SK752686A3 - Activation method of by genetic technology gained heterologous proteins with content of disulfidic bridges of eukaryotic origin after expression in prokaryotic organisms - Google Patents

Description

Method for activation of heterologous proteins obtained by genetic technology containing disulfide bridges of eukaryotic origin after expression in prokaryotic organisms

Technical field

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

BACKGROUND OF THE INVENTION

When expressing heterologous proteins in prokaryotic organisms, these proteins often form inactive, sparingly soluble aggregates in the target cells. refractile bodies, 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 the formation of large amounts of enzymes in a cell results in the aggregation of enzymes in the cell to 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 methods, activation can be achieved in various 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 composition, for example guanidine hydrochloride or urea in high concentration, or by adding strongly acidic agents, for example a mixture of glycine and phosphoric acid. Further excipients which may be mentioned 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 carry out the purification in the next step in a conventional manner, for example by gel chromatography or ion exchange chromatography. Thereafter, the material obtained is strongly diluted to reduce the concentration of the denaturing agent. When guanidine hydrochloride is used, the sample must 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, Journal 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 reflective 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 bridge formation, b) the protein is sulfonated, then dissolved in a solution in a weak denaturing agent and an S-sulfonate group are converted to a reduced and oxidized form, for example by GSH / GSSG to form -SS- groups, or c) the weak denaturing agent solution is directly treated with a sulfhydryl-type reagent, for example GSH / GSSG. A typical example of the above problems is t-PA.

The major component of the blood protein matrix is polymeric fibrin. This protein is dissolved by the action of a plasmid that is formed from plaminogen by activation ΐζν. 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 can be increased by adding more than ten times the above 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 to 2019, Nieuwenhiuzen et al., Biochemica et Biophysica Acta 755, (1983), 531-533. The factor of stimulation with BrCN-type cleavage products 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 enzymatic stimulatability of the product as such. The fact that cells of prokaryotic organisms do not usually produce active proteins of eukaryotic origin is also shown, for example, in The Journal 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 tris-HCl pH 8 0.10 in a mixture with sodium chloride, ethylenediaminetetraacetic acid and Tween 80. After dialysis, the material is centrifuged and the plasminogen activator can be detected in the supernatant. The t-PA thus obtained is proteolytically active, and cannot be stimulated by cleavage products after treatment with bromine cyanide on fibrin (BrCN-PSP), as described in J. H. 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 connected by 2.2 x 10 methods, with only one of these structures corresponding to the natural state. Methods known to the art for activating t-PA have resulted in the obtaining of proteolytically 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 upon expression in prokaryotic organisms by cell disruption, solubilization based on 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 concentration of GSSG 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 conventionally 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. Mixtures of the above denaturing agents may also be used. 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.

The steps of the activation process of reoxidation and activation or previous and subsequent cells, dissolution and optionally one of the activation and / or subsequent initiation according to the example in the step, for example or greater of the prior art, European patent purification is as described above. 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 above-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, 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 may also be achieved.

Cell disruption can be accomplished by known methods, for example by sonication, dispersion at elevated pressure, or lysozyme, preferably a buffer solution which allows a neutral to weakly acidic pH, for example 0.1 mol / l tris-HCl, is used as the suspension medium. 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 non-β-PA-degrading substances, but as far as possible dissolving cellular proteins such as water or phosphate buffer with optional mild surfactants such as triton, 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. The compounds of formula I can also be used as well

R ² -CO-NRR ¹ (I) wherein

R @ ³ and R @ ³ denote a hydrogen atom or an alkyl radical having from 1 to carbon atoms and n1

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

As reducing agents from the group of mercaptans, for example, ethanol, for example treitone and / or reduced glutathione GSH or -mercapto at a concentration of 50 to 400 mmol / l, dithiodithioerythritol (DTE) or dithiothreitol (DTT), for example at 80 to 400 mmol / l. 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 the denaturant 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. Purification after modification of the thiol groups has the advantage that the protein is protected against oxidation by the action of air and is therefore stable over a wide pH range, while facilitating 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 mmol / l, and at a denaturing agent concentration which is not yet denatured.

