HRP921075A2 - Process for activating heterologous, eucariotic proteins, genetically engineered and presenting disulphide bridges after their expression in procaryotic cells - Google Patents

Abstract

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

Description

The proposed invention relates to a procedure for activating genetically prepared, eukaryotic proteins, which contain disulfide bridges, after expression in prokaryotes.

During the expression of heterologous proteins in prokaryotes, these proteins often form inactive, hard-to-dissolve aggregates (i.e. "refractile bodies") in host cells, which are also contaminated with host cell proteins. It is assumed that the formation of such "refractile bodies" is a consequence of the resulting high protein concentration in the cell during expression. It is known that when large amounts of enzymes are produced in the cell, enzymes aggregate into insoluble, high-molecular, mostly inactive particles. Before we use such proteins, for example for therapeutic purposes, they need to be purified and converted into an active form.

According to known procedures, the activation of such proteins, which appear as aggregates, can proceed in several stages (cf. e.g. B. R. Jaenicke, FEBS Federation of European Biochemical Societies, Vol. 52 (1979) 187 to 198; R. Rudolph, Biochemistry 18 ( 1979) 5572 to 5575):

In the first stage, solubilization is achieved by the addition of denaturing agents, eg guanidine hydrochloride or urea in high concentration, or by the addition of strongly acidic agents, eg a mixture of glycine and phosphoric acid. Reducing SH-reagents (e.g. dithioerythritol, DTE) and EDTA have proven to be useful as additional auxiliary substances, e.g. when renaturing LDH. If the protein is contaminated with host cell proteins, the next step is purification by methods that are known and common in themselves, eg gel or ion exchange chromatography. Then we dilute strongly to reduce the concentration of denaturing agents. When using guanidine hydrochloride, dilute to a value of 0.5 mol/l. In the case of enzymes with simple SH-groups, the addition of agents that protect the SH-groups has proven to be suitable (see, e.g., B. R. Jaenicke, Journal Polymer Science, Part C (1967) 2143 to 2160).

In EP-A-0114506 they describe procedures for isolation, purification and activation of some heterologous expression products from bacterial cultures; for activation, they translate solutions of "refractile bodies" in a strong denaturing agent a) directly into a solution in a weaker denaturing agent, and then subject them to oxidizing conditions for the re-formation of disulfide bridges; b) the protein is sulfonated, and then converted into a solution in a weaker denaturing agent and the S-sulfonate groups are converted into their reduced and oxidized forms by treatment with a sulfhydryl reagent, for example with GSH/GSSG they are converted into -S-S-groups; or c) solution in a weaker denaturing agent directly treated with a sulfhydryl reagent, eg with GSH/GSSG. A notable example where the problems presented earlier occur is t-PA.

The main component of the protein matrix of clotted blood is the polymer fibrin. That protein matrix is dissolved by plasmin, which is formed from plasminogen through activation with the so-called plasminogen activators, for example with t-PA (tissue-type plasminogen activator). The enzymatic activity of natural or eukaryotic prepared t-PA (catalytic activation of plasminogen to plasmin) is very low in the absence of fibrin or fibrin cleavage products (FSP) and can be significantly enhanced (by more than a factor of 10) in the presence of these stimulators. That so-called stimulatory efficacy is a decisive advantage of t-PA compared to other known plasminogen activators such as urokinase or streptokinase (see, e.g., M. Hoylaerts et al., J. Biol. Chem. 257 (1982) 2912 to 2919; Nieuwenhiuzen et al. ., Biochimica et Biophysica Acta 755 (1983) 531 to 533). The stimulation factor with BrCN-splitting products is therefore stated differently in the literature and marked with up to 35.

t-PA-nast, a non-glycosylated product is also formed in genetically manipulated prokaryotes (after c-DNA insertion); such a product does not belong to the stimulation of the effectiveness of t-PA from eukaryotes. It is possible that this results from the fact that the redox conditions in prokaryotic cells are so different from the conditions in eukaryotic cells from which the gene originates, that an inactive product is initially formed, which is easily explained by the fact that, for example, the numerous S-S bridges that a natural active molecule contains , connected in the wrong way or not formed at all. The therapeutic use of t-PA requires not only enzymatic activity as such, but also its stimulation. The fact that the prokaryotic cell probably does not achieve the right conditions for shaping the activity of eukaryotic proteins in a proper way was shown for other substances in The EMBO Journal 4, Nr. 3 (1985) 775 to 780.

