AU607083B2

AU607083B2 - Process for the activation of genetically engineered, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes

Info

Publication number: AU607083B2
Application number: AU41321/89A
Authority: AU
Inventors: Stephan Fischer; Ralf Mattes; Rainer Rudolph
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1985-10-23
Filing date: 1989-09-13
Publication date: 1991-02-21
Anticipated expiration: 2006-10-23
Also published as: KR870700601A; FI933868A0; DK175091B1; PT83609A; SK752686A3; WO1987002673A2; DK175109B1; CZ752686A3; DE3537708C2; YU47185B; HRP921075A2; IL80325A; DE3537708A1; EP0393725A1; ES2020498A4; IE62634B1; EP0219874A3; SI8611796A; FI872753A0; AU590029B2

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

Q;3 COMMONWEALTH OF AUSTRALIA PATENT ACT 1952 COMPLETE SPECIFICATION FOR OFFICE USE CLASS INT. CLASS Application Number: Lodged: Complete Specification Lodged Accepted Published: Priority: S Related Art: This document contains the am ildments mad under Section 49 and is correct for Sprinting.

S*

NAME OF APPLICANT ADDRESS OF APPLICANT NAME(S) OF INVENTOR(S) ADDRESS FOR SERVICE: BOEHRINGER MANNHEIM GmbH Sandhofer Strasse 112-132, D-6800 Mannheim-Waldhof, FEDERAL REPUBLIC OF GERMANY Rainer RUDOLPH Stephan FISCHER Ralf MATTES DAVIES COLLISON, Patent Attorneys 1 Little Collins Street, Melbourne. Vic. 3000.

4 4 COMPLETE SPECIFICATION FOR THE INVENTION ENTITLED: "PROCESS FOR THE ACTIVATION OF GENETICALLY ENGINEERED, HETEROLOGOUS, DISULPHIDE BRIDGE-CONTAINING EUKARYOTIC PROTEINS AFTER EXPRESSION IN PROKARYOTES" The following statement is a full description of this invention, including the best method of performing it known to us: -1 I I Description 100 The invention concerns a process for the activation of genetically engineered, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes.

In the case of the expression of heterologous proteins in prokaryotes, in the host cells these proteins often form inactive, sparingly soluble aggregates (socalled "refraclie bodies") which,. in addition, are also contaminated with proteins of the host cells. It is assumed that the formation of such "refractile bodies" is a result of the high protein concentrations in the cell arising in the case of the expression. It is known that,, in the case of the formation of large amounts of enzymes in the cell, the aggregation of the enzymes to insoluble, high molecular, mostly inactive particles takes place Before such proteins can be used, e.g. for therape'utic purposes, they must consequently be purified and converted into their active form.

According to known processes, a reactivation of such proteins present as aggregates takes place in several steps (cf, e.g. R, Jaenicke, FEBS Federation of European Biochemical Socities, Vol. 52 (1979) 187 to 198; R. Rudolph et al., Biochemistry 18 (1979) 5572 to 5575).

In the first step,, a solubilisation is achieved by the addition of strong denaturing agents, for example guanidine hydrochloride or urea, in high concentration or by the addition of strongly acidic agents, for example -2- *000 0 0 *0 0 00 0 0 0* *000 0 0 *0 0 00 0 0 0 0

C

glycine/phosphoric acid mixtures. As further adjuvants,.

there have proved useful reducing SH reagents (e.g.

dithioerythritol, DTE) and EDTA, for example in the renaturing of IDH., Insofar as the protein is contaminated by proteins of the host cells, as next step there follows a purification with per se known and usual methods, e.g.

gel or ion exchanger chromatography. Subsequently, it is highly diluted in order that the concentration of the denaturing agent becomes smaller. In the case of the use of guanidine hydrochloride, it is thereby diluted to values below 0.5 mole/l. In the case of enzymes with free SH groups, the addition of agents protecting the SH groups proves to be advantageous e.g. R. Jaenicke, Journal Polymer Science, Part C, 16 (1967) 2143 to 2160).

In EP-A-0114506, processes are described for the isolation,: purification and reactivation of some heterologous expression products from bacterial cultures; for the reactivation,, the solutions of the "refractile bodies" Sin a strong denaturing agent are a) transferred directly into a solution of a weaker denaturing agent which is then subjected to oxidising Sconditions for the reformation of disulphide bridges; b) the protein is sulphonated, then transferred into a solution in a weak denaturing agent and the S-sulphonate groups are converted into group by treatment with a sulphhydryl :-eagent in its reduced and oxidised form, e.g. with GSH/GSSG; or c) the solution in a weak denaturing agent is treated directly with the sulphhydryl 3 L-

L

r Z i t 0S 0

S.