In 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 approximately 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 such that not only activation to the usual biological activity is achieved, but additionally the stimulus as above is achieved in an amount that far exceeds that of t-PA at a higher factor. 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 stated, the percentages are by weight and temperature data are 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 0.1 mol / l mixture of tris-HCl at 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 wet E. coli cell mass in 1.5 liters of 0.1 mol / l, pH 6.5 and 20 mmol / l EDTA are homogenized on an Ultra-Turrax for 10 seconds, and then 0, 25 mg / ml lysozyme. After 30 minutes of incubation 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 (Sorvall GSA, 2 hours at 13,000 rpm, 21,000 xg, 4 ° C), the pellet is mixed with 1.2 liters of 0.1M tris-HCl at pH 6.5, 20 mmol. / 1 EDTA and 2.5%

Triton-X-100 and then homogenized. The material is then centrifuged repeatedly (Sorvall GSA, 30 minutes at 13,000 rpm, 27,000 x g, 4'C) to pellet. is mixed with 1.3 L of Tris-HCl, pH 6.5, 0.1 mol / L 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, 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 bands containing t-PA are identified by Western blotting and quantitatively determined by densitometry. Using this method, a strong t-PA band with a molecular weight of about 60 units can be detected. The proportion of t-PA in the total protein of the refractive bodies is approximately 21%.

b) Solubilization / reduction of refractive bodies

Refractive bodies with a protein concentration of 1 to 5 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 / ml. 1 EDTA for 2 to 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 to 20,000 rpm and 35,000 to 50,000 x g). 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 4 ° C.

c) Reoxidation / activation

Reoxidation / activation is performed at a dilution of 1:50 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 1-arginine, 2 mmol / l GSH and 0.2 mmol / l GSSG. After 17 to 24 hours of activation at about 20 ° C, the efficacy is compared to that of native glycosylated t-PA from eukaryotic organisms and the yield of the substance.

The yield, based on the total protein content of the refractive bodies, is in the range: 2.5 ± 0.5%, the stimulus being 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 reoxidation is performed at a dilution of 1: 100 in a solution containing 0.1 mol / l tris-HCl at pH 10.5, 1 mmol / l EDTA, 1 mg / ml BSA, 0.3 mol / l 1- arginine and the amount of GSSG shown in the following table. In addition, a residual concentration of 0.06 mol / l guanidine hydrochloride and 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 / 1) 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

A sample of refractory bodies (450 OD / g) 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 hours at room temperature. The insoluble material, e.g., 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 (superfine) gel 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 at pH 10.5 + 1 mmol / l EDTA + 0.5 mol / l 1-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 V. Nieuwenhuizon et al., Biochemica et Biophysica Acta 755 (1983) 531-533. The% potency and the stimulatability as a factor are determined by 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 + 1.0 mmol / 1 EDTA + 1.0 mg / ml BSA + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG

(a) 1-arginine

1-Arginine effectiveness stimulatability (Mol / l) (%) (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 At this try to consider that 1-arginine method

is the inhibition of t-PA. 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) effectiveness (Mol / l) (%) 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

Rexidation with in 0.1 mol / l tris-HCl with pH 10.5, 1.0 mmol / L mmol / L GSSG. EDTA, 1.0 mg / mL BSA, 5 mmol / L GSH, 0.2

(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

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

(d) Dimethylurea Dimethylurea (mol / l) potency (%) (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

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

The re-oxidation 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) Formamide formamide (mol / l) potency (%)

0 42 0.5 59 1 175 1.5 245 2 325 2.5 423 3 444 4 416 5 341

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

(C) Acetamide

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

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

(e) Butyramide Butyramide (mol / l) Efficiency (%)

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 / L 1-arginine + 1.0 mg / ml BSA + 0.5 mmol / L GSH + 0.5 mmol / L GSSG

PH efficiency 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

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 / l 1-arginine + 1.0 mg / ml BSA

a) +1 mmol / L GSH

GSSG effectiveness stimulatability

(Mmol / 1) (%) (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 GSH (Mmol / 1) GSSG effectiveness (%) stimulatability (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)

The reoxidation is carried out in a mixture

0.1 mol / l tris-HCl with pH 10.5 +1.0 mmol / l EDTA stimulation (factor) + 1.0 mg / ml BSA + 0.5 mol / l 1-arginine + 0.5 mmol / 1 GSH + 0.5 mmol / 1 GSSG dilution efficiency (%)

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 yield on BSA addition

The reoxidation is carried out in a mixture

0.1 mol / l Tris-HCl, pH 10.5 1.0 mmol / 1 EDTA 0.5 mol / l 1-Arginine 0.5 mmol / 1 GSH 0.5 mmol / 1 GSSG

BSA (mg / ml)

0,5 efficiency (%)

100

102

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 reoxiation at room temperature in 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.

