Improved peptide function from random mutagenesis over short 'windows'.
We have applied random mutagenesis over short contiguous residue tracts ('windows') within an active peptide (the alpha-peptide of beta-galactosidase) such that all window residues are replaced simultaneously. A novel technique using mixed synthetic oligonucleotides and selection against an EcoK restriction site has allowed the construction of libraries of mutants for two separate windows, sites A and B. Mutant phenotypes can be easily assessed in vivo by a complementation test, and panels of mutants have been quantitatively tested in vitro. This 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 upmutations for standard or altered environments. Window mutagenesis can in principle be applied to any protein--protein or protein--ligand interaction.
Dunn
I S
IS
CSIRO Division of Biotechnology, North Ryde, NSW, Australia.
Cowan
R
R
Jennings
P A
PA
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics; D004274:DNA, Recombinant / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005696:Galactosidases / Q000096:biosynthesis*; D005818:Genetic Engineering / Q000379:methods*; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011897:Random Allocation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D005696:Galactosidases; D001616:beta-Galactosidase