S S 0 reagent, e.g. with GSH/GSSG. A typical example in which the above-discussed problems arisesis t-PA.

The main component of the protein matrix of coagulated 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. by t-PA (tissue-type plasminogen activator). The enzymatic activity of natural t-PA or of t-PA obtained by genetic engineering from eukaryotes (catalytic activation of plasminogen to plasmin) is very low in the absence of fibrin or fibrin cleavage products (FCP) but can be substantially increased in the.'presende of these stimul&aors (by more than a factor of 10), This so-called stimulatability of the activity is a decisive advantage of -t-PA incomparison with other known plasminogen activators, such as urokinase or streptokinase (cf. e.g.

M. Hoylaerts et al., J. Biol. Chem..,, 25 (1982) 2912 to 2019; Nieuwenhiuzen et Biochemica et Biophysita Acta 755 (1983) 531 to 533). Therefore, the factor of the stimulatability with BrCN cleavage products is variously given in the literature and given a value of up to A t-PA-like, non-glycosilated product is also fbrmed in genetically manipulated prokaryotes (after introduction of the c-DNA); however, such a product does not have the stimulatability of the activity of a t-PA from eukaryotes. It is conceivable that the reason for this is that the redox conditions inthe prokaryote: cell i-

S

SS S 0 50 5*

S

55.55.

t differ in such a way from eukaryote cells from which the gene originates that, ab initio, a non-active product is formed which, for example,, could be due to the fact that numerous SS bridges which the natural active molecule contains are linked in a false way or are not even formed, However, for the therapeutic use of t-PA,! there is necessary not only the enzymatic activity as such but, in addition, also its stimulatability. Regarding the fact that the prokaryote cells presumably do not provide the correct conditions in order to form the activity of S. eukaryotic proteins in the correct way, reference is Smade to other substances in the EMBO Jpurnal 4, No.3 (1985) 775 to 780.

t According to EB-A-0093639, for the reactivation of t-PA,, the cell pellets obtained from E. coli are suspended in 6 mole/l. guanidine hydrochloride,, treated S* with ultrasonics, incubated and subsequently dialysed for four hours against a solution of tris-HC1 (pH sodium chloride, EDA and Tween 80. After dialysis, it is centrifuged,, whereby the plasminogen activator activity S is to be found in the supernatant, t-PA renatured in this way is admittedly proteolytically active but shows no measureable stimulatability by BrCN cleavage products (BrCN-fgP) of fibrin according to the process described in J.H. Vereijen, Thromb. Haemostas., 48 260-269 (1982).

For the reactivation of denatured proteins, no generally useable process is known from the state of

C

the art; this applies quite especially for t-PA because the native protein possesses a very complex structure; it contains a free thiol group and 17 SS bridges which theoretically can be linked in 2.2 x 1020 different ways, whereby only one structure corresponds to the native state. The processes known from the state of the art for the reactivation of t-PA but which shows no measurable stimulatability; an activation process which leads to stimulatable t-PA is not known.

Therefore, it is an object of the present invention to make available a process for the complete activation of genetically engineered, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes.

15 According to the embodiment of the present invention broadly claimed herein, there is provided a process for the autivation of genetically engineered, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes by cell 20 digestion and separation of insoluble components, solubilisation of said insoluble components under denaturing and reducing conditions, and reactivation under oxidising conditions in the presence of GSH, characterised in that the reducing/denaturing agent is separated off, the thiol groups of the proteins are converted into mixed disulphides of protein and glutathione by addition of GSSG under denaturing conditions, and the reactivation step is carried out at a 6 pH value of 7 to 10.5, a GSH concentration of 0.5 to mmole/1, and with a non-denaturing concentration of the denaturing agent.

As denaturing agent, there can as a rule, be used a denaturing agent usually employed for activation under oxidising conditions or arginine; amongst the known denaturing agents, there is preferred guanidine hydrochloride or urea or its derivatives. Furthermore, arginine has proved to be useful. Furthermore, mixtures 6f these denaturing agents can be used. This activation step ispreferably also carried out in the presence of a foreign protein; as such,. there is, as a rule, suitable any foreign protein, so long as it is not proteolytically S* active; bovine serum albumin (BSA) is preferably used, 15 in an amount of 1 to 3 mg./ml. The addition of BSA brings about a slight increase of the yield and stabilisation of the protein (probably by protection against o •O surface denaturing and/or proteolytic breakdown.

The other process conditions can correspond to 20 the known and usual conditions for the reactivation steps from the prior art. The period of the activation (incubation) preferably amounts to 20 to 48 hours at room temperature. The half life time of the activation lies, in the presence of 0.5 mmole/1, reduced (GSH) and oxidised (GSSG) glutathione,at about 10 to 15 hours at 20 0 C, In the case-of a longer incubation (48 hours) under reoxidation conditions, as a rule the stimulatability by CNBr-FCP decreases. The activation step is preferably carried out in the presence of EDTA, whereby 7 the most expedient ooncentration amounts to about 1 mmole/1, EDTA.