According to EP-A-0093639 to activate t-PA, cell pellets obtained from E. coli are suspended in 6 mol/l guanidine hydrochloride, treated with ultrasound, incubated and then dialyzed for 4 hours against Tris-HCl solution (pH=8.0) , sodium chloride, EDTA and Tween 80. After dialysis, it is centrifuged, and the effectiveness of plasminogen activation is found in the upper layer (supernatant). The t-PA thus naturated is indeed proteolytically active, although it does not show measurable stimulation with BrCN-products, it separates (BrCN-FSP) fibrin in the sense of the procedure described in J. H. Verheijen, Thromb. Haemostas., 48, (3), 260-269 (1982).

For activating denatured proteins, no generally applicable procedure is known from the state of the art; this is especially true for t-PA, which has a native protein with a very complex structure; contains a simple thiol group and 17 SS-bridges that can theoretically connect in 2.2x1020 different ways, with only one structure corresponding to the native state. From the state of the art, known procedures for activating t-PA otherwise lead to proteolytically active t-PA that does not show measurable stimulation; the activation process leading to stimulated t-PA is not yet known.

The task of the proposed invention is to provide us with a procedure for the complete activation of genetically prepared, heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes; we solve this task with the object of the proposed invention.

The subject of the invention is a procedure for activating genetically prepared, heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes, after cell disintegration, using solubilization under denaturing and reducing conditions, and activation (renaturation) under oxidizing conditions in the presence of GSH/GSSG, indicated with the fact that at the activation stage we work at a pH value of 9 to 12, GSH-concentration 0.1 to 20 mmol/l, GSSG-concentration 0.01 to 3 mmol/l, and with a non-denaturing concentration of the denaturing agent. As a denaturing agent, we can generally use a common denaturing agent or arginine for activation under oxidizing conditions; among the known denaturing agents, it is preferable to use guanidine hydrochloride or urea or its derivatives. In addition, arginine has also proven to be suitable. Furthermore, a mixture of these denaturing agents can be used. Preferably, we perform this stage of activation also in the presence of a foreign protein; as a rule, any foreign protein is suitable if it is proteolytically active; preferably we use bovine serum albumin (BSA), for example in the amount of 1 to 3 mg/ml. Addition of BSA may cause a small gain increase and protein stabilization (probably protection against surface denaturing and/or proteolytic degradation).

Other process conditions may correspond to conditions that are known and common for reactivating stages from the state of the art. The duration of activation (incubation) is preferably 20 to 48 hours at room temperature. The half-time of activation in the presence of 0.5 mmol/l of reduced (GSH) and oxidized (GSSG) glutathione is about 10 to 15 hours at 20°C. During further incubation (48 hours) under reoxidation conditions, stimulation with CNBr-FSB generally decreases. The activation stage is preferably carried out in the presence of EDTA, with the best planned concentration being around 1 mmol/l EDTA.

The stages of the process that we perform before or after the activation stage (reoxidation/activation) such as cell disintegration, solubilization (solubilization/reduction) and in the given example one or more cleaning operations, which we perform before and/or after the activation stage, can be performed using a technique that is known , that is, common for procedures of this type, e.g. from EP-0114506, EP-A-0093619; to achieve an optimal result with respect to gain and activation, it may be considered to perform the steps individually or all steps of the process using one or more of the implementation methods explained in the proposed description. Thus, it is possible to carry out the activation stage in terms of the invention, in the mixture obtained after decomposition without earlier denaturation and/or reduction, albeit with a low gain. The expression is carried out in prokaryotes, preferably in P. putida and particularly advantageously in E. coli. The method according to the invention is also suitable when we experiment in other prokaryotes (eg in Bacilla).

