from eukaryotic organisms that can be activated by the method of the invention after expression in prokaryotic organisms is β-interferon.

The practice of the process according to the invention will be explained in the following examples. Unless otherwise indicated, percentages refer to weight percent and temperature data to degrees Celsius.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. Figures 1 and 2 show the efficacy with and without the addition of CNBr-FSP in a standard experiment after 17 hours of reoxidation at room temperature in a mixture of 0.1 mol / l tris-HCl pH 10.5 + 1.0 mmol / l EDTA + 0 5 mol / l 1-arginine + 1 mg / ml BSA + 0.5 mmol / l GSH + + 0.5 mmol / l GSSG. In FIG. 1 and 2, curve A is the potency of the presence of CNBr-FSP and curve B is the potency without CNBr-FSP.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

a) Preparation of refractive bodies

100 g of E. coli wet cell mass in 1.5 L of 0.1 mol / l, pH 6.5 and 20 mmol / l EDTA are homogenized on an Ultra-Turrax for 10 seconds, then 0 , 25 mg / ml lysozyme. After incubation for 30 minutes at room temperature, the mixture is again homogenized and cooled to 3 ° C. The cells are disrupted by dispersion at a high pressure of 550 kg / cm ² . Then 300 ml of 0.1 mol / l tris-HCl at pH 6.5 and 20 mmol / l EDTA are added. After centrifugation 5 (Sorvall GSA, 2 hours at 13,000 rpm),

21000 xg, 4 ° C) is mixed with 1.2 L of 0.1 M Tris-HCl at pH 6.5, 20 mmol / L EDTA and 2.5% Triton-X-100, and then is homogenized. Centrifuge the material (Sorvall GSA, 30 minutes at 10 13,000 rpm, 27,000 xg, 4 ° C) and mix the pellet with 1.3 L of 0.1 mol / l tris-HCl, pH 6.5. and 20 mmol / l EDTA and 0.5% Triton-X-100 and the material is homogenized. The material is then centrifuged once more (Sorvall GSA, 30 minutes at 13000 rpm, 15 27 000 xg, 4 ° C) and the pellets are homogenized in 1 liter of 0.1 mol / l tris-HCl at pH 6, 5 and 20 mmol / l EDTA, this centrifugation is performed three times.

The t-PA content of the refractive bodies in the resulting material is determined by SDS-PAGE, the p-20s containing t-PA are identified by West blotting and quantitatively determined by densitometry. Using this method, it is possible to detect a strong band containing t-PA with a molecular weight of about 60 k units. The proportion of t-PA in total protein of refractory bodies is approximately 21%.

b) Solubilization / reduction of refractive bodies

Refractive bodies with a protein concentration of 1 to mg / ml are incubated in 0.1 mol / l tris-HCl at pH 8.6, 6 mol / l guanidine hydrochloride, 0.15 to 0.4 mol / l DTE and 1 mmol / l. EDTA for 2-3 hours at room temperature. Thereafter, insoluble material such as cell fragments and the like are separated by centrifugation (e.g. Sorvall SS 34, 30 minutes at 4 ° C, 15,000-20,000 rpm and 35,000-50,000 xg). The pH of the supernatant is adjusted to pH 3 with concentrated hydrochloric acid. The denaturing and reducing agents are separated by dialysis against 0.01 mol / l hydrochloric acid at a temperature of ⁴⁰ te 4 ° C.

c) Reoxidation / activation

Reoxidation / activation is performed at a 1:50 dilution of 45 to 1: 200 in a solution containing 0.1 mol / l tris-HCl at pH 10.5, 1 mmol / l EDTA, 1 mg / ml BSA, 0.5 mol / L of 1-arginine, 2 mmol / L of GSH and 0.2 mmol / L of GSSG. After 17 to 24 hours of activation at about 20 ° C, the efficacy is compared to that of native 50 glycosylated t-PA from eukaryotic organisms and the yield of this substance. The yield based on the total protein content of the refractive bodies is in the range of: 2.5 ± 0.5%, the stimulatability is 10 ± 5.

The yield based on the proportion of t-PA in the refractive bodies is approximately 12%.

