IE862683L - Activating closed gene products - 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

62631 - 2- The invention concerns a process for the activation of gene-technologically produced, disulphide bridge-containing eukaryotic proteins after expression in prokaryotes« 5 In the case of the expression of heterologous proteins in prokaryotes„ in the host cells these proteins often form inactive, sparingly soluble aggregates (so-called "refraccile bodies") whichs in addition, are also contaminated with proteins of the 10 host cells. It is assumed that the formation of such "refractile bodies" is a result of the high protein concentration 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 cells, the 15 aggregation of the enzymes to insoluble„ high molecular„ mostly inactive particles takes place. Before such proteins can be used* e.g. for therapeutic purposes,, they must consequently be purified and converted into their active form. 20 According to known processes., a reactivation of such proteins present as aggregates takes place in several steps (cf» e.g. R. Jaenickes, FEBS Federation of European Biochemical Societies, Vol- 52 (1979) 187 to 198; R. Rudolph et al.» Biochemistry IjJ (1979) 25 5572 to 5575).

In the first step«, a solubilisation is achieved by addition of strong denaturing agents, for example guanidine hydrochloride or urea, in high concentration or by addition of strongly acidic agents, for example 30 glycine/phosphoric acid mixtures. As further adjuvants, there have proved useful reducing SH reagents (e.g. dithioerythricol, DTE) and EDTA, for example in the renacuring of LDH. Insofar as the protein is contaminated by proteins of the host cells., as next step there 35 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 use of guanidine hydrochloride*, it is thereby diluted to values below 0.5 mo1/X » In the case of enzymes with free SH groups, the addition of agents protecting SH groups proves to be advantageous (cf.. e.g. R. Jaenickes Journal Polymer Sciencea Part Cs 16 (1967) 2143 to 2160).

In EP-A-0114506, processes are described for the isolations purification and reactivation of some heterologous expression products from bacterial cultures for the reactivations the solutions of the "refractile bodies" in a strong denaturing agent are a) transferred directly into a solution of a weaker denaturing agent which is then subjected to oxidising conditions for the reformation of disulphide bridges % b) the protein, is sulphonated s then transferred into a solution in a weak denaturing agent and the S-sulphonate groups are converted into -S-S- groups by treatment with a sulphhydryl reagent in its reduced and oxidised forms, e.g. with GSH/GSSGs or c) the solution in a weak denaturing agent is treated directly with the sulphhydryl reagents, e.g. with GSH/GSSG. A typical example in which the above-discussed problems arises is t-PA« In T„w. Odorzynski ec al«19795 The Journal of Biological Chemistry, 254(10),, 4291-4295 and in C.F. Dudos at al., 1982, The Journal of Biological Chemistry j, 257(16) 9866-98715l processes are described for the reactivation of natural proteins,, such as e.g. trypsinogen.

The main component of the protein matrix of coagulated blood is polymeric fibrin.. This protein matrix is dissolved, by plasmin which is formed from -4« 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 gene-technologically from eukaryotes 5 (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 presence of these stimulators (by more than the factor of 10). This so-called stimulatability of the 10 activity is a decisive advantage of t-PA in comparison with other known plasminogen activators, such as urokinase or streptokinase (cf» e.g. M. Hoylaerts et al., J. Biol. Chem.,, 257 (1982) 291 to 2019; Nieuwenhiuzen et al., Biochemica et Biophysica Acta 755 (1983) 531 15 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 35.

A t-PA-like, non-glycosilated product is also formed in genetically manipulated prokaryotes (after 20 introduction of the c - DMA); 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 in the prokaryote cell differ in such a way from the eukaryote 25 cells from which the gene originates that, ab initio, a non-active product is formed whichs for example ? could be due to the face that numerous SS bridges which the natural active molecule contains are linked in a false way or are not even formed. However „ for 30 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 eukaryotic 35 proteins in the correct way, reference is made to other -5- substances in The EMBO Journal 4, No. 3 (1985) 775 to 780.

According to EP-A-0093639„ for the reactivation of t-PA9 the cell pellets obtained from E. coli are 5 suspended in 6 raol/1 guanidine hydrochloridetreated with ultrasonics, incubated and subsequently dialysed for four hours against a solution of tris-HCl (pH = 8.0), sodium chloride, EDTA and Tween 80, After dialysis, it is cencrifugeds whereby the plasminogen activator 10 activity is to be found in the supernatant. t-PA renatured in this way is admittedly proreolytically active but shows no measurable stimulatability by BrCH cleavage products (BrCN-FCP) of fibrin according to the process described in J.H. Verheijen., Thrombi 15 Haemostas«, 48 (3), 260-269 (1982).

