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US 5,453,363 AGranted
Process for the activation of t-PA or Ing after genetic expression in prokaryotes
AssigneeBoehringer Mannheim GmbH
InventorsRainer Rudolph, Stephan Fischer, Ralf Mattes
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Abstract
A process for the activation of t-PA or IgG after expression in prokaryotes is described. The process includes cell digestion, solubilization under denaturing and reducing conditions and activation under oxidizing conditions in the presence of GSH/GSSG.
Claims20
A process for activating a tissue type plasminogen activator molecule, comprising:
- (i) expressing said tissue type plasminogen activator molecule in a prokaryote to form refractile bodies;
- (ii) digesting prokaryotic cells which contain said refractile bodies;
- (iii) solubilizing said refractile bodies under conditions which reduce and denature said molecule;
- and
- (iv) oxidizing said solubilized, reduced and denatured molecule by contact with (a) reduced glutathione (GSH)/oxidized glutathione (GSSG) at a pH of from 9 to 12, wherein GSH is at a concentration of from 0.01 to 20 mmol/liter, GSSG is at a concentration of from 0.01 to 3 mmol/liter, and (b) a denaturing agent present at a non-denaturing concentration, wherein said denaturing agent is selected from the group consisting of arginine, guanidine hydrochloride, and a compound of formula R.sub.2 --CO--NRR.sub.1, wherein R and R.sub.1 are H or C.sub.1 -C.sub.4 alkyl, and R.sub.2 is H, NHR.sub.1, or C.sub.1 -C.sub.3 alkyl;
- so as to activate said molecule.
3 more elements
A process for activating a recombinantly produced molecule selected from the group consisting of (a) a tissue type plasminogen activator molecule and (b) an IgG molecule, comprising:
- (i) expressing said molecule in a prokaryote, to form refractile bodies,
- (ii) digesting prokaryotic cells which contain said refractile bodies,
- (iii) solubilizing said refractile bodies with an agent which reduces and denatures said molecule,
- (iv) removing said agent,
- (v) adding oxidized glutathione (GSSG) to said solubilized, reduced, and denatured molecule, under denaturing conditions, so as to convert thiol groups in said molecule into mixed disulphides, and GSSG into glutathione,
- (vi) removing any unreacted GSSG, and
- (vii) oxidizing said solubilized, reduced, and denatured mixed disulphide containing molecule by adding reduced glutathione (GSH) at a concentration of from 0.5 mmole/liter to 5 mmole/liter, at a pH of from 7 to 10.5, and a non-denaturing concentration of a denaturing agent selected from the group consisting of arginine, guanidine hydrochloride, and a compound of formula R.sub.2 --CO--NRR.sub.1, wherein R and R.sub.1 are H or C.sub.1 -C.sub.4 alkyl, and R.sub.2 is H, NHR.sub.1, C.sub.1 -C.sub.3 alkyl;
- so as to activate said molecule.
5 more elements
Dependencies are inferred from the claim text.