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

Refractive bodies are incubated at a protein concentration of 1.25 mg / ml in a mixture containing 0.1 mol / l tris-HCl at pH 8.6, 6 mol / l guanidine hydrochloride, 0.2 mol / l DTE and 1 mmol / ml. 1 EDTA at room temperature for 2 hours. Immediately thereafter, reo65 xidation is performed at a 1: 100 dilution in a solution containing 0.1 mol / l tris-HCl, pH 10.5, 1 mmol / l EDTA, 1 mg / ml

BSA, 0.3 mol / 11-arginine and the amount of GSSG shown in the following table. In addition, a residual concentration of 0.06 mol / l of guanidine hydrochloride and a DTE of 2 mmol / l is determined in the activation mixture.

Dependence of activation yield on GSSG concentration in activation without separation of denaturing / reducing agent

GSSG (mmol / L) yield (%) stimulatability (Factor) 0.2 0 1 0.13 4.0 5 1.49 1.4 6 1.28 5.4 7 1.04 5.8 9 0.98 5.2 10 1.77 10.0 15 0 20 0 -

Line yield - yield of effective t-PA based on total protein content of refractive bodies.

Example 2

Sample refractive. The body (450 ODwo / ml) is incubated in a mixture containing 1 ml of 0.1 mol / l tris-HCl, pH 8.6, 6 mol / l guanidine hydrochloride and 0.15 to 0.2 mol / l DTE, 2 to 3 mol / l. 3 hours at room temperature. The insoluble material, for example, cell fragments, is then separated by centrifugation at 17,000 rpm for 20 minutes. The denaturing and reducing agent is then removed by filtration on a Sephadex G 25 gel (competing) in 0.01 M HCl. The sample is then diluted 5 to 10 times. The reduced material is stored at -20 ° C in 0.01 M HCl.

Example 3

The following tables summarize the effect of various parameters of the method of the invention on t-PA activation and stimulatability. In these reoxidation experiments, the solubilized, reduced protein was used without further purification.

The reduced protein in 0.01 mol HCl was activated by diluting 1:10 to 1: 500 in reoxidation buffer. Activation was detected at room temperature after incubation for 22 to 48 hours at the same temperature. The efficacy of reoxidated protein refers to standard reoxidation, which is considered 100% in a solution containing

0.1 mol / l tris-HCl with pH 10.5 + 1 mmol / l EDTA + 0.5 mol / 11-arginine + 1.0 mg / ml BSA + 0.5 mmol / l GSH (reduced glutathione) + 0.5 mmol / l GSSG (glutathione disulfide).

Stimulability is calculated as E + CNBrFSP / E-CNBrFSP by the method described by W. Nieuwenhuizon et al., Biochemica et Biophysica Acta 755 (1983) 531-533. The% potency and the stimulatability as a factor are determined according to the method of J. N. Verheijen Thromb. Hacmostas. 48 (3), 266-269 (1982).

The results:

1. Dependence of the activation yield on the additions of 1-arginine guanidine hydrochloride

The reaction is carried out in a mixture

0.1 mol / l tris-HCl with pH 10.5 + 1.0 mmol / l EDTA + 1.0 mg / ml BSA + 0.5 mmol / l GS + 0.5 mmol / l GSSG

a) 1-arginine

1-arginine (mol / l) effectiveness (%) stimulatability (Factor) 0 4 2.5 0.25 98 7.5 0.5 100 21.9 0.5 27 16.3 1.0 23 3.5

In this experiment, it should be considered that 1-arginine causes t-PA inhibition. Thus, the decrease in the activation yield at a higher concentration of this compound must be added to the inhibition.

(b) Guanidine hydrochloride (Gdn.HCl)

(Gdn.HCl) (mol / l) effectiveness (%) 0 11 0.25 22 0.5 53 0.75 58 1.0 12

2. Dependence of the yield of activation on the addition of urea or its derivatives

Reoxidation is carried out in a mixture of 0.1 mol / l tris-HCl, pH 10.5, 1.0 mmol / l EDTA, 1.0 mg / ml BSA, 5 mmol / l GSH, 0.2 mmol / l GSSG.