The process steps preceding and following the activation step (reoxidation/activation), such as cell digestion, solubilisation (solubilisation/reduction) and possibly one or more of the purification operations preceding and/or following the activation step, can be carried out according to known and usual methods for such prooesses from the prior art, e.g. from EP-A *oo@: 10 0114506 and EP-A-0095619; however, for a result which is So, optimal with regard to yield and activation, it can be expedient to carry out individual or all process steps having regard to one or more of the here-described process embodiments. In particular, it is also possible to carry-out' the step of activation qccording to the invention in the mixture obtained after digestion without previous denaturing and/or reduction but with lower yield.

The expression is carried out in prokaryotes, preferably in P. putida and especially in E. coli. However, the process according to the invention is just as suitable when one expresses in other prokaryotes Bacilli).

The cell digestion can be carried out by methods usual herefor, e.g. by means of ultrasonics, high pressure dispersion or lysozyme; it is preferably carried out in a buffer solution suitable for the adjustment of a neutral to weakly acidic pH value as suspension medium, such as e.g. in O.1 mole/I, tris HC1. After the cell digestion, the insoluble components ("refractile bodies") are -8separated off in any desired way, preferably by centrifuging off at comparatively high g values and comparatively long contrifuging timee or by filtration. After washing with agents which do not disturb t-PA but possibly dissolve foreign proteins, e.g. water,. phosphate buffer solution,. possibly with the addition of mild detergents, such as Triton,. the precipitate (pellet) is subjected to the solubilisation (solubilisation/reduction).

The solubilisation preferably takes place in the alkaline pH range, especially at pH 8,6 0.4 and in the presence of a reducing agent of the mercaptan group and of a denaturing agent.

As denaturing agent, there can be used the denaturing agents known and usual from the state of the art, e.g. from EP-A-0114506,. and especially guanidine hydrochloride or urea. The concentration of guanidine hydrochloride expediently amounts to about 6 mole/l.,, that of urea to about 8 mole/l.. Compounds of the general formula I can also be used:

R

2 -CO-NRRi (J) in which R and R 1 signify H or an alkyl with 1-4C atoms, and R 2 signifies H or NHR 1 or alkyl with 1-3 C atoms.

i As reducing agent from the mercapto group, there can be used reduced glutathione (GSH) or 2-mercaptoethanol, e.g. in a concentration of about 50 to 400 S mmole/1. and/or especially DTE (dithioerythritol) or DTT (dithiothreitol), in a concentration of about to 400 mmole/l. The solubilisation expediently takes place at room temperature over a period (incubation) of 1 to several hours, preferably of two hours. For the S .prevention of oxidation of the reducing agent by 0 eS 0 e 0* S 0 9a

S

l atmospheric oxygen,, it can be expedient to add EDTA.

Besides the solubilising/reduction, the solubilising step also has a purification effect since a large part of material (foreign proteins) not cross-reacting immunologically with t-PA goes into solution.

After the solubilisation and before the activation step, per se known and usual purification steps can be introduced; as purification methods, there come into question, e.g. steric exclusion chromatography 10 (SEC)(in the presence of guanidine hydrochloride or urea) o* or ion exchangers (in the presence of urea or its derivatives); a non-specific reoxidation can be prevented by addition of a reducing agent 2-mercaptoethanol) or by pH value 4.5 (cf. e.g. R. Rudolph, Biochem. Soc.

Transactions, 15 (1985) 308 to 311). If DTE was used in the preceding solubilisation step,, this must be separated off in the purification step. The purification can take place e.g. by SEC over.Sephadex G 100 in the pres ence of guanidine hydrochloride and of a reducing agent, of GSH, at a pH of 1 to 4 (in this step,.

a large amount of foreign protein can be separated off); or by separating off of the denaturing/reducing agent by desalination over Sephadex G 25 in 0.01 mole/1.

HC1 or 0.1 mole/1, acetic acid. Alternatively, the separating off of the denaturing/reducing agent is possible by dialysis against the same solutions.

A further purification step can follow the reactivation step; such a purification takes place by r means of dialysis or also a subsequent isolation of the activated t-PA, for example by affinity chromatography, for example over Lys-Sepharose.

The process of this embodiment of the invention depends upon the formation of the mixed disulphide bridge-containing eukaryotic proteins and glutathione (in the following abbreviated t-PASSG). This can simplify not only the separation of foreign proteins in the denatured state but also the further purification of the native protein. A purification after modification of the thiol groups has the advantage that the protein is protected against air oxidation and thus is stable in a greater pH range and a change of the nett charging simplifies the purification. In particular, a separation from non-modified protein can advantageously be carried out by ion exchanger treatment.