Cell disintegration can be carried out in the usual way and with usual procedures, for example using ultrasound, high-pressure dispersion or lysozyme; it is preferable to perform this in a buffer solution suitable for the formation of a neutral or slightly acidic pH value, as a suspension medium, for example in 0.1 mol/l Tris-HCl. After cell disintegration, we separate the insoluble part ("refractile bodies") in a suitable way, preferably by centrifugation at higher g-values and a longer centrifugation time or by filtration. After washing with agents that do not damage t-PA foreign cellular proteins, if possible dissolve them, for example in water, phosphate buffer solution and in the given example with the addition of mild detergents such as triton, subject the precipitate (pellet) to solubilization (solubilization/reduction). Solubilization preferably takes place in the alkaline pH range, especially at pH 8.6 + 0.4 and in the presence of a reducing agent from the mercaptan group and a denaturing agent.

As denaturing agents for solubilization, we can use known and common denaturing agents known from the state of the art, for example from EP-A-0114506, especially guanidine hydrochloride or urea. The concentration of guanidine hydrochloride is about 6 moles/l, urea about 8 moles/l. We can also use compounds of the general formula I.

As reductants from the group of mercaptans, we can use, for example, reduced glutathione (GSH) or 2-mercaptoethanol, for example in a concentration of about 50 to 400 mmol/l dithiothreitol and/or especially DTE (dithiothreitol) or DTT (dithiothreitol), for example in a concentration of about 80 to 400 mmol/l. Solubilization takes place at room temperature during (incubation) 1 to several hours, preferably two hours. To prevent oxidation of the reductant by oxygen from the air, it is possible to use EDTA. In addition to solubilization/reduction, the solubilization step also has a cleaning effect because most of the material that does not immunologically cross-react with t-PA (foreign proteins) does not go into solution.

After solubilization and before the activation step, we can carry out self-known and usual cleaning steps; as cleaning methods come into consideration, for example, steric separation chromatography (SEC) (in the presence of guanidine hydrochloride or urea) or ion exchangers (in the presence of urea or its derivatives); non-specific reoxidation can be prevented by adding a reductant (eg 2-mercaptoethanol) or by using a pH-value ≤ 4.5 (cf. eg R. Rudolph, Biochem. Soc. Transactions 13 (1985) 308 to 311). If we use DTE in the earlier stage of solubilization, it should be removed in the cleaning stage. Purification can be carried out, for example, with SEC over Sephadex G 100 in the presence of guanidine hydrochloride and a reductant, for example GSH, at pH 1 to 4 (at this level we can separate a large amount of foreign protein); or by separating the denaturing-educting agent by desalting over Sephadex G 25 in 0.01 mol/l HCl or 0.1 mol/l acetic acid. Separation of the denaturing-reducing agent can alternatively be carried out by dialysis against the same solution. The reactivation stage can be joined by the next stage of cleaning; as a rule, such purification proceeds by means of dialysis or by subsequent isolation of activated t-PA, for example by affinity chromatography, e.g. over Lys-Sepharose.

The next embodiment of the invention relates to the formation of mixed disulfides of genetically prepared, heterologous, eukaryotic proteins containing disulfide bridges and glutathione

(hereinafter abbreviated as t-PASSG). This can facilitate the separation of foreign proteins in the denatured state as well as further purification of the native protein. Cleaning by modification of thiol groups has the advantage that the protein is protected from oxidation from the air and is thus stable in a wider pH range, and the change in net charge facilitates cleaning. Separation of the unmodified protein can be carried out particularly conveniently by ion exchange treatment.

For the formation of mixed disulfides, we incubate the dialyzed, reduced protein from which the denaturing and reducing agents have been purified, with a diluted, for example, 0.2M solution of GSSG, which contains the denaturing agent. Activation begins after the separation of the denaturing and oxidizing agents at a pH value of 7 to 10.5, a GSH concentration of 0.5 to 5 mmol/l, and with a non-denaturing concentration of the denaturing agent.

