SELECTION OF STREPTOMYCES-GRISEUS PROTEASE-B MUTANTS WITH DESIRED ALTERATIONS IN PRIMARY SPECIFICITY USING A LIBRARY SCREENING STRATEGY

Streptomyces griseus protease B (SGPB) has primary specificity for lar ge hydrophobic residues. The protease is secreted in a premature form, and autocatalytic removal of the propeptide is essential for activity We genetically substituted the P1 Leu at the premature junction of SG PB with Phe, Met, or Val and monitored expression levels in Escherichi a coli. Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity An E. col i expression library containing 29,952 possible SGPB mutants was const ructed with variations at seven sites involved in conferring primary s pecificity A rapid, visual screening strategy was used to detect activ e protease secretion. The expression library was screened, in conjunct ion with the different premature junction sequences, for those variant s producing increased proteolytic activity. The sequences of the isola ted mutant genes were determined; the substrate specificities and ther mostabilities of the corresponding proteases were investigated. Mutant s isolated from the screen with the wild-type premature junction exhib ited substrate specificities and thermostabilities similar to wild-typ e. The screen with Phe at the premature junction P1 site resulted in t he isolation of mutant proteases with increased thermostabilities (up to an order of magnitude increase in half-life at 55 degrees C), while a protease with broad substrate specificity was isolated from the Val screen. Proteases isolated from the screen with Met at the premature junction P1 site exhibited dramatic increases in activity towards a sy nthetic substrate with Met at the P1 site. The results suggest that th e substrate specificity of recombinant SGPB is constrained by the sequ ence of the premature junction; active protease production is dependen t on the efficiency of the self-processive premature junction cleavage . With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities e ngineered at the premature junction. (C) 1996 Academic Press Limited