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

Journal of Molecular Biology
S S Sidhu, T J Borgford

Abstract

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 sit...Continue Reading

Citations

Apr 2, 1999·European Journal of Biochemistry·H Czapinska, J Otlewski
Dec 26, 2007·Biochimica Et Biophysica Acta·Ting-Wai Lee, Michael N G James
Jul 12, 2002·Medicinal Research Reviews·Claudiu T SupuranBrian W Clare
Oct 1, 2010·Biomolecular Concepts·Ilya V DemidyukSergey V Kostrov
May 29, 1999·Applied and Environmental Microbiology·P BressollierB Verneuil
Jun 16, 2004·Biochemistry·Koman JoeAndrew J Bennet
Aug 1, 1997·Current Opinion in Structural Biology·P Kast, D Hilvert
Aug 1, 1997·Current Opinion in Structural Biology·H Zhao, F H Arnold
Jan 7, 1998·Trends in Biotechnology·O Kuchner, F H Arnold
Mar 14, 2000·Chemistry & Biology·R J ElliottT J Borgford

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