Selection for improved subtiligases by phage display.

ABSTRACT: Engineering enzyme activity has been challenging because of uncertainties in structure-function relationships and difficulties in screening a large number of mutant enzymes. A product capture strategy using phage display is presented here for the selection of improved enzymes from a large library of variants (>10(9) independently derived mutants). Subtiligase, a double mutant of subtilisin BPN' that catalyzes the ligation of peptides, was displayed on phage. Twenty-five active site residues were randomly mutated in groups of four or five to yield six different libraries that were independently sorted. Variants that ligated a biotin peptide onto their own extended N termini were selectively captured. Mutant subtiligases were identified that had increased ligase activity. The selection also

SUBMITTER: Atwell S 

PROVIDER: S-EPMC22237 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature