Selection of Streptomyces griseus protease B mutants with desired alterations in primary specificity using a library...

Streptomyces griseus protease B (SGPB) has primary specificity for large hydrophobic residues. The protease is secreted in a promature form, and autocatalytic removal of the propeptide is essential for activity. We genetically substituted the P1 Leu at the promature junction of SGPB with Phe, Met, or Val and monitored expression levels in Escherichia coli. Substitution with Phe had no effect on active SGPB production; substitution with Met or Val abolished proteolytic activity. An E. coli expression library containing 29,952 possible SGPB mutants was constructed with variations at seven sites involved in conferring primary specificity. A rapid, visual screening strategy was used to detect active protease secretion. The expression library was screened, in conjunction with the different promature junction sequences, for those variants producing increased proteolytic activity. The sequences of the isolated mutant genes were determined; the substrate specificities and thermostabilities of the corresponding protease were investigated. Mutants isolated from the screen with the wild-type promature junction exhibited substrate specificities and thermostabilities similar to wild-type. The screen with Phe at the promature junction P1 site resulted in the 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 Val screen. Proteases isolated from the screen with Met at the promature junction P1 site exhibited dramatic increases in activity towards a synthetic substrate with Met at P1 site. The results suggests that the substrate specificity of recombinant SGPB is constrained by the sequence of the promature junction; active protease production is dependent on the efficiency of the self-processive promature junction cleavage. With an efficient screening strategy, this relationship can be used to isolate catalytically active proteases with desired specificities engineered at the promature junction.