(a) Urea

urea (mol / 1) effectiveness (%) 0 1 0.5 20 1 59 1.5 126 2 162 2.5 141 3 72 4 12 5 0

(b) Methylurea

methylurea (Mol / 1) effectiveness (%) 0.5 22 1 174 1.5 313 2 375 2.5 332 3 215 4 12 5 0

(c) Ethylurea

ethyl urea (mol / 1) effectiveness (%) 0.5 46 1 212 1.5 323 2 300 2.5 107 3 19 4 0 5 0

c) Acetamide

acetamide (mol / l) effectiveness (%) 5 0.5 72 1 134 1.5 207 2 261 10 2.5 204 3 237 4 198 5 141

(d) Dimethylurea

Dimethylurea (mol / l) effectiveness (%) 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

d) Propionamide

propionamide (Mol / 1) effectiveness (%) 20 0.5 95 1 99 1.5 197 2 150 2.5 101 25 3 39 4 2 5 0

3. Dependence of the yield of activation on the addition of aliphatic acid amides

Reoxidation is carried out in a mixture of 0.1 M tris-HCl pH 10.5, 1.0 mmol / l EDTA, 1.0 mg / ml BSA, 5 mmol / l GSH, 0.2 mmol / l GSSG.

(a) Formamide

formamide (Mol / 1) effectiveness (%) 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 / l) effectiveness (%) 0.5 100 1 135 1.5 304 2 389 2.5 466 3 452 4 425 5 121

e) Butyramide

butyramide effectiveness (Mol / 1) (%) 0.5 55 1 52 1.5 17 2 0

4. Dependence of activation yield on pH

The reoxidation is carried out in a mixture

0.1 mol / l tris-HCl pH 10.5 + 1.0 mmol / l EDTA + 0.5 mol / 11-arginine +1.0 mg / ml BSA + 0.5 mmol / l GSH + 0, 5 mM GSSG.

50 pH effectiveness (%) stimulation (factor) 7 1 8 22 3.0 55 9 89 13.6 10 105 20.3 11 95 21.3

5. Dependence of activation yield on concentration

GSII / GSSG

The reoxidation is carried out in a mixture

0.1 mol / l tris-HCl pH 10.5 +1.0 mmol / l EDTA + 0.5 mol / 11-arginine +1.0 mg / ml BSA

a) + 1 mmol / L GSH

GSSG (mmol / l) effectiveness (%) 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 mmolA GSSG

GSH (mmol / l) effectiveness (%) 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

6. Dependence of activation yield on protein concentration during reoxidation (1:20 to 1: 500 dilution)

Reoxidation is carried out in a mixture of 0.1 mol / l tris-HCl pH 10.5 + 1.0 mmol / l EDTA + 1.0 mg / ml BSA + 0.5 mol / l l-arginine + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG.

dilution effectiveness (%) 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

7. Dependence of activation gain on BSA addition

The reoxidation is carried out in a mixture

0.1 mol / l tris-HCl with pH 10.5 + 1.0 mmol / l EDTA + 0.5 mol / 11-arginine + 0.5 mmol / l GSH + 0.5 mmol / l GSSG.

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

In FIG. Figures 1 and 2 show the efficacy with and without the addition of CNBr-FSP in a standard experiment after 17 hours of reoxidation at room temperature in a mixture of 0.1 mol / l tris-HCl pH 10.5 + 1.0 mmol / l EDTA + 0 5 mol / 11-arginine + 1 mg / ml BSA + 0.5 mmol / l GSH + + 0.5 mmol / l GSSG. In FIG. Ia represents curvature A efficiency of the presence of CNBr-FSP and curve B activity without CNBr-FSP.

Example 4

Activation of t-PA in the formation of mixtures of t-PA and GSSG disulfides

Refractive bodies are obtained by the method of any of the preceding examples. The reduction of these bodies is carried out for 2 hours at room temperature in a mixture containing 0.1 mol / l tris-HCl at pH 8.6 + 1.0 mmol / l EDTA, 6 mol / l Gdn / HCl and 0.2 molA DTE at a protein concentration of about 1 mg / ml.

Protein reduced and dialyzed against 0.01 mol / l HCl is diluted 1: 1 with 0.1 mol / l tris pH 9.3, 9 molA urea and 0.2 mol / l GSSG, and then incubated 5 hours at room temperature.

After acidification with concentrated hydrochloric acid to pH 3, the mixture is dialyzed against 0.1 M HCl at 4 ° C. After dialysis, the total protein concentration is 0.33 mg / ml. Using the thus obtained tPASSG, optimal reactivation conditions can be determined.

a) Optimal pH to activate t-PASSG

In the following description 1) no GSSG was used and 2) activation was determined after 17 hours incubation at room temperature. Activation takes place at a dilution of 1: 100 in a mixture containing 0.1 mol / tris, 1 mmol / l EDTA, 0.5 molA-arginine, 1 mg / ml BSA and 2 mmol / l GSH, simultaneously changing the pH.