At all other reaction stages, the activation of the protein through the formation of mixed disulfides with GSSG derived forms corresponds to the activation of the earlier part of the invention. In this embodiment, the optimal pH is 8.5, the gain is approximately twice as much and the activated protein is stable for a long time in the renaturing buffer.

In terms of the invention, we were lucky enough to activate t-PA from prokaryotes in such a way that not only activation of normal biological activity was achieved, but on top of that we also achieved stimulation in the previously defined sense, which far exceeds the activation of native t-PA and is already by a factor of 10, and can exceed and factor 50. The next eukaryotic protein that can be activated in terms of the invention after expression in the prokaryote is β-interferon.

The following examples further illustrate the invention and do not limit it.

Unless otherwise stated, data in percentages refer to mass percentages, data on temperature in degrees Celsius.

EXAMPLE 1

Preparation of "refractile bodies"

We homogenized 100g of wet cell mass of E. coli taken in 1.51 0.1 mol/l Tris/HCl (pH 6.5) in 20 mol/l EDTA (Ultra-Turax, 10 sec.) and added 0.25 mg /ml lysosomes. After 30 minutes of incubation at room temperature, we homogenized again and cooled to 3°C. Cell disintegration was achieved by high-pressure dispersion (550 kg/cm2). Then we rinsed with 300 ml of 0.1 mol/l Tris/HCl 9pH 6.5 and 20 mmol/l EDTA. After centrifugation (Sorvall GSA, 2 hours at 13,000 rpm, 21,000 g, 4°C), the pellet was collected in 1.2 l of 1.2 mol/l Tris/HCl (pH 6.5), 20 mmol/ lEDTA and 2.5% Triton-x-100 and homogenized. After centrifugation again, (Sorvall GSA, 30 min. at 13000 U/min., 27000g, 4°C) we took the pellet in 1.3 l 0.1 tris/HCl (pH 6.5), 20 mmol/l EDTA and 0.5 Triton-x-l and homogenized. Three more times we performed alternate centrifugation (Sorvall GSA, 30 minutes at 13,000 rpm, 27,000 g, 4°C) and pellet homogenization in 1 1 0.1 mol/l Tris/HCl (pH 6.5) and 20 mmol/l EDTA.

We quantified the content of "refractile bodies" t-PA bands in the preparations with SDS-PAGE, identifying t-PA bands with "Western-blotting" and densitometric analysis. "Refractile bodies" show on SDS-PAGE and "Western-blotting" a strong t-PA band with a molecular mass of about 60 kDa. The share of t-PA in the total protein content of "refractile bodies" is about 21%.

Solubilization/reduction of "refractile bodies"

"Refractile bodies" with a protein concentration of 1 to 5 mg/ml were incubated in 0.1 mol/l Tris/HCl (pH 8.6), 6 mol/l guanidine hydrochloride, 0.15 to 0.4 mol/l DTE and 1 mmol/l EDTA, 2 to 3 hours rpi at room temperature. Then we centrifuged insoluble material (fragments of cell walls, etc.) (eg Sorvall SS 34, 30 minutes at 15,000 to 20,000 rpm, 35,000 to 50,000 g, 4°C). The pH value of the upper layer (supernatant) was adjusted to pH 3 using concentrated hydrochloric acid. The denaturing and reducing agents were then separated by dialysis against 0.01 mol/l HCl at 4°C.

Reoxidation/activation

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

Gain with respect to total protein content in "refractile bodies": 2.5+/-0.5%

stimulation: 10+/-5

Gain with regard to the share of t-PA in "refractile bodies": Ca. 12%.

Reoxidation/activation without separation of the denaturing/reducing agent

"Refractile bodies" were incubated at a protein concentration of 1.25 mg/ml in 0.1 mol/l Tris/HCl (pH 8.6), 6 mol/l guanidine hydrochloride, 0.2 mol/l DTE and 1 mmol /l EDTA for 2 hours at room temperature. Then we introduced reoxidation with a 1:100 dilution in 0.1 mol/l Tris/HCl (pH 10.5), 1 mmol/l EDTA, 1 mg/ml BSA, 0.3 mol/l, L-arginine, and with the amounts of GSSG specified in the tables. Additionally, there was a remaining concentration of 0.06 mol/l guanidine hydrochloride and mmol/1 DTE in the activating residue.