For the reactivation of denatured proteins, no generally usable process is known from the state of; the art; this applies quite especially for t-PA because the native protein possesses a very complex structure 20 it contains a free thiol group and 17 SS bridges which 20 theoretically can be linked in 2.2 x 10 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 admittedly lead 25 to a proceolycically active t-PA but which shows no measurable stimulatability; an activation process which leads to stimulatable t-PA is not known.

Therefore» it is the task of the present invention to make available a process for the complete 30 activation of gene-technologically produced„ heterologous „ disulphide bridge-containing eukaryocic proteins after expression in prokaryotes; this task is solved with the subject of the present invention., The subject of the invention is a process for 35 the activation of heterologous„ disulphide bridge- -6- containing eukaryotic proteins produced gene-technological ly by expression in prokaryotes by a) digestion of the prokaryote cells , b) solubilisation of the eukaryotic proteins under 5 denaturing and reducing conditions, c) separation of the reducing/denaturing agent, d) reactivation under oxidising conditions by e) conversion of the thiol groups of the solubilised proteins into the mixed disulphides of protein and 10 glutathione by addition of GSSG under denaturing conditions, f) formation of active protein from the mixed disulphides at a GSH concentration of 0.5 to 5 mmol/1, a pH value of 7 to 10.5 and in the 15 presence of a non-denaturing concentration of a denaturing agent.

Preferred embodiments of this process are the subject of the subsidiary claims- As denaturing agent, there can, as a rule, be 20 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 suitable- Furthermore s 25 mixtures of these denaturing agents can be used- This activation step is preferably 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 active; bovine serum albumin (BSA) 30 is preferably used, e.g. 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 surface denaturing and/or proteolytic breakdown). -7- The other process conditions can correspond to 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 5 room temperature - 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 EDTA9 whereby the most expedient concentration amounts to 10 about 1 mmol/1 EDTA» The process steps preceding and following the activation step (reoxidation/activation)» such as cell digestion,, solubilisation (solubi lis at ion/reduction) and possibly one or more of the purification operations 15 preceding and/or following the activation stepj, can be carried out according to known and usual methods for such processes from the prior arcs e«g» from EP-A-0114506 and EP-A-0093619; however, for a result which is optimal with regard to yield and activations it can 20 be expedient to carry out individual or all process steps having regard to one or more of the here-described process embodiments. The expression is carried out in prokaryotes, preferably in P, putida and especially in E. coli- However., the process according to the 25 invention is just as suitable when one expresses in other prokaryotes (e.g. Bacilli).

The cell digestion can be carried out by methods usual here for 5, e»g» by means of ultrasonics s high pressure dispersion or lysosyme; it is preferably 30 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 0.1 mol/1 tris-HCl. After the cell digestion,, the insoluble components ("reftactile bodies") are separated off in any desired 35 ways preferably by cantrifuging off at comparatively high g values and comparatively long centrifuging times 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 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 arts e.g. from EP-A-0114506 , and especially guanidine hydrochloride or urea. The concentration of guanidine hydrochloride expediently amounts to about 6 mol/l«, that of urea to about 8 mol/1. Compounds of the general formula I can also be used.

As reducing agent from the mercapto group, there can be used e.g.. reduced glutathione (GSH) or 2-mercaptoethanol, e.g. in a concentration of about 50 to 400 mmol/1 and/or especially DTE (dithioerythritol) or DTT (dithiothreitol), e.g. in a concentration of about 80 to 400 mmol/1. The solubilisation expediently takes place at room temperature over a period (incubation) of 1 to several hours„ preferably of two hours. For the prevention of the oxidation of the reducing agent by atmospheric oxygens, it can also be expedient to add EDTA. Besides the solubi1 ising/reduction, the solubilising step also has a purification effect since a large part of the foreign proteins does not go into solution.

After the solubilisation. there takes place the separating off of the reduction and of the denaturing agents? a non-specific reoxidation can be prevented by -9- addition of a reducing agent (e.g. 2-mercaptoethanol) or by pH values 4.5 (cf. e.g. R. Rudolph* Biochem. Soc. Transactions, 1_3 (1985) 308 to 311). The separating off of the denaturing/reducing agents takes place 5 by desalination over Sephadex G 25 in 0.01 mol/1 HC1 or 0.1 mol/1 acetic acid. Alternatively,, the separating off of the denaturing/reducing agents is possible by dialysis against the same solutions.

A further purification step can follow the 10 reactivation step; as a rules such a purification takes place by means of dialysis or also a subsequent isolation of the activated tPAs for example by affinity chromatography's for example over Lys-Sepharose.