PH yield (%) stimulatability 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 is determined in% of active t-PA based on the amount of protein used.

(b) Reproducibility of results for t-PASSG activation

Under the same activation conditions, different yields could be observed at different measurements, which could be due, among other things, to changes in the t-PA standard. In order to verify the breadth of this error, all activation data after dilution at a ratio of 1: 100 and 1: 200 in a mixture containing 0.1 mol / l tris-HCl at pH 8.5.1 mmol / l EDTA were summarized, 0.5 mol / l-arginine, 1 mg / ml BSA and 2 mmol / l GSH.

SK 278317 Β6

attempt yield (%) stimulatability 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

c) Dependence of the activation yield on the amount of urea

The solution from b) was used, activation was carried out for 17 hours at 0 ° C. Dependence of activation on urea

urea (M) effectiveness (%) 0 13 0.5 100 1 200 1.5 100

mean value 5.1 + 1.9 11.3 + 3.8

c) Stability of the activated protein

Activation was performed by diluting 1: 200 in a mixture containing 0.1 mol / l tris-HCl, 1 mmol / l EDTA, 0.5 mol / l 1-arginine, 1 mg / ml BSA and 2 mmol / l GSH.

Example 5

Activation of genetically engineered β-interferon

Refractive bodies were obtained as described above. The reduction / solubilization of these bodies was performed as follows: the pellet was incubated for 3 hours at 25 ° C in 10 ml of a mixture, 9.1 mol of tris-HCl pH 8.6, 6 mol / l Gdn / HCl, 1 mmol / l EDTA and 0.2 mol / L DTE and after 30 minutes the mixture was centrifuged at 4 ° C and at 48,000 xg and the pH of the supernatant was adjusted to 3 by addition of concentrated hydrochloric acid. Then, filtration was performed on a Sephadex G25 P gel in 0.01 M HCl.

The eluate was monitored at 280 nm and the extinction, protein concentration and reactivity were determined.

The activation of the standard (100%) is carried out in a mixture containing 0.1 mol of tris-HCl, pH 10.5, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG and 0.25 mol / l. 11-arginine.

a) Activation versus time

The eluate was diluted 1:50 with a mixture containing 0.1 mol of tris-HCl, pH 8.5, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG and 0.25 mol / l. 11-arginine.

d) Dependence of the activation yield on the amount of formamide

Activation is carried out in the same mixture as in b), the samples are monitored after 17 hours of activation at 0 ° C.

Activation dependence on formamide

formamide (M) effectiveness (%) 0 13 1 13 2 13 3 0 4 0

e) Dependence of activation yield on redox buffer

The eluate is diluted 1:50 with a mixture containing 0.1 mol of tris-HCl, pH 8.5, 1 mmol / l EDTA, 0.25 mmol / l 1-arginine, and the samples are monitored after 17 hours of activation at 0 C.

activation time effectiveness (Hrs.) (%) 1 15 3 15 20 2

b) Dependence of activation yield on addition of 1-arginine

The eluate is diluted 1:50 with a mixture containing 0.1 mol of tris-HCl, pH 8.5, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG, and then the mixture is activated with 20 ml. hours at 0 ° C

Dependence of activation on the amount of 1-arginine

1-Arginine effectiveness (M) (%) 0 8 0.25 8 0.5 15 0.75 15

Dependence of activation on GSH / GSSG ratio

GSH (mM) GSSH (mM) efficiency (%) 1 0.5 6 5 0.5 13 10 0.5 25 20 0.5 25 5 0.1 13 5 0.5 13 5 1.0 13 5 5 6

f) Dependence of activation yield on protein concentration

The eluate is diluted 1: 10 to 1: 100 with 0.1 M Tris-HCl pH 8.5, 1 mM EDTA, 0.5 mM GSSG, 5 mM GSH and 0.25 mmol / L 1-arginine. , the assay is performed after activation for 17 hours at 0 ° C.

Dependence of cp activation

cp (mg / ml) efficiency (%) 0,018 13 0,036 13 0,072 13 0,108 8 0,180 10

SK 278317 Β6

g) Dependence of activation yield on BSA addition

The eluate was diluted 1: 5 with 0.1 M tris-HCl pH 8.5, 1 mM EDTA, 0.5 mM GSSG, 5 mM GSH and 0.25 mmol / L 1-arginine and the result was determined after 17 hours of activation at 0 ° C.