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

[image]

ʹ= gain of active t-PA with respect to total protein content in "refractile bodies".

EXAMPLE 2

The preparation RB ("refractile bodies") (450 OD550/ml) was incubated in 1 ml of 0.1 mol/l Tris/HCl (pH=8.6) guanidine hydrochloride and 0.15 to 0.2 mol/l DTE 2 to 3 hours at room temperature. Insoluble material (fragments of cell walls, etc.) was then separated by centrifugation (20 minutes at 17,000 rpm). Denaturing and reducing agents were removed by gel filtration through Sephadex G 25 (superfine) in 0.01 mol/l HCl. In doing so, we diluted the sample by a factor of about 5 to 10. We stored the reduced material in 0.01 mol/l HCl at -20°C.

EXAMPLE 3

The following tables summarize the influence of various parameters in terms of the invention on the activation and stimulation of t-PA. For these reoxidation experiments, according to example 1, we did not further purify the solubilized, reduced protein.

The reduced protein (in 0.01 mol HCl) was diluted 1:10 to 1:500 and activated in the "reoxidation buffer". We finished the activation after 22 to 40 hours of incubation at room temperature.

The activity of the reoxidized protein refers to "standard reoxidation" (=100%) in:

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

+ 0.5 mol/l L-arginine

+ 1 mg/ml BSA

+ 0.5 mmol/l GSH (reduced glutathione)

+ 0.5 mmol/l GSSG (glutathione disulfide)

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

We got the following results:

1. Dependence of activation utilization after addition of L-arginine guanidine hydrochloride

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

+ 1 mmol/l EDTA

+ 1 mg/ml BSA

+ 0.5 mmol/l GSH

+ 0.5 mmol/l GSSG

a) L-arginine

[image]

In this experiment, it should be noted that t-PA is inhibited by 1-arginine. Therefore, the decrease in activated gain should be corrected at higher concentrations of L-arginine due to inhibition.

b) Guanidine hydrochloride (Gdn. HCl)

[image]

2. Dependence of activation utilization on the addition of urea and urea derivatives.

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

a) Urea

[image]

b) Methylurea

[image] c) Ethylurea

[image]

d) Dimethylurea

[image]

3. Dependence of activation utilization on fatty acid amide addition.

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

a) Formamide

[image]

b) Methylformamide

[image]

c) Acetamide

[image]

d) Propionamide

[image]

e) Butyramide

[image]

4. Dependence of activation utilization on pH value.

Reoxidation in 0.1 mol/l Tris/HCl + mmol/l EDTA

+ 0.5 mol/l L-arginine

+ 1 mg/ml BSA

+ 0.5 mmol/l GSH

+ 0.5 mmol/l GSSG

[image]

5. Dependence of activation utilization on GSH/GSSG concentration.

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

+ 1 mmol/l EDTA

+ 0.5 mol/l L-arginine

+ 1 mg/ml BSA

a) + 1 mmol/l GSH

[image]

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

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

+ 0 mmol/l EDTA

+ 0.5 mol/l L-arginine

+ 1 mg/ml BSA

+ 0.5 mmol/l GSH

+ 0.5 mmol/l GSSG

[image]

7. Dependence of activation utilization on BSA addition

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

+ 1 mmol/l EDTA

+ 0.5 mol/l L-arginine

+ 0.5 mmol/l GSH

+ 0.5 mmol/l GSSG

[image]

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

EXAMPLE 4

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

Usable "refractile bodies" only obtained according to one of the earlier examples. The reduction of "refractile bodies" was carried out after a two-hour incubation at room temperature in 0.1 mol/l Tris/HCl, pH 8.6, 1 mmol/l EDTA, 6 mol/l Gdn/HCl, 0.2 mol/l DTE at a protein concentration of about 1 mg/ml.

