Improved peptide function from random mutagenesis over short ‘windows’

Abstract

We have applied random mutagenesis over short contiguous residue tracts (‘windows’) within an active peptide (the α-peptide of β-galactosidase) such that all window residues are replaced simultaneously. A novel technique using mixed synthetic oligonucleotides and selection against an EcoKrestriction site has allowed the construction of libraries of mutants for two separate windows, sites A and B. Mutant phenotypes can be easily assessed in vivoby a complementation test, and panels of mutants have been quantitatively tested in vivoThis allowed the rapid probing of structural requirements for each site. The two windows yielded markedly disparate results. Site B was much less stringent in its sequence requirements for significant function than Site A, and mutants with improved function were isolated at Site B alone. In addition, one Site B mutant with wild-type levels of activity showed enhanced stability to heat or a protein denaturant. We propose that short tracts with the characteristics of Site B constitute ‘secondary’ interaction sites which are more tolerant of sequence diversity. Random manipulation of such secondary sites is thus more likely to yield up-mutations for standard or altered environments. Window mutagenesis can in principle be applied to any protein-protein or protein-Ugand interaction.