EP0253823A1

EP0253823A1 - Process for activating heterologous, eucaryotic proteins genetically engineered and presenting disulphide bridges after their expression in procaryotic cells

Info

Publication number: EP0253823A1
Application number: EP86906320A
Authority: EP
Inventors: Stephan Fischer; Ralf Mattes; Rainer Rudolph
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1985-10-23
Filing date: 1986-10-23
Publication date: 1988-01-27
Also published as: SK278317B6; ES2020498T3; DK200001897A; IL80325A0; EP0393725A1; PT83609A; PT83609B; CZ752686A3; DE3537708C2; KR870700601A; EP0393725B1; WO1987002673A2; HUT43643A; ES2020498A4; JPH04218387A; SK752686A3; DE3650449D1; FI94050C; ZA868012B; JPH0824594B2

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

Process for the activation of heterologous eukaryotic proteins produced by genetic engineering and having disulfide bridges after expression in prokaryotes

Description

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

When heterologous proteins are expressed in procarytes, these proteins often form inactive, poorly soluble aggregates (so-called “refractile bodies”) in the host cells, which are also contaminated with proteins from the host cells. It is assumed that the formation of such "refractile bodies" is a consequence of the high protein concentration in the cell that occurs during expression. It is known that when large amounts of enzyme are formed in the cell, the enzymes are aggregated into insoluble, high-molecular, mostly inactive particles. Before such proteins, e.g. B. for therapeutic purposes, they must therefore be cleaned and converted into their active form.

Known methods can be used to reactivate such proteins, which are present as aggregates, in several steps (see, for example, BR Jaenicke, FEBS Federation of European Biochemical Societies, Vol. 52 (1979) 187 to 198; R. Rudolph et al ., Bioche istry 1_8_ (1979) 5572 to 5575):

In the first step, solubilization is achieved by adding strong denaturing agents, for example guanidine hydrochloride or urea in high concentration, or by adding strongly acidic agents, for example glycine / phosphoric acid mixtures. Other auxiliary substances have decorative SH reagents (e.g. dithioerythritol, DTE) and EDTA, for example in the renaturation of LDH. If the protein is contaminated by proteins of the host cell, the next step is a purification using conventional methods known per se, e.g. B. gel or ion exchange chromatography. Then it is diluted strongly so that the concentration of the denaturing agent is reduced. If guanidine hydrochloride is used, it is diluted to values below 0.5 mol / 1. In the case of enzymes with free SH groups, the addition of agents protecting SH groups has proven advantageous (cf., for example, BR Jaenicke, Journal Polymer Science, Part C 16 (1967) 2143 to 2160).

EP-A-0114506 describes processes for the isolation, purification and reactivation of some heterologous expression products from bacterial cultures; for the reactivation, the solutions of the "refractile bodies" in a strong denaturing agent are a) transferred directly into a solution in a weaker denaturing agent, which is then subjected to oxidizing conditions to re-form disulfide bridges; b) sulfonating the protein, then transferring it to a solution in a weak denaturant, and treating the S-sulfonate groups by treating them with a sulfhydryl reagent in its reduced and oxidized form, e.g. B. with GSH / GSSG, transferred to -S-S groups; or c) the solution in a weak denaturant directly with the sulfhydryl reagent, e.g. B. with GSH / GSSG, treated. A typical example in which the problems set out above occur is t-PA.

The main component of the protein matrix of clotted Blood is polymeric fibrin. This protein matrix is dissolved by plasmin, which is formed from plasminogen via activation by the so-called plasminogen activators, e.g. B. by t-PA (tissue plasminogen activator, tissue-type plasminogen activator). The enzymatic activity of natural t-PA or genetically engineered from eukaryotes (catalytic activation of plasminogen to plasmin) is very low in the absence of fibrin or fibrin cleavage products (FSP), but can be increased significantly in the presence of these stimulators (by more than the factor 10). This so-called stimulability of the activity is a decisive advantage of t-PA over other known plasminogen activators, such as urokinase or streptokinase (cf. e.g. BM Hoylaerts et al., J. Biol. Chem. 257 (1982) 2912 to 2019; Nieuwenhiuzen et al., Biochemica et Biophysica Acta 755 (1983) 531 to 533). The factor of stimulability with BrCN cleavage products is therefore given in various ways in the literature and numbered up to 35.