Preferably, for the formation of the mixed i disulphides, the analyzed, reduced protein, purified from denaturing and reducing agents, is incubated with a diluted, e.g. 0.2 mole/l solution of CSSG containing a denaturing agent. The activation takes place after separation off of the denaturing and oxidation agent at a pH level of 7 to 10.5, at a GSH concentration of 0.5 to mmole/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 mixed disulphides with 11

L-%

I

10 *0 0

S

oo o•0 with GSSG corresponds to the embodimental forms for the activation of the previously mentioned part of the invention. Ip the case of this embodimental form, the pH optimum lies at 8.5, the yield is about twice as high and the activated protein is stable in the regeneration buffer over a longer time.

According to the invention, it is possible so to activate t-PA from prokaryotes that there is achieved not only anactivation of the normal biological activity but, in addition,. a stimulatability in the above-defined sense is achieved which far excess the stimulatability of the native t-PA and is greater than a factor of and can even exceed a factor of A further eukaryotic protein which, according to the invention,. can be activated after expression in prokaryotes is @-interferon.

The following Examples further ecplain the invention without limiting it thereto. If not stated otherwise, statements of percentage refer to percent by weight and statements of temperature to degrees Celsius.

Example 1 a) Preparation of the "refrachile bodies" 100 g. E. coli moist cell mass, taken up in 1. of 01 mole/I. tris-HC1 (pH 6.5) and 20 mmole/1.

of EDTA were homogenised (Ultra-Turrax, 10 sec.) and 0.25 mag./ml. .ysozyme added thereto. After 50 min.

incubation at room temperature,. it was again homo- -12- 0050 00 50 S O 55 00 o 00 5505 00 5* 5 00 00 5 5 0

L

I

1 g

S..

2

C

S

genised and cooled to 30C. The cell digestion was achieved by high pressure dispersion (500 kg./cm 2 Subsequently, it was after-rinsed with 500 ml. of 0.1 mole/i, tris/HCl (pH 6.5) and 20 mmole/1. EDTA.

After centrifuging (2 hrs. at 27000 g, 4 0 the pellet was taken up in 1.3 1. of 0.1 mole/1. tris/HC1 (pH mmole/l. EDTA and 2 5f Triton X-100 and homogenised.

After renewed centrifuging (30 min. at 27000 g, the pellet was taken up in 1.3 1. of 0.1 mole/i. tris/HCl (pH 20 mmole/l. EDTA and 0.5% Triton X-100 and homogenised. Alternatingly centrifuging (30 min at at 27000 g, 40C.) and homogenisation of the pellets in 1 1, of 0.1 mole/i. tris/HC1 (pH 6.5) and 20 mmole/1l EDTA was further carried out three times.

The t-PA(content of the "refrac4le bodies" preparations was quantified by the SDS-PAGE, idehtification of the t-PA bands by "Western blotting" and densitometric analysis. I n the case of the SDS-PAGE and "Western blotting",, the "refractile bodies" show a strong t-PA band with a molecular weight of about 60 kDa.

The t-PA proportion of the total protein content of the "refrachile bodies" amounts to about 21%.

b) Solubilising/reduction of the "refractile bodies" "Refractile bodies" were incubated at a protein concentration of 1 to 5 mg./ml. in 0.1 mole/I, of tris/HC1 (pH 6 mole/1, guanidine hydrochloride, 0.15 to 0.4 mole/1. DTE and 1 mmole/l. EDTA for 2 to 3 hrs. at room temperature. Thereafter, insoluble -13- 11 10 o4* 4 aterial (cell wall fragments etc.) was centrifuged off 30 min, at 35000 to 50000 g, 4 0 The pH of the supernatant was adjusted with conc. HC1 to pH 3, Denaturing and reducing agents were separated off by dialysis against 0.01 mole/i. HC1 at 4°C.

c) Reoxidation/activation Reoxidation/activation took place by means of a 1:50 to 1:200 dilution in 0.1 mole/1..tris/HC1 (pH 10.5), 1 mmole/l. EDTA,. 1 mg./ml. BSA-, 0.5 mole/1.

L-arginine, 2 mmole/1. GSH, 0.2 mmole/l. GSSG. After 17 to 24 hours activation at about 30 0 there was determined the activity and the yield in comparison with the activity of native glycosilated t-PA from eukaryonts.