The reduced protein, dialyzed against 0.01 mol/l HCl, was diluted 1:1 with 0.1 mol/l Tris, pH 9.3, 9 mol/l urea and 0.2 mol/l GSSG and incubated for 5 hours at room temperature. After acidification with concentrated HCl to pH 3, dialysis against 0.1 mol/l HCl followed at 4°C. After dialysis, the total protein concentration was 0.33 mg/ml. With the t-PASSG prepared in this way, we achieved optimal conditions.

a) pH-optimum of t-PASSG activation

In this example, as well as in the following optimal experiments, we (1) did not use GSSG and (2) the activation was completed after 17 hours of incubation at room temperature. Activation was carried out with a 1:100 dilution in 0.1 mol/l Tris, 1 mmol/l EDTA, 0.5 mol/l L-arginine, 1 mg/ml BSA in 2 mmol/l GSH at varying pH values.

[image]

We determined the gain as a percentage of active t-PA with regard to the amount of protein used.

b) Reproducibility of activation results from t-PA

Under identical activating conditions, we observed for different types of measurements, different gains, which are conditioned, among other things, by the use of standard t-PA. To clarify this width, we put all activating data for a 1:100 or 1:200 dilution in 0.1 mol/l Tris/HCl, pH 8.5, 1 mmol/l EDTA, 0.5 mol/l L-arginine, 1 mg/ml BSA and 2 mmol/l GSH.

[image]

c) Stability of the activated protein

In the above examples, activation was carried out with a 1:200 dilution in 0.1 mol/l Tris/HCl, 1 mmol/l EDTA, 0.5 mol/l L-arginine, 1 mg/ml BSA and 2 mmol/l GSH.

EXAMPLE 5

Activation of genetically engineered interferon-β

We produced "refractile bodies" according to the above methods. The reduction/solubilization of "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/l Gdn/HCl, 1 mmol/l EDTA and 0.2 mol/l DTE and after 30 minutes of centrifugation at 4°C and 48000 g, we adjusted the pH of the upper layer (supernatant) to about 3 using concentrated HCl. Then we performed gel filtration through Sephadex G 25 F in 0.01 mol/l HCl.

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

Standard activation (100%) was performed in 0.1 mol/l Tris/HCl, pH 10.5, 1 mmol/l EDTA, 5 mmol/l GSH, 0.5 mmol/l GSSG and 0.25 mol/l L-arginine.

a) Time dependence of activation

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

[image]

b) Dependence of activated gain on L-arginine supplementation

The eluate was diluted 1:50 with 0.1 mol/l Tris/HCl, pH 8.5, 1 mmol/l EDTA, 5 mmol/l GSH, 0.5 mmol/l GSSG and activated for 20 hours at 0°C.

Dependence of activation on L-arginine

[image]

c) Dependence of gain on urea addition

The activating solution corresponded to the one from point b), but the activation was carried out for 17 hours at 0°C.

Dependence of activation on urea

[image]

d) Dependence of the activation gain on the addition of formamide

Activation as under b; we checked the samples after 17 hours of activation at 0°C.

Dependence of activation on formamide

[image]

e) Dependence of the activation gain on the redox buffer

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

Dependence of activation on GSH/GSSG

f) [image] Dependence of activation gain on protein concentration

We diluted the eluate 1:10 to 1:100 in 0.1; Tris/HCl, pH 8.5, 1 mM EDTA, 5 mM GSSG and 0.25 M L-arginine, and we controlled after 17 hours of incubation at 0°C.

cp-dependence of activation

[image]

g) Dependence of activation gain on BSA addition

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

BSA-dependent activation

[image]

h) Dependence of activation gain on pH

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

pH-dependence of activation

[image]

Claims (23)