A t-PA-like, non-glycosylated product is also formed in genetically manipulated prokaryotes (after the c-DNA has been introduced); such a product does not have the ability to stimulate the activity of a t-PA from eukaryotes. It is conceivable that this can be attributed to the fact that the redox conditions in the prokaryotic cell are so different from the eukaryotic cell from which the gene originates that an inactive product is formed from the beginning, which could be due, for example, to this that the numerous SS bridges that the natural active molecule contains are incorrectly linked or not formed at all. For the therapeutic The use of t-PA not only requires the enzymatic activity as such, but also its stimulability. The fact that the prokaryotic cell presumably does not create the right conditions in order to develop the activity of eukaryotic proteins in the right way is pointed out for other substances in The EMBO Journal 4_, No. 3 (1985) 775 to 780.

According to EP-A-0093639 to reactivate t-PA, the cell pellets obtained from E. coli are suspended in 6 mol / 1 guanidine hydrochloride, treated with ultrasound, incubated and then for four hours against a solution of Tris-HCl (pH = 8.0), sodium chloride, EDTA and Tween 80 dialyzed. After dialysis, centrifugation is carried out, the plasminogen activator activity being found in the supernatant. In this way, renatured t-PA is proteolytically active, but shows no measurable stimulation by BrCN fission products (BrCN-FSP) of fibrin, according to the method described in J. H. Verheijen, Thro b. Haemostas., 48, (3), 260-269 (1982).

No generally applicable method is known from the prior art for reactivating denatured proteins; this is especially true for t-PA because the native protein has a very complex structure; it contains a free thiol group and 17 SS bridges, which theoretically are 2.2 x 10 20 different

Wise men can be linked, whereby only one structure corresponds to the native state. The methods known from the prior art for reactivating t-PA lead to a proteolytically active t-PA, but it shows no measurable stimulability; an activation method which leads to stimulable t-PA is not known. The object of the present invention is therefore to provide a method for the complete activation of genetically engineered, heterogeneous eukaryotic proteins containing disulfide bridges after expression in prokaryotes; this object is achieved with the subject matter of the present invention.

The invention relates to a method for activating genetically engineered, heterologous, disulfide bridging eukaryotic proteins after expression in prokaryotes according to claim 1 by cell disruption, solubilization under denaturing and reducing conditions and activation (renaturation) under oxidizing conditions in the presence of GSH / GSSG, which is characterized in that in the stage of activation at a pH of 9 to 12, a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol / l, and works with a non-denaturing concentration of the denaturing agent.

Preferred embodiments of this method are the subject of the dependent claims.

A denaturing agent or arginine usually used for activations under oxidizing conditions can generally be used as the denaturing agent; guanidine hydrochloride or urea or its derivatives is preferably used among the known denaturing agents. Arginine has also proven to be suitable. Mixtures of these denaturants can also be used. This activation step is preferably also carried out in the presence of a foreign protein; suitable as such usually any foreign protein, as long as it is not proteolytically active; preferably bovine serum albumin (BSA) is used, e.g. B. in an amount of 1 to 3 mg / ml. The addition of BSA causes a slight increase in the yield and stabilization of the protein (probably through protection against surface denaturation and / or proteolytic degradation).

The other process conditions can correspond to the conditions known and customary for reactivation stages from the prior art. The duration of the activation (incubation) is preferably 20 to 48 hours at room temperature. The half-life of the activation in the presence of 0.5 mmol / l reduced (GSH) and oxidized (GSSG) glutathione is about 10 to 15 hours at 20 ° C. With a longer incubation (48 hours) under reoxidation conditions, the stimulability with CNBr-FSP usually decreases. The activation stage is preferably carried out in the presence of EDTA, the most appropriate concentration being approximately 1 mmol / l EDTA.

The process steps preceding and following the activation stage (reoxidation / activation), such as cell disruption, solubilization (solubilization / reduction) and optionally one or more cleaning operations preceding and / or following the activation stage can be carried out according to the prior art, e.g. B. from EP-A-0114506, EP-A-0093619, methods known and customary for such processes can be carried out; However, for an optimal result with regard to yield and activation, it can be expedient to carry out individual or all process steps taking into account one or more of the process configurations explained here. In particular it is also possible to carry out the activation step according to the invention in the mixture obtained after the digestion without prior denaturation and / or reduction, but with a low yield. Expression is carried out in prokaryotes, preferably in P. putida, and in particular in E. coli. However, the method according to the invention is also suitable if expression is carried out in other prokaryotes (eg Bacilli).

The cell disruption can be carried out by methods customary for this purpose, e.g. B. by means of ultrasound, high pressure dispersion or lysozyme; it is preferably carried out as a suspension medium in a buffer solution suitable for adjusting a neutral to weakly acidic pH, such as, for. B. in 0.1 mol / 1 Tris-HCl. After cell disruption, the insoluble constituents ("refractile bodies") are separated in any manner, preferably by centrifuging at higher g numbers and a longer centrifugation time or by filtration. After washing with agents that do not interfere with t-PA, but dissolve foreign cell proteins as much as possible, e.g. B. water, phosphate buffer solution, optionally with the addition of mild detergents such as Triton, the precipitation (pellet) is subjected to solubilization (solubilization / reduction). The solubilization is preferably carried out in the alkaline pH range, in particular 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 from the prior art, for. B. from EP-A-0114506, known and conventional denaturing agents be, and especially guanidine hydrochloride or urea. The concentration of guanidine hydrochloride is advantageously about 6 mol / 1, that of urea about 8 mol / 1. Compounds of the general formula I can also be used.

As a reducing agent from the mercaptan group z. B. reduced glutathione (GSH) or 2-mercaptoethanol, z. B. in a concentration of about 50 to 400 mmol / l and / or in particular DTE (dithioerythritol) or DTT (dithiothreitol), for. B. in a concentration of about 80 to 400 mmol / l. The solubilization expediently takes place at room temperature for a period (incubation) of 1 to several hours, preferably of two hours. To prevent oxidation of the reducing agent by atmospheric oxygen, it can also be expedient to add EDTA. In addition to solubilization / reduction, the solubilization stage also has a cleaning effect, since a large part of material (foreign proteins) that does not cross-react with t-PA (foreign proteins) does not dissolve.

After the solubilization and before the activation stage, known and customary cleaning stages can be inserted; come as cleaning methods such. B. steric exclusion chromatography (SEC) (in the presence of guanidine hydrochloride or urea) or ion exchanger (in the presence of urea or its derivatives) in question; unspecific reoxidation can be prevented by adding a reducing agent (eg 2-mercaptoethanol) or by pH values of 4.5 (see eg BR Rudolph, Biochem. Soc. Transactions 1_3_ (1985) 308 to 311). If DTE was used in the previous solubilization stage, this can be separated in a cleaning stage. The cleaning can e.g. B. by SEC over Sephadex G 100 in the presence of guanidine hydrochloride and a Reduk¬ tion agent, eg. B. from GSH at a pH of 1 to 4 (in this step a large amount of the foreign protein can be separated); or by removing the denaturing / reducing agents by desalting over Sephadex G 25 in 0.01 mol / 1 HC1 or 0.1 mol / 1 acetic acid. Alternatively, the denaturing / reducing agents can be separated off by dialysis against the same solutions.

Another cleaning step can follow the reactivation stage; Such purification is generally carried out by means of dialysis, or else a subsequent isolation of the activated tPA, for example by affinity chromatography, for example using Lys-Sepharose.

Another embodiment of the invention is based on the formation of the mixed disulfides of genetically engineered, heterologous, disulfide bridging eukaryotic proteins and glutathione (hereinafter abbreviated t-PASSG). This can facilitate both the separation of foreign proteins in the denatured state and the further purification of the native protein. Purification after modification of the thiol groups has the advantage that the protein is protected against air oxidation and is therefore stable in a larger pH range, and a change in the net charge makes cleaning easier. In particular, separation from the unmodified protein can advantageously be carried out by ion exchange treatment.

To form the mixed disulfides, the dialysis Incubated, reduced protein, purified from denaturing and reducing agents, incubated with a diluted, for example 0.2 mol / l, solution of GSSG containing a denaturing agent. The activation was carried out after removal of the denaturing and oxidizing agents at a pH 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 .

In all other reaction steps, the activation of the protein via the formation of the mixed disulfides with GSSG corresponds to the embodiments for the activation of the aforementioned part of the invention. In this embodiment, the pH optimum is 8.5, the yield is approximately twice as high and the activated protein is stable in the renaturation buffer over a long period of time.

According to the invention it is possible to activate t-PA from prokaryotes in such a way that not only activation of the normal biological activity is achieved, but also a stimulability in the sense defined above is achieved which far exceeds the stimulability of the native t-PA and is greater than a factor 10 is, may even exceed a factor of 50.

Another eukaryotic protein that can be activated according to the invention after expression in prokaryotes is β-interferon.

The following examples illustrate the invention in more detail without restricting it. Unless otherwise stated, percentages relate to percentages by weight and temperatures refer to degrees Celsius. Example 1

a) Preparation of the "refractile bodies"

100 g of E. coli wet cell mass, taken up in 1.5 1, 0.1 mol / 1 Tris / HCl (pH 6.5) and 20 mmol / l EDTA were homogenized (Ultra-Turrax, 10 seconds) and 0.25 mg / ml lysozyme added. After 30 minutes of incubation at room temperature, the mixture was homogenized again and cooled to 3 ° C. The cell disruption was achieved by high pressure dispersion (550 kg / cm ² ). It was then rinsed with 300 ml of 0.1 mol / l Tris / HCl (pH 6.5) and 20 mmol / l EDTA. After centrifugation (.2 hours at 27,000 g, 4 ° C) the pellet was in 1.3 1 0.1 mol / l Tris / HCl (pH 6.5), 20 mmol / l EDTA and 2.5% Triton -x-100 recorded and homogenized. After renewed centrifugation (30 minutes at 27,000 g, 4 ° C.), the pellet was dissolved in 1.3 l 0.1 mol / l Tris / HCl (pH 6.5), 20 mmol / l EDTA and 0.5 % Triton-x-100 added and homogenized. Alternating centrifugation (30 min at 27,000 g, 4 ° C) and homogenization of the pellet in 1 1 0.1 mol / l Tris / HCl (pH 6.5) and 20 mmol / l EDTA was carried out three more times.

The t-PA content of the "refractile bodies" preparations was quantified by SDS-PAGE, identification of the t-PA bands by "Western blotting" and densitometric analysis. In SDS-PAGE and "Western blotting" the "refractile bodies" show a strong t-PA band with a molecular weight of approx. 60 kDa. The t-PA portion of the total protein content of the "refractile bodies" is approximately 21%. b) solubilization / reduction of the "refractile bodies"

"Refractile bodies" were at a protein concentration of 1 to 5 mg / ml 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 incubated for 2 to 3 hours at room temperature. Insoluble material (cell wall fragments, etc.) was then centrifuged off (for example 30 minutes at 35,000 to 50,000 g, 4 ° C.). The pH of the supernatant was conc. HCl adjusted to pH 3. Denaturing and reducing agents were then removed by dialysis against 0.01 mol / l HCl at 4 ° C.

c) reoxidation / activation

Reoxidation / activation was carried out by 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 activation for 17 to 24 hours at approx. 20 ° C., the activity and, compared to the activity of native glycosylated t-PA, the yield from eukaryotes were determined.

Yield based on the total protein content of the "refractile bodies": 2.5 +/- 0.5% stimulability: 10 +/- 5

Yield based on the t-PA portion of the "refractile bodies": approx. 12% d) reoxidation / activation without removal of the denaturing / reducing agents

"Refractile bodies" were at a protein concentration of 1.25 mg / ml in 0.1 mol / l Tris / HCl (pH 8.6), 6 ml / 1 guanidine hydrochloride, 0.2 mol / l DTE and 1 mmol / l EDTA incubated for 2 hours at room temperature. Thereafter, reoxidation was immediately carried out by 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 amounts of GSSG indicated in the table. In addition, there was a residual concentration of 0.06 mol / l guanidine hydrochloride and 2 mmol / l DTE in the activation mixture.

The activation yield is dependent on the GSSG concentration when activated without removing the denaturing / reducing agents.

GSSG yield 'stimulability

(mmol / l) (%) (factor)

0.2 0 -

1 0.13 4.0

5 1.49 7.4

6 1.28 5.4

7 1.04 5.8

9 0.98 5.2

10 1.77 10.0

15 0 -

20 0 _

'= Yield of active t-PA based on the total protein content of the "refractile bodies". Example 2

An RB ("refractile bodies") preparation (approx. 5 mg) was dissolved in 1 ml 0.1 mol / l Tris / HCl (pH = 8.6), 6 mol / l guanidine hydrochloride and 0.15-0 , 2 mol / l DTE incubated for 2 to 3 hours at room temperature. Insoluble material (cell wall fragments, etc.) was then separated by centrifugation (20 minutes at 17,000 g). Denaturing and reducing agents were removed by gel filtration over Sephadex G 25 (superfine) in 0.01 mol / l HCl. The sample was diluted by a factor of 5 to 10. The reduced material in 0.01 mol / l HCl was stored at -20 ° C.

Example 3

The influence of various parameters according to the invention on the activation and stimulability of t-PA is summarized in the following tables. For these reoxidation experiments, the reduced protein solubilized according to Example 2 was not further pre-purified.

The reduced protein (in 0.01 mol / l HCl) was activated by dilution from 1:10 to 1: 500 in "reoxidation buffer". Activation was determined after 22 to 48 hours of incubation at room temperature. The activity of the reoxidized protein refers to a "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).

The stimulability is calculated from Δ ^E + cκBrF _S P ' ^{/ ΔE} -CNBrFS (cf. W. Nieuwenhuizen et al., Biochimica et Biophysica Acta 755 (1983) 531 to 533). The activity (in percent) and the stimulability (factor) was according to JH Verheijen Thromb. Haemostas. 48 (3), 266-269, (1982).

The following results were obtained:

1. Dependence of the activation yield on the addition of L-arginine or 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

L-arginine) activity stimulability

(mol / l) (%) (factor)

0 4 2.5

0.25 98 7.5

0.5 100 21.9

0.75 27 16.3

1.0 23 3.5 In this experiment it should be borne in mind that t-PA is inhibited by L-arginine. The decrease in the activation yield at higher L-arginine concentrations must therefore be corrected for the inhibition.

b) Guanidine hydrochloride (Gdn / HCl)

(Gdn / HCl) activity

(mol / l) (%)

0 11

0.25 22

0.5 53

0.75 58

1.0 12

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

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

a) urea

Urea activity

(mol / l) (%)

0 1 0.5 20

1 59 1.5 126

2 162 2.5 141

3 72

4 12

5 0 b) methyl urea

Methyl urea activity

(mol / l) (%)

0.5 22

1 174

1.5 313

2,375

2.5 332

3 215

4 12

5 0

c) ethyl urea

Ethyl urea activity

(mol / l) (%)