The formation of the mixed disulphides of gene-15 technologically produced* heterologous® disulphide bridge-containing eukaryotic proteins with glutathione (in the following abbreviated to c-PASSG) simplifies not only the separation of foreign proteins in the denatured state but also the further purification of 20 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 particulars a separation 25 from non-modified protein can advantageously be carried out by ion exchanger treatment» For the formation of the mixed disulphides 2, the dialyseds, reduced proteins, purified from denaturing and reducing agents, is incubated with a diluted, e.g. 30 0.2 mol/1„ solution of GSSG containing a denaturing agent. The activation takes place after separation off of the denaturing and oxidation agent at a pH value of 7 to 10-5»» at a GSH concentration of 0™5 to 5 mmol/1 and with a non-denaturing concentration of the 35 denaturing agent.

The pH optimum lies at 8 - 5s the yield is about twice as high as in the case of a reactivation with GSH/GSSG without separate formation of the mixed disulphides and the activated protein is stable in the regeneration buffer over a longer time.

According to the inventions, it is possible so to activate t-PA from prokaryotes that there is achieved not only an activation of the normal biological activity but, in addition, a stimulatability in the above-defined sense is also achieved which far exceeds the stimulatability of the native t-PA and is greater than a factor of 10 and can even exceed a factor of 50.

A further eukaryotic protein which„ according to the invention., can be activated after expression in prokaryotes is ^-interferon™ The following Examples explain the invention in more detail 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 "refractile bodies" 100 g E. coli moist cell mass, taken up in 1.5 1 of 0.1 mol/1 tris-HCl (pH 6.5) and 20 mmol/1 of EDTA were homogenised (U1tra-Turrax,, 10 sec-) and 0.25 mg/ml lysozytne added thereto. After 30 min» incubation at room temperature., it was again homogenised and cooled to 3°C. The cell digestion was achieved by high 2 pressure dispersion (500 kg/cm ). Subsequently., it was after-rinsed with 300 ml of 0.1 mol/1 tris/HCl (pH 6.5) and 20 mao 1/1 EDTA.. After centrifuging (2 hrs. at 27000 gs 4°C)s, the pellet was taken up in 1.3 1 of 0.1 mol/1 tris/HCl (pH 6.5), 20 mmol/1 EDTA and 2.5% Triton X-100 and homogenised. After renewed centrifuge ing (30 rain, at 27000 g«, 4°C) s the pellet was taken up in 1.3 1 of 0.1 mol/1 tris/HCl (pH 6.5)s 20 mmol/1 EDTA and 0.5%. Triton X-100 and homogenised. Alternating centrifuging (30 min» at 27000 g« 4°C) and homogenisation of the pellets in 1 1 of 0.1 mol/1 Tris/HCl (pH 6.5) and 20 mmol/1 EDTA was carried out a further three times.

The t-PA content of the "refractile bodies" preparations was quantified by SDS-PAGE5, identification of the t-PA bands by "Western blotting" and desensito-metric analysis. In the case of 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 "refractile bodies" amounts to about 21%. b) Activation of t-PA via the mixed disulphides of t-PA and glutathione.

The "refractile bodies" used were obtained according to a). The reduction of the "refractile bodies" was carried out by 2 hours incubation at room temperature in 0.1 mol/1 tris/HCl j, pH 8»69 1 mmol/1 EDTAS 6 mol/1 Gdn.HCl, 0.2 mol/1 DTE at a protein concentration of about 1 mg/inl.

The reduced protein dialysed against 0.01 mol/1 HC1 was diluted in the ratio of lil with 0.1 mol/1 tris 5, pH 9»3S 9 mol/1 urea and 0„2 mol/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 mol/1 HC1 at 4°C.

After the dialysis,, the total protein concentration amounted to 0.3 mg/ml. The optimum reactivation conditions were determined with the so-prepared t-PASSG. c) pH optimum of the activation of t-PASSG Here, as in the following optimising experiments, there was (1) used no GSSG and (2) the activation was determined after 17 hours incubation at room temperature. Activation took place by a 1:100 dilution in 0.1 mol/1 tris ,s 1 mmol/1 EDTA., 0.5 mol/1 L-arginine, 1 mg/ml BSA and 2 mmol/1 GSH with variation of the pH value. pH yield (%) stimulatability 6 0.04 3.3 6. 5 0.37 9.5 7 1.35 11.4 7.5 5.66 7.1 8 7.32 8.2 9 CO 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 % of active t-PAs referred to the amount of protein used. d) 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 lilOO or Is 200 dilution* respectively? in 0.1 mol/1 tris/HCl, pH 8.5, 1 mmol/1 EDTA, 0.5 mol/1 L-arginine„ 1 mg/ml BSA and 2 mmol/1 GSH, are summarised. -13- experiment yield (%) stimulatability 1 8.65 9 o 2 4.47 9.3 3 4.49 9.7 4 8.50 6.5 5 3 .45 17.2 6 4.32 8.3 7 3.29 14.0 8 3.54 13.4 Q ✓ 5.07 16.4 average 5#1 +/„ 1>9 Um3 +/„ 3o8 value e) Stability of the activated protein Activation took place in the said Example by a 15 Is 200 dilution in 0.1 mol/1 tris/HCl, 1 mmol/1 EDTA, 0-5 mol/1 L-arginines, 1 mg/ml BSA and 2 mmol/1 GSH. time pH 9.5 (h) yield (%) stim. 1 0 20 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 25 239 2.28 14.3 ift k-

Claims (21)

-14 Patent Claims

1. „ Process for the activation of heterologous, disulphide bridge-containing eukaryotic proteins produced gene-technologically by expression in prokaryotes by a) digestion of the prokaryote cells. b) solubilisation of the eukaryotic proteins under denaturing and reducing conditions., c) separation of the reducing/denaturing agents9 d) reactivation under oxidising conditions by e) conversion of the thiol groups of the solubilised proteins into the mixed disulphides of protein and glutathione by addition of GSSG under denaturing conditions s f) formation of active protein from the mixed disulphides at a GSH concentration of 0.. 5 to 5 mmol/1, a pH value of 7 to 10.5 and in the presence of a non-denaturing concentration of a denaturing agent.

2. Process according to claim 19 characterised in that one carries out the expression in E. coli or P. putida.

3. Process according to claim 1 or 2, characterised in that., in the reactivation steps as denaturing agents, one uses arginine, guanidine hydrochloride and/or at least one compound of the general formula R^-CO-NRR-j (I) in which R and R^ signify H or alkyl with 4 C-atoms and R2 signifies H or NHR^ or alkyl with 1 to 3 C-atoms.

4. Process according to claim 3, characterised in that the concentration of arginine and/or guanidine hydrochloride amounts to 0-1 to 1.0 mol/1, especially 0*25 to 0*8 mol/1.

5. Process according to claim 3, characterised in that the concentration of the compound of the general formula I amounts to 0.5 to 4 mol/1» especially 1 to 3.5 mol/1.

6. Process according to one of the preceding claims„ characterised in that,, in the step of reactivation, one works in the presence of a non-proteolytically effective protein* especially in the presence of bovine serum albumin.

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

8. Process according to claim 7, characterised in that one carries out the digestion in a dilute aqueous buffer solution, especially in 0.1 mol/1 tris s at a neutral to weakly acidic pH value.

9. Process according to one of the preceding claims, characterised in that, after the cell digestion, one separates off the insoluble components,

10. Process according to one of the preceding claims, characterised in that, in the step of solubilisation., one works at an alkaline pH value in the presence of a reducing agent of the mercapto group and in the presence of a denaturing agent.

11. Process according to claim 10, characterised in that one works in the presence of guanidine hydrochloride and/or of the general formula I as denaturing agent,

12. Process according to claim 11, characterised in that the concentration of guanidine hydrochloride amounts to 6 mol/1, that of the compound of the general formula I to 8 mol/1.

13. Process according to one of claims 10 to 12, characterised that one works in the presence of DTE, p-mercaptoethanol» cysteine or GSH.

14. Process according to one of the preceding claims,,, characterised in that one carries out purification and separation of reducing, oxidising and denaturing agents by means of steric exclusion chromatography or dialysis.

15. » Process according to one of the preceding claims, characterised in that* after the step of reactivation,, one carries out a purification step by means of dialysis.

16. - Process according to one of claims 1 to 15,? characterised in that one uses t-PA as gene-technologically produced eukaryotic protein.

17. Stimulatable.. non-glycosilated t-PA, obtainable according to the process according to one of claims 1 co 16.

18. » Process according to claim 6* characterised in chat one separates the mixed disulphide of protein and glutathione from the non-modified protein by ion exchanger treatment-

19. Process according to claim 1 for the activation of gene-technogically produced, heterologous eukaryotic proteins containing disulphide bridges, substantially as hereinbefore described and exemplified.

20. Gene-technologically produced, heterologous eukaryotic proteins,, whenever produced by the process according to any preceding claim.

21. Stimulatable? non-glvcosilated t-PA, whenever produced by the process according to any of claims 1 to 16f 18 or 19. F. R„ KELLY A CO. AGENTS FOR THE APPLICANTS.