1. Procedure for activating genetically prepared heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes with cell disintegration, solubilizer under denaturing conditions and reducing conditions, and activation under oxidizing conditions in the presence of GSH/GSSG, indicated by the fact that at the activation stage we work with a pH value of 9 to 12, a GSH concentration of 0.1 to 20 mmol/l, a GSSG concentration of 0.01 to 3 mmol/l, a non-denaturing concentration of a denaturing agent. 2. The method according to claim 1, characterized in that in the activation stage the pH value is 9.5 to 11. 3. The method according to one of claims 1 or 2, characterized in that in the activation stage the concentration of GSH is 0.2 to 10 mmol/1 and/or the concentration of GSSG is 0.05 to 1 mniol/l. 4. The procedure according to one of the earlier requirements, indicated by the fact that after solubilization and before activation, we perform a cleaning step. 5. The method according to one of claims 1 to 3, indicated by the fact that we perform activation without earlier separation of the denaturing/reducing agent, whereby the reaction solution after denaturation/reduction is diluted with an active buffer and during the next activation the concentration of GSSG exceeds the remaining residual concentration of DTE. 6. A variant of the procedure for activating genetically prepared, heterologous, eukaryotic proteins containing disulfide bridges, after expression in prokaryotes with cell disintegration, solubilization under denaturing and reducing conditions, and activation under oxidizing conditions in the presence of GSH, indicated that the reducing/denaturing agent separately, with the addition of GSSG under denaturing conditions, we convert the thiol groups of proteins into mixed disulfides of proteins and glutathione, and in the activation stage we work at a pH value of 7 to 10.5 and a GSH concentration of 0.5 to 5 mmol/1 and at non-denaturing concentrations of the denaturing agent . 7. The method according to one of claims 1 to 6, characterized in that we carry out the expression in E. coli or P. putida. 8. The method according to one of claims 1 to 7, characterized in that in the activation stage, as a denaturing agent, we use arginine, guanidine hydrochloride and/or at least one compound of the general formula R2-CO-NRR1(I), in which R and R1 , denote hydrogen: or alkyl with 1 to 4 carbon atoms, and R 2 denotes hydrogen or NHR 1 , or alkyl with 1 to 3 carbon atoms. 9. The method according to claim 8, characterized in that the concentration of arginine and/or guanidine hydrochloride is 0.1 to 1.0 mol/l, particularly preferably 0.25 to 0.8 mol/l. 10. The method according to claim 8, characterized in that the concentration of the compound of general formula I is 0.5 to 4 mol/1, particularly preferably 1 to 3.5 nol/1. 11. The method according to one of the earlier claims, characterized by the fact that in the activation step we work in the presence of a non-proteolytically effective protein, it is particularly advantageous in the presence of bovine serum albumin. 12. The procedure according to one of the previous claims, characterized in that cell disintegration is carried out using ultrasound, high-pressure dispersion or lysosomes. 13. The method according to claim 12, indicated by the fact that we carry out cell disintegration in a diluted aqueous buffer solution, particularly preferably in 0.1 mol/l Tris, at a neutral or slightly acidic pH value. 14. The method according to one of the previous claims, characterized in that after cell disintegration, we separate the insoluble ingredients. 15. The procedure according to one of the earlier requirements, indicated by the fact that in the solubilization stage we work at an alkaline pH value in the presence of a reducing agent from the mercapto group and in the presence of a denaturing agent. 16. The method according to claim 15, characterized in that we work in the presence of guanidine hydrochloride and/or a compound of general formula I as a denaturing agent. 17. The method according to claim 16, characterized in that the concentration of guanidine hydrochloride is 6 mol/l, the concentration of the compound of general formula I is 8 mol/l. 18. The method according to one of claims 15 to 17, characterized in that we work in the presence of DTE, β-mercaptoethanol or GSH. 19. The method according to one of the earlier claims, characterized in that the purification and separation of reducing, oxidizing and denaturing agents is carried out by means of steric exclusion chromatography or by means of dialysis. 20. The method according to one of the earlier claims, characterized in that after the activation step, we perform a cleaning step using dialysis. 21. The method according to one of claims 1 to 20, characterized in that we use t-PA as a genetically prepared eukaryotic protein. 22. The method according to claim 1 to 20, characterized in that interferon-β is used as a genetically prepared eukaryotic protein. 23. The method according to claim 6, characterized in that we separate the mixed. protein and glutathione disulfide using ion-exchange treatment from unmodified protein.