Random mutagenesis of staphylococcal nuclease and phage display selection.
Multiple cycles of mutagenesis and phage display selection have