Yield .ndferred to the total protein content of the "refractile bodies": 2.5 stimulatability: 10 Yield referred to the t-PA portion of the "refractile bodies":. about 12%.

d) Reoxidation/activation without separation of the denaturing/reducing agent "Refractile bodies" were incubated at room temperature at a protein concentration of 1.25 mg./ml.

in 0.1 mole/1, tris/HC1 (pH 6 mole/i, guanidine hydrochloride, 0.2 mole/1. DTE and 1 mmole/l. EDTA for 2 hours at room temperature. Thereafter, reoxidation was immediately initiated by a 1:100 dilution in 0.1 mole/l, tris/HC1 (pH 10.5), 1 mmole/1. EDTA, -14-

S..

4 S

S

S 4 .9.

'S

S

I

S

a~ *OS e0

S

0e 0* *0 00 @0 1 mg./ml. BSA, 0.3 mole/1. L-arginine and the amounts of GSSG given in the Table. In addition, in the activation batch was present a residual concentration of 0.06 mole/i, guanidine hydrochloride and 2 mmole/l. DTE.

Dependency of the activation yield upon the GSSG concentration in the case of activation without separation of the denaturing/reducing agent GSSG yield' stimulatability (mmole/l.) (factor) 0.2 O 1 0.13 1.49 7.4 6 1.28 5.4 7 1,04 5.8 9 0.98 5.2 10 1.77 10.0 o 20 0 yield of active t-PA, referred to the total protein content of the "refractile bodies".

Example 2 An RB (refractile bodies") preparation (about 5 mg.) was incubated for 2 to 5 hours at room temperature in 1 ml. of 0.1 mole/., tris/HC1 (pH 8.6), 6 mole/i, guanidine hydrochloride and 0.15 0.2 mole/i, DTE, Insoluble material (cell wall fragments etc.) were thereafter separated off by centrifuging (20 minutes at 17000 Denaturing and reducing agents were 0 0 *0 00 00 0 00 *000 00 *0 0 *0 00 00 0 0 0 h

I

ti removed by gel filtration over Sephadex G 25 (superfine) in 0..01 mole/i. HC1, The sample was thereby diluted by about the factor 5 to 10. The reduced material was stored at -200C, in 0.01 mole/i. HC1.

Example 3 In the following Tables is summarised the influence of various parameters according to the invention on the activation and stimulatability. For these reoxidation experiments, the solubilised, reduced protein according to Example 2 was not further prepurified.

The reduced protein (in 0.01 mple/l. HC1) was activated by dilution of 1:10 to 1:500 in "reoxidation buffer", The activation was determined at room temperature after 22 to 48 hours incubation. The activity of the reoxidised protein refers to a "standard reoxidation" 100%) in: 0.1 mole/1, tris/HC1 (pH 10.5) L mmole/l. EDTA 0.5 mole/., L-arginine 1 mg./mli: BSA 0.5 mmole/l. GSH (reduced glutathione) 0.5 mmole/1. GSSG (glutathione disulphide).

o* The s timulatability is calculated from E+ CNBr FCP A ECNBr FCP (cf. W. Nieuwenhuizen et al., Biochemica et Biophysica Acta 75 (1983) 551 to 533). The activity (in percent) and the stimulatability (factor) was determined according to J.H. Verheijen Thromb. Haemostas. 48(3), 266-269(1982).

The following results were obtained: -16-

I

1. Dependency of the activation yield upon the addition of L-erginine or guanidine hydrochloride.

Reoxidatipn in 0.1 mole/1, tr±I/HCl (pH 10.5) 1 mmole/l. EDTA 1 mg./ml. BSA 0.5 mmole/l,

GSH

0.5 mmole/l. GSSG a) L-arginine 10 4 a @6 4 rd 200 4* 4 go 4, 40 4 S 0 04* *0 L-arginine activity stimulatability (mol.e/1.) (factor) 0 4 0,25 98 100 21.9 0.75 27 16.3 1.0 23 In the case of this experiment,, it is to be borne in mind that t-PA is inhibited by L-arginine. The drop of the activity yield at higher L-earginine concentrations ieitberefore,. to be corrected with regard to the inhibition.

b) Guanidine hydrochloride (Gdn/HC1) (Gdn/HC1 activity (mole/1.) O 11 0.25 22 53 0.75 58 12 -17- Dependency of the activation yield upon the addition of urea and urea derivatives.

Reoxidation in 0.1 mole/i. tris (pH 10.5), 1 mmole/1. EDTA,. 1 mg./ml, BSA, 5 mmole/l. GSH, 0.2 mmole/1. GSSG a) Urea, urea (mole/i.) activity

W%

S

OS*S@S

S

OS S 06

S.

S S

S

555555 5

*SOS

555 0 1 1.5 2 2.5 3 4 1 59 126 162 14.1 72 12 0 b) Methylures

S

S.

5~ .5 S methylurea (mole/i.) activity

W%

o.5 1 2 5 3 4 22 174 315 375 332 215 12 0 -18c) Ethyiurea ethylurea (mole/i.) activity 1 2 3 4 46 212 323 300 107 19 0 0

S

0e

S

S**S

S

5 S. SO

S

d) Dimethlurea dimethylurea (mole/i.) activity

(W)

stimulatability (factor) 0.5 1 2 3 5 167 256 283 177 78 23 4 2 8.8 8.9 9.4 7.7 8.9 9.9 8.6 3. Dependency of the activation yield upon the addition of fatty acid amides: Reoxidation in 0.1 mole/i. tris (pH 10.5),9 1 mmole/l. EDTA,* 1 mg./ml. BSA 5 mmole/1. GSH, 0.2 nmole/.. GSSG -19-

L-

a) Formamide formamide (mole/i.) ac tivity

W%

0 1 2 3 4 4.2 59 175 245 325 4.23 444 416 341

C

OeSOe* 4 C we a.

I. *9 a.

Ca *e C

C

ewe b) riethylforinamide me thy iformamide (mole/i.) activity

W%

0.5

I

2 2,5 3 4 100 135 304 389 4.66 452 425 121 h CC

C

C C C a ~w *Ce

C

CO

c) Acetamide acetamide activity (mole/i.) W%

C

I

C

we 0 C 1 2 3 4 72 134 207 26 1 204 237 198 14.1

L

j~ miii I No w d) Propionamide propionamide (mole/I.) activity

I

ii i 1 99 197 2 150 101 3 39 4 2 5 0 butyramide. activity (mole/i.)

W

1 52 1.5 17 2 0 0 0* 0* 0 000

S..

0000

S

e) Butyramide 4. Dependency of the activation yield on the pH value Reoxidation in 0.1 mole/i. tris/HCI 1 mmole/l. EDTA 0.5 mole/i. L-arginine 1 mg./ml. BSA 0.5 mnole/l. GSH 0.5 mmole/1. GSSG 55 @0 5 5 000000 S S pH activity stimulatability (factor) 7 1 8 22 9 89 13.6 105 20.3 1i 95 21.3 -21- Dependency of the activation yield upon the GSH/GSSG concentration Reoxidation in 0.1 mole/i. tris/HCl, pH 10.5 1 mmole/l. EDTA 0.5 mole/i. L-arginine 1 mg./ml. BSA a) 1 znmoie/1. GSH 6*SSSS

S

0* 0

OS

S.

S. SO S S

S

0*O*Os

S

*0*S

S..

(GSSG) activity stimulatability 0.1 239 14,.9 0.2 273 15.3 0.5 193 13.3 1 198 12.5 517 2.1 10 0 20 0 5055 0 5~ S~

S.

SO S 0 55

S.

50 0 b) 0.2 mrnole/1. GSSG .0 5 0 S (GSH) activity stirulatability (minole/l.) (factor.) 0.05 15 2.2 0.1 40 3.8 0.2 112 6.8 142 7.4 1 273 6.8 260 7.9 143 6.3 55 5.1 -22r 6. Dependency of the activation yield upon the protein concentration in the case of the reoridation (dilution 1:20 1:500) Reoxidation on 0,1 mole/l. tris/HC1 (pH 10.5) 1 mmole/l. EDTA 0.5 mole/l. L-arginine 1 mg./ml. BSA 0.5 mmole/I. GSH 0.5 mmole/1, GSSG or

C

oo~ rogo o

S

0060

C

dilution activity stimulatability (factor) 1:10 29 15.3 1:20 45 25.4 I.:.50 69 37.9 1:100 100 37.9 1:200 79 52.7 1:500 29 28.7 0eSO o S C. o

S.

Cooo*

O

CC..

0

S

7, Dependency of the. activation yield upon the addition of BSA Reoxidation in 0.1 mole/i. tris/HC1 (pH 10.5) 1 mmole/1. EDTA 0.5 mole L-arginine 0.5!mmole/l. GSH 0.5 mmole/1. GSSG BSA activity 0 47 83 1 100 3 102 52 -23-

I

I i; 9 9.

S

SVC

S

.9 The Figures 1 and 2 show the activity with and without CNBr-FCP in the standard test after 17 hours reoxidation at room temperature in 0,1 mole/1, tris/HC1 (iH 10,5) 1 mmole/1. EDTA 0.5 mole/i. L-arginine 1 mg./ml. BSA 0.5 mmole/1, GSH 0.5 mmole/l. GSSG.

In the Figs. 1 and the curves signify the activity in the presence of CNBr-FCP, the curves (B) the activity without CNBr-FCP, Example 4 Activation of t-PA via the mixed disulphides of t-PA and glutathione.

The "refractile bodies" used were obtained according to one of the preceding Examples. The reduction of the "refractile bodies" was carried out by 2 hours incubation at room temperature in 0.1 mole/lI tris/HCI, pH 8.6, 1 mmole/1. EDTA,: 6 mole/i. Gdn.HCI,. 0.2 mole/i.

DTE at a protein concentration of about 1 mg./ml.

The reduced protein dialysed against 0.01 mole/i.

HCI was diluted in the ratio of 1:1 with 0.1 mole/1.

tris, pH 9.35 9 mole/i. urea and 0.2 mole/1. GSSG and incubated for 5 hours at room temperature, After acidification with cone, HC1 to pH 3, there took place dialysis against 0.01 mole/1. HC1 at Bfter the dialysis, the total protein concentration amounted to 0.33 mg./ml. The optimum reactivation conditions were determined with the so-prppared t-PASSG.

a) pH optimum of the activation of t-PASSG Here, as in the following optimising experiments, there was used no GSSG and the activation S S 09 9 99 9 9 u S. U

S

9 9 -24was determined after 17 hours incubation at room temperature. Activation took place by a 1:100 dilution in 0.1 mole/i. tris, 1 mmole/1. EDTA, 0.5 mole/l. arginine, 1 mg./ml. BSA and 2 mmole/l. GSH with variation of the pH value.

pH yield ()stimulatability 6 0.04 3.3 0.37 7 1.35 11.14 5.66 7.1 8 7.32 8.2 8.5 8.65 9 8.59 8.-7 9.5 8.32 11.7 6.15 12. 10.5 3.-07 11.2 0~ 20 The yield was determined in of active, t-PA.

referred to the amount of proteiai used.

b) Reproducibility of the results of the activation of t-PASSG in the case of identical activation conditions,..

in the case of different measurement series there are observed different yields which, inter alia, are caused by variations of the standard t-PA's. For the clarification of the breadth of error, all activation data after 1:100 or 1:200 dilution in 0.1 mole/l.

tris/HC1, pH 8.5, 1 miole/l. EDTA, 0.5 mole/l. Larginine, 1 mg./ml. BSA and 2 mmole/1., GSH, are assembled.

R

experiment yield stimulatability 8.65 4.47 4.49 8.50 3.45 4.32 3.29 3.54 5.07 9.3 9.7 17.2 8.3 14.0 13.4 16.4 0 0* a o 0* average 5.1 1.9 11.3 5.8 value c) Stability of the activated protein Activation took plade in the said Example by a 1:200 dilution in 0.1 mole/1.,tris/HC1, 1 mmole/l, EDTA,.

mole/1. L-arginine, 1 mg../ml. BSA and 2 mmole/l. GSH .time pH yield stim.

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 Activation of genetically engineered interferon-0 "Refractile bodies" were produced according to the previously mentioned methods. The reduction/solubilisation of the "refractile bodies" was carried out as follows: The pellet was incubated for 3 hours at 25 0

C.

in 10 ml,. 9.1 mole/1, tris/HC1, pH 8.6, 6 mole/i.

-26- 2

S

4 *5 I. *5 0

S

5*4* 9 Gdn,.HC1., 1 mmoe/ 11 EDTA and 0,2 mole/i. DTE and af ter minutes centrifuging at 4~ 0 and 4~8000 g, the pH of the supernatant was adjusted to about 5 with concentrated NC.. Subsequently, there was carried out a gel filtration over Sephadex G25F? in 00.01 mole/i. HC1.

The eluate was investigated for conductivity,.

protein concentration and reactivatability.

The activit7y of the reoxidised protein refers- to a ""standard activation" 100%) in 0.1 mole/i. tris/HC1, PH 10.5, 1 mmole/l. EDTA,: 5 mmole/1. GSHI 0.5 mmole/l.

GSSG and 0..25 mole/i. L-arginine.

a) Dependency of the activation yield on the addition of L-arginine The eluate was- diluted 1-:50 with 0.1 mole/i 9 tris/HCI,, pH 1 mmole/l. EDTA,. 5 mmoie/l. GSH, mmole/l. GSSG and activated for 20 hours at 00C.

L-Arginine dependency of the activation L-arginine (mol-e/i.) acti~ity 0 8 0.25 8 0.5 0.75 b) Dependency of the activation yield on the addition of urea The activation solution corresponded to that of point a) but it was activated for 17 hours at OO 4

S

OS

S

4 0* 9 4* 4.

t C 'I .4 4 0 -27- Urea dependency of the activation urea (mole/i..) activity W~ 0 13 100 1 200 100 00 0 000000 0 *0 00 000 00 0 00 0 0 0 c) Dependency of the activation yield upon the addition of formamide Activation as in the samples were investigated after 17 hours activation at 000.

Formamide dependency of the activation .ffrmamide (mole/i.) activity 0 13 .1 13 2 13 3 0 4 0 d) Dependency of the activation yield on the redox buffer The eluate was diluted 1:50 in 0.1 mole/l.

tris/HCl,: pH 8.5, 1 mmole/l. EDTA and 0.25 mole/i. Larginine and the samples investigated after 17 hours activation at 0 0

C.