0.5 46

1 212

1.5 323

2,300

2.5 107

3 19

4 0

5 0

d) dimethyl urea

Dimethyl urea activity stimulability (mol / l) (%) (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 fatty acid amides:

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

Formamide activity

(mol / l) (%)

0 42

0.5 59

1 175

1.5 245

2 325

2.5 423

3,444

4 416

5,341

b) methylformamide

Methylformamide activity

(mol / l) (%)

0.5 100

1 135

1.5 304

2 389

2.5 466

3 452

4,425

5 121

c) acetamide

Acetamide activity

(mol / l) (%)

0.5 72

1 134

1.5 207

2 261

2.5 204

3,237

4 198

5 141 d) propionamide

Propionamide activity

(mol / l) (%)

0.5 95

1 99

1.5 197

2 150

2.5 101

3 39

4 2

5 0

e) butyramide

Butyramide activity

(mol / l) (%)

0.5 55

1 52

1.5 17

2 0

Dependence of the activation yield on the pH value

Reoxidation in 0.1 mol / l Tris / HCl + 1 mmol / l EDTA + 0.5 mol / l L-arginine + 1 mg / ml BSA + 0.5 mmol / l GSH + 0.5 mmol / l GSSG

pH activity stimulability

(%) (Factor)

7 1 __

8 22 3.0

9 89 13.6

10 105 20.3

11 95 21.3

Dependence of the activation yield on the 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

(GSSG) activity stimulability

(mmol / l) (%) (factor)

0.1 239 14.9

0.2 273 15.3

0.5 193 13.3

1 198 12.5

5 17 2.1

10 0

20 0 * ~

b) + 0.2 mmol / l GSSG

(GSH) activity stimulability

(mmol / l) (%) (factor)

0.05 15 2.2

0.1 40 3.8

0.2 112 6.8

0.5 142 7.4

1,273 6.8

5,260 7.9

10 143 6.3

20 55 5.1

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

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

+ 0.5 mmol / l GSH

+ 0.5 mmol / l GSSG

Dilution activity stimulability

(%) (Factor)

1:10 29 15.3

1:20 45 25.4

1:50 69 37.9

1: 100 100 37.9

1: 200 79 52.7

1: 500 29 28.7 7. Dependence of the activation yield on the addition of BSA

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

BSA activity

(mg / ml) (%)

0 47

0.5 83

1 100

3 102

5 52

Figures 1 and 2 show the activity with and without CNBr-FSP in the 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-arginine + 1 mg / ml BSA + 0.5 mmol / l GSH + 0.5 mmol / l GSSG. 1 and 2, curves (A) denote the activity in the presence of CNBr-FSP, and curves (B) denote the activity without CNBr-FSP.

Example 4

Activation of t-PA via the mixed disulfides of t-PA and glutathione.

The "refractile bodies" used were obtained according to one of the previous examples. The reduction of "Refractile bodies" was incubated for 2 hours at room temperature in 0.1 mol / l Tris / HCl, pH 8.6, 1 mmol / 1 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 conc. HCl to pH 3 was followed by dialysis against 0.01 mol / l HCl at 4 ° C. After dialysis, the total protein concentration was 0.33 mg / ml. The optimal reactivation conditions were determined with the t-PASSG prepared in this way.

a) pH optimum of the activation of t-PASSG

Here, as in the following optimization experiments, (1) no GSSG was used and (2) the activation was determined after 17 hours of incubation at room temperature. Activation was carried out by a 1: 100 dilution in 0.1 mol / l Tris, 1 mmol / l EDTA, 0.5 mol / l L-arginine, 1 mg / ml BSA and 2 mmol / l GSH with variation of the pH .

pH yield (%) stimulability

6 0.04 3.3

6.5 0.37 9.5

7 1.35 11.4

7.5 5.66 7.1

8 7.32 8.2

8.5 8.65 7.0

9 8.59 8.7

9.5 8.32 11.7

10 6.15 12.5

10.5 3.07 11.2 The yield was determined in% active t-PA based on the amount of protein used.

b) Reproducibility of the results of the activation of t-PASSG

With identical activation conditions, different yields are observed in different series of measurements, which include are caused by fluctuations in the standard t-PA. To clarify this range of errors, all activation data after 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 combined.