Example 4

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

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 mol / 1 DTE at a protein concentration of approximately 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 mol / l urea and 0.2 mol / l GSSG, and then Incubate for 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 ΐ-PASSG thus obtained, 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 mol / l 1-arginine, 1 mg / ml BSA and 2 mmol / l GSH, simultaneously changing 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 data for activation 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 of 1-arginine, 1 mg / ml of BSA and 2 mmol / l of GSH.

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

mean value 5.1 ± 1.9 11.3 ± 3.8

c) Stability of the activated protein

Activation was performed by dilution 1: 200 mixed with EDTA, 0.5 mol / L sahom 0,1 1-arginine, mol / l 1 mg / ml tris-HCl, 1 mmol / L BSA and 2 mM GSH. Example 1 d 5

Activation of genetically engineered β-interferon

Refractive bodies were obtained as described above. The reduction / solubilization of these bodies was performed as follows: pellet. was incubated for 3 hours at 25 ° C in 10 ml of a mixture of 9.1 mol of tris-HCl, pH 8.6, 6 mol / l Gdn / HCl, 1 mmol / l EDTA and 0.2 mol / l DTE. The mixture was centrifuged at 4 ° C for 30 minutes and the pH of the supernatant was adjusted to pH 3 by addition of concentrated hydrochloric acid. Filtration was then 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. 1-arginine.

a) Activation versus time

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 0.25 mol / l. 1-arginine.

activation time (H) effectiveness (%) 1 15 3 15 20 2

b) Dependence of the activation yield on the 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 (M) efficiency (%)

0,258

0,515

.7515

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.

Urea Activation Dependency Urea (M) Efficiency (%)

0.5

1.5

100

200

100

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

Activation shall be carried out in the same mixture as in paragraph (b), samples shall be monitored after 17 hours of activation at 0 C.

Dependence of activation on formamide formamide efficacy (M)

e) Dependence of activation yield on redox buffer

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

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, determined 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

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 resultant provides 17 hours of activation at 0 ° C.

- 27 BSA Activation Dependence

BSA (mg / ml) potency (%)

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 determines 17 hours of activation at 0 ° C.

Dependence of activation on pH

PH efficiency (%)

6.5

7.5

8.5

9.5

10.5

100?! / 4516 'N |

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Claims (21)

PATENT CLAIMS A method of activating genetically engineered heterologous proteins containing disulphide bridges of eukaryotic origin after expression in prokaryotic organisms after cell disruption by solubilization and subsequent denaturation and reduction upon activation under oxidative conditions in the presence of GSH / GSSG, characterized in that activation is performed 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 that 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 to 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. The 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 GSSG concentration exceeds the residual DTE concentration upon subsequent activation. 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-glutathione disulfides, the activation being carried out at a pH of 7 to 10.5, at a GSH concentration of 0.5 to 5 mmol / l and 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. Process according to Claims 1 to 6, characterized in that, in the activation step, arginine, guanidine hydrochloride and / or at least one compound of the formula I • R ² -CO-NRR ¹ ( I) where R a r! R @ 1 is hydrogen or C1 -C6 alkyl 4 carbon atoms; 9 “1 R represents a hydrogen atom, an NHR 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 does not have a 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 a neutral or slightly acidic pH. Method according to claims 1 to 11, characterized in that the insoluble fraction is separated after the cells have been disrupted. Process according to one of Claims 1 to 12, characterized in that in the solubilization step the process is carried out at alkaline pH in the presence of a reducing agent containing mercapto groups 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 mol / l. Method according to claims 13 to 15, characterized in that the process is carried out in the presence of DTE, β-mercaptoethanol or GSH. The 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 steric chromatography or dialysis. Method according to one of Claims 1 to 17, characterized in that, after activation, a cleaning step is carried out, the cleaning step being carried out by dialysis. The method according to claims 1 to 18, characterized in that the genetically engineered eukaryotic protein t-PA is used. The method according to claims 1 to 19, characterized in that the genetically engineered eukaryotic protein interferon β is used. 21. The method of claim 6, wherein the mixed protein disulfide and glutathione are separated from the unmodified protein by ion exchanger treatment.