GSH/GSSG dependency of the activation GSH. (mmole/1.) GSSG (mmole/l.) 'activity W~ 6 0.5 13 0.5 0.5 0.1 13 0.5 13 1.0 13 6 -28e) Dependency of the activation yield on the addition of BSA The eluate was diluted 1:50 in 0.1 mole/i. tris/HC1, pH 8.5, 1 mmole/1. EDTA, 5 mmole/1. GSH, 0.5 mmole/1. GSSG and 0.25 mole/i. L-arginine and investigated after 17 hours activation at OOC.

BSA dependency of the activa:tion BSA activity 0 13 1. 13 2 13 r i

I

z

J

s 6 0 0* S* 6:*6*6 1

OO

*0 6• e o oo go f) Dependency of the activation yield on the pH The eli:ate was diluted 1:50 in 0.1 mole/l.

tris/HC1,, 1 mmole/1. EDTA, 5 mmole/l. GSH,. 0.5 mmole/1.

GSSG and 0.25 mole/i. L-arginine and investigated after 17 hours activation at OOC.

pH dependency of the activation pH activity 6.5 o 6 13 10.5 100 -29-

Claims

1. A process for the activation of genetically engineered, heterologous, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes by cell digestion and separation of insoluble components, solubilisation of said insoluble components under denaturing and reducing conditions, and reactivation under oxidising conditions in the presence of GSH, characterised in that the reducing/denaturing agent is separated off, the thiol groups of the proteins are converted into mixed S S disulphides of protein and glutathione by addition of GSSG under denaturing conditions, and the reactivation step is carried out at a pH value of 7 to 10.5, a GSH concentration of 0.5 to 5 mmole/l., and with a S non-denaturing concentration of the denaturing agent. S2. Process according to claim 1, characterised in that the mixed disulphide of protein and glutathione is separated from the non-modified protein by ion exchanger treatment.

3. Process according to claim 1 or claim 2, characterised in that the expression is carried out in E_ coli or P. putida.

4. Process according to any one of the claims 1 to 3, characterised in that the denaturing agent used in the reactivation step is selected from arginine, guanidine hydrochloride and/or at least one compound of the general formula R 2 -CO-NRR 1 in which R and R 1 signify H or alkyl with 1 to 4 C-atoms and R 2 signifies H or NHR 1 or alkyl with 1 to 3 C-atoms. i I 31 Process according to claim 4, characterised in that the concentration of arginine and/or guanidine hydrochloride amounts to 0.1 -o 1.0 mole/i., especially 0.25 to 0.8 mole/l.

6. Process according to claim 4 characterised in that the concentration of the compound of general formula I amounts to 0.5 to 4 mole/i., especially 1 to 3.5 mole/i. S* 7. Process according to any one of the preceding claims, characterised in that the reactivation step is carried out in the presence of a non-proteolytically effective protein, especially in the presence of bovine 6 serum albumin.

8. Process according to any one of the preceding claims, characterised in that the cell digestion is carried out by means of ultrasonics, high pressure dispersion or lysozyme.

9. Process according to any one of the preceding claims, characterised in that the solubilisation step is carried out at an alkaline pH value in the presence of a reducing agent of the mercapto group and in the presence of a denaturing agent. Process according to claim 9 characterised in that the solubilisation step is carried out in the presence of guanidine hydrochloride and/or compound of general formula I as denaturing agent.

11. Process according to claim 10, characterised in that the concent:ation of guanidine hydrochloride amounts to 6 mole/i., that of the compound of general formula I to 8 mole/i. 1 .1 32

12. Process according to any one of claims 9 to 11, characterised in that the solubilisation step is carried out in the presence of DTE, P-mercapto-ethanol, cysteine or GSH.

13. Process according to any one of the preceding claims, characterised in that purification and separation of reducing, oxidising and denaturing S agents is carried out by means of steric exclusion chromatography or dialysis. S* 14. Process according to any one of the preceding claims, characterised in that after the reactivation step, a purification step is carried out by means of dialysis. Process according to any one of claims 1 to 14, characterised in that the genetically engineered eukaryotic protein is t-PA. *C

16. Stimulatable (as herein defined), non-glycosilated tPA, obtainable according to the process according to any one of claims 1 to

17. Process according to any one of claims 1 to 14, characterised in that the genetically engineered produced eukaryotic protein is interferon P.

18. A process according to claim 1 or a product obtainable according to said process, substantially as hereinbefore described. 33 Dated this 13th day of September, 1989. BOEHRINGER MANNHEIM GMBH By its Patent Attorneys DAVIES COLLISON X A S .9 S 4 H