PROCESS FOR THE ACTIVATION OF GENETICALLY ENGINEERED, HETEROLOGOUS, DISULPHIDE BRIDGE-CONTAINING EUKARYOTIC PROTEINS AFTER EXPRESSION IN PROKARYOTES

摘要

To activate heterologous, procaryotic proteins genetically engineered and presenting disulphide bridges after their expression in procaryotic cells, cellular disintegration, solubilisation under denaturating and reducing conditions and activation under oxidating conditions in the presence of GSH/GSSG, either one works during the activation stage with a pH value between 9 and 12, a GSH concentration between 0.1 and 20 mmol/l, a GSSG concentration between 0.01 and 3 mmol/l and a non-denaturating concentration of the denaturating agent, or one separates the reducing and denaturating agents, transforms the thiol groups of the proteins in the mixed disulphides of protein and glutathione by adding GSSG under denaturating conditions and sets during the activation stage a pH value between 7 and 10.5, a GSH concentration between 0.5 and 5 mmol/l and a non-denaturating concentration of the denaturating agent. This process is particularly useful to obtain t-PA and interferon beta .