RENATURATION OF RECOMBINANT PROTEINS PRODUCED AS INCLUSION-BODIES

Expression of recombinant proteins in Escherichia coli often results i n the formation of insoluble inclusion bodies. Within the last few yea rs specific methods and strategies have been developed to prepare acti ve proteins from these inclusion bodies. These methods include (i) iso lation of inclusion bodies after disintegration of cells by mechanical forces and purification by washing with detergent solutions or low co ncentrations of denaturant, (ii) solubilization of inclusion bodies wi th high concentrations of urea or guanidine-hydrochloride in combinati on with reducing reagents, and (iii) renaturation of the proteins incl uding formation of native disulphide bonds. Renatured and native disul phide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulp hide interchange starting from mixed disulphides containing peptides. The final yield of renatured proteins can be increased by adding low c oncentrations of denaturant during renaturation.