Trial yield (%) stimulability

Mean 5.1 +/- 1.9 11.3 +/- 3.8

σ) Stability of the activated protein

In the example mentioned, activation was carried out by 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 .

Time PH 9.5

(h) Education Forehead.

(%)

1 0

6 0.89 15.5

23 2.43 23.1

47 2.83 23.6

71 2.62 21.5

215 2.21 22.6

239 2.28 14.3

Example 5

Activation of genetically engineered interferon-ß.

"Refractile bodies" were produced using the aforementioned methods. The reduction / solubilization of the "refractile bodies" was carried out as follows: The 'pellet was 3 hours at 25 ° C in 10 ml 9.1 mol / l Tris / HCl, pH 8.6, 6 mol / l Gdn'HCl, 1 mmol / l EDTA and 0.2 mol / l DTE and after 30 minutes centrifugation at 4 ° C. and 48,000 g the pH of the supernatant was adjusted to about 3 with concentrated HCl. Then gel filtration was carried out over Sephadex G25 F in 0.01 mol / l HCl.

The eluate was examined for conductivity, protein concentration and reactivability. The activity of the reoxidized protein relates to a "standard activation" (= 100%) 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) Dependence of the activation yield for the addition of L-arginine

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 at 0 ° C. for 20 hours .

L-arginine dependence of activation

L-arginine (mol / l) activity (%)

0 8

0, 25 8

0, 5 15

0, 75 15

b) dependence of the activation yield on the addition of urea

The activation solution corresponded to that of point a); however, it was activated at 0 ° C for 17 hours.

Urea dependence of activation

Urea (mol / l) activity (%)

Ö 13

0.5 100

1,200

1.5 100 c) dependence of the activation yield on the addition of formamide

Activation as in a); the samples were examined after 17 hours activation at 0 ° C.

Formamide dependence of the activation of formamide (mol / l) activity

0 13 1 13 2 13 3 0

4 0

d) dependence of the activation yield on the redox buffer

The eluate was 1:50 in 0.1 mol / l Tris / HCl, pH 8.5,

Diluted 1 mmol / l EDTA and 0.25 mol / l L-arginine and the

Samples examined after 17 hours activation at 0 ° C.

GSH / GSSG dependency of the activation

GSH (mmol / l) GSSG (mmol / l) activity (%) _

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 e) dependence of the activation yield on the addition of BSA

The eluate was 1:50 in 0.1 mol / l Tris / HCl, pH 8.5, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG and 0.25 mol / l L- Diluted arginine and examined after 17 hours of activation at 0 ° C.

BSA dependence of activation BSA (mg / ml) activity (%)

0 13 1 13 2 25 5 13

f) Dependence of the activation yield on the pH

The eluate was diluted 1:50 in 0.1 mol / l Tris / HCl, 1 mmol / l EDTA, 5 mmol / l GSH, 0.5 mmol / l GSSG and 0.25 mol / l L-arginine and after 17 Hours of activation examined at 0 ° C.

pH dependence of the activation pH activity (%)

10.5 100

Claims

Patent claims

1. A process for activating genetically engineered, heterologous eukaryotic proteins containing disulfide bridges after expression in prokaryotes by cell disruption, solubilization under denaturing and reducing conditions and reactivation under oxidizing conditions in the presence of GSH / GSSG, characterized in that in the reactivation stage at a pH of 9 to 12, a GSH concentration of 0.1 to 20 mmol / l, a GSSG concentration of 0.01 to 3 mmol / l and a non-denaturing concentration of the denaturing agent works.

2. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that in the reactivation stage, the pH is 9.5 to 11.

