title: Random mutagenesis suggests that sequence errors are not a major cause of variation in the activity of individual molecules of β-galactosidase.
abstract: Wild-type Escherichia coli lacZ was subjected to error-prone PCR to generate two plasmid-encoded gene libraries containing approximately 2.6 (SD 1.9) nucleotide exchanges resulting in 1.8 (SD 1.4) amino-acid substitutions. The libraries were used, along with a plasmid containing wild-type lacZ, to transform E. coli lacking genomic lacZ. Cells expressing functional β-galactosidase were identified by blue/white screening. Cell lysates containing the populations of heterogeneously mutagenized β-galactosidase were subjected to single molecule assays using a capillary electrophoresis laser-induced fluorescence-based protocol. There was no significant difference in the average catalytic rate between the random mutagenized and wild-type enzyme populations. Furthermore, there was no clear pattern between error rates and the variances in the population catalytic rates. This suggests that random sequence errors are not a substantial source of the catalytic heterogeneity of this enzyme.
authors.affiliation: Chemistry Department, University of Winnipeg, 515 Portage Avenue, Winnipeg, MB, Canada. d.craig@uwinnipeg.ca
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Craig
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwab
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015723:Gene Library; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D001616:beta-Galactosidase / Q000235:genetics*
chemical_list: D001616:beta-Galactosidase