Activation dependence on BSA

BSA (mg / ml) efficiency (%) 0 13 1 13 2 25 5 13

h) Dependence of activation yield on pH

The eluate was diluted 1: 5 with 0.1 M tris-HCl pH 8.5, 1 mM EDTA, 0.5 mM GSSG, 5 mM GSH and 0.25 mmol / L 1-arginine and the result was determined after 17 hours of ativation at 0 ° C.

Dependence of activation on pH

pH efficiency (%) 6.5 0 7.5 6 8.5 13 9.5 50 10.5 100

Claims (21)

PATENT CLAIMS A method for activating genetically engineered, heterologous proteins containing disulphide bridges of eukaryotic origin after expression in prokaryotic organisms, cell disruption by solubilization and subsequent denaturation and reduction, upon activation under oxidative conditions in the presence of GSH / GSSG, characterized in that activation it is carried out at a pH of 9-12, at a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol / l, and at a denaturing agent concentration which does not yet lead to protein denaturation. Method according to claim 1, characterized in that the activation is carried out at a pH in the range of 9.5 to 11. Method according to claims 1 and 2, characterized in that the activation is carried out at a GSH concentration of 0.2 to 10 mmol / l and / or at a GSSG concentration of 0.05 to 1 mmol / l. Method according to claim 1 to 3, characterized in that the activation is carried out without prior separation of the denaturing / reducing agent, wherein the reaction solution is diluted with the activation buffer after the denaturation / reducing and the subsequent GSSG concentration exceeds the residual DTE concentration, Activation method according to claim 1, characterized in that the reducing / denaturing agent is separated, by the addition of GSSG under denaturing conditions, the thiol groups of the proteins are converted to mixed protein disulfides and glutathione, the activation is carried out at pH 7 to 10.5, at GSH concentration 0.5 to 5 mmol / l and at a denaturant concentration that does not yet lead to protein denaturation. Method according to claims 1 to 5, characterized in that E. coli or P. putida microorganisms are used for expression. The process according to claims 1 to 8, characterized in that in the activation step, as the denaturant, arginine, guanidine hydrochloride and / or at least one compound of the general formula (I), 10-CO-NRR ¹ (T) are used as the denaturant. R @ ¹ and R @ ¹ denote a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms; R ² represents a hydrogen atom, an NHR ¹ radical or an alkylated radical having 1 to 3 carbon atoms. Process according to claim 7, characterized in that the concentration of arginine and / or guanidine hydrochloride is 0.1 to 1.8 mol / l, preferably 0.25 to 0.8 mol / l. Process according to claim 7, characterized in that the compound of the formula (I) is used in a concentration of 0.5 to 4 mol / l, preferably 1 to 3.5 mol / l. Method according to claims 1 to 9, characterized in that in the activation step the process is carried out in the presence of a protein which has no proteolytic effect, in particular in the presence of bovine serum albumin. Method according to claim 10, characterized in that the disruption of the cells is carried out in a dilute aqueous buffer solution, preferably in 0.1 mol / l tris buffer at neutral or weakly pH. Method according to claims 1 to 11, characterized in that an insoluble fraction is separated after the cells have been disrupted. Process according to claims 1 to 12, characterized in that the step of the solubilization step is carried out at alkaline pH in the presence of a mercapto-containing reducing agent in the presence of a denaturing agent. Process according to claim 13, characterized in that the process is carried out in the presence of guanidine hydrochloride and / or a compound of the formula (I) as a denaturing agent. Process according to Claim 14, characterized in that guanidine hydrochloride is used in a concentration of 6 mol / l and the compound of the formula (I) in a concentration of 8 mmol / l. A process according to claims 13 to 15, characterized in that the process is carried out in the presence of DTE, β-mercaptoethanol or GSH. Process according to claims 1 to 16, characterized in that the purification and separation of the reducing, oxidizing or denaturing agent is carried out by spherical chromatography or dialysis. Method according to claims 1 to 17, characterized in that after activation a purification step is arranged, the purification is carried out by dialysis. The method according to claims 1 to 18, characterized in that the genetically engineered eukarvotic protein t-PA is used. Method according to claims 1 to 19, characterized in that it is used genetically 276 engineered eukaryotic protein interferon β · 21. The method of claim 6, wherein the mixed protein disulfide and glutathione are separated from the unmodified protein by treatment with 5 ion exchangers.