3. The method according to any one of claims 1 and 2, so that the GSH concentration in the reactivation step is 0.2 to 10 mmol / l and / or the GSSG concentration is 0.05 to 1 mmol / l.

4. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that one carries out a cleaning step after the solubilization and before the reactivation.

5. The method according to any one of claims 1 to 3, characterized in that the reactivation without prior separation the denaturing / reducing agent is carried out, the reaction solution after denaturing / reduction being diluted with reactivation buffer and in the subsequent reactivation the GSSG concentration exceeds the remaining residual concentration of DTE.

Modified method for activating genetically engineered, heterologous, disulfide bridges having eukaryotic proteins after expression in prokaryotes by cell disruption, solubilization under denaturing and reducing conditions, and reactivation under oxidizing conditions in the presence of GSH, characterized in that the reduction - / Denaturing agent is separated, by adding GSSG under denaturing conditions, the thiol groups of the proteins are converted into the mixed disulfides of protein and glutathione, in the reactivation step at a pH of 7 to 10.5 and a GSH concentration of 0 , 5 to 5 mmol / l and works with a non-denaturing concentration of the denaturing agent.

Method according to one of claims 1 to 6, that the expression in E. coli or P. putida is carried out.

Process according to one of Claims 1 to 7, characterized in that arginine, guanidine hydrochloride is used as the denaturing agent in the reactivation step and / or at least one compound of the general formula R _^ -CO-NRR. (I), in which R and RH or alkyl having 1 to 4 carbon atoms and R_H or NHR. or alkyl having 1 to 3 carbon atoms.

9. The method according to claim 8, that the concentration of arginine and / or guanidine hydrochloride is 0.1 to 1.0 mol / l, in particular 0.25 to 0.8 mol / l.

10. The method according to claim 8, d a d u r c h g e k e n n z e i c h n e t that the concentration of the compound of general formula I is 0.5 to 4 mol / l, in particular 1 to 3.5 mol / l.

11. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that in the reactivation step in the presence of a non-proteolytically active protein, in particular in the presence of

Bovine serum albumin, works.

12. The method according to any one of the preceding claims, that the cell disruption is carried out by means of ultrasound, high-pressure dispersion or lysozyme.

13. The method according to claim 12, characterized in that the digestion is carried out in a dilute aqueous buffer solution, in particular in 0.1 mol / l Tris, at a neutral to weakly acidic pH.

14. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that one separates the insoluble components after cell disruption.

15. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that one works in the solubilization step at an alkaline pH 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, d a d u r c h g e k e n n z e i c h n e t that one works in the presence of guanidine hydrochloride and / or compound of general formula I as denaturing agent.

17. The method according to claim 16, so that the concentration of guanidine hydrochloride is 6 mol / l, that of the compound of the general formula I 8 mol / l.

18. The method according to any one of claims 15 to 17, d a d u r c h g e k e n n z e i c h n e t that one works in the presence of DTE, ß-mercaptoethanol, cysteine or GSH.

19. The method according to any one of the preceding claims, characterized in that cleaning and separation of reducing, oxidizing or denaturing agents is carried out by means of steric exclusion chromatography or dialysis.

20. The method according to any one of the preceding claims, d a d u r c h g e k e n n z e i c h n e t that one carries out a cleaning step by means of dialysis after the reactivation stage.

21. The method according to any one of claims 1 to 20, d a d u r c h g e k e n n z e i c h n e t that t-PA is used as the genetically engineered eukaryotic protein.

22. Stimulable non-glycosylated tPA, obtainable by the method according to one of claims 1 to 21.

23. The method according to any one of claims 1 to 20, d a d u r c h g e k e n n z e i c h n e t that interferon ß is used as the genetically engineered eukaryotic protein.

24. The method according to claim 6, d a d u r c h g e k e n n z e i c h n e t that the mixed disulfide of protein and glutathione is separated from the unmodified protein by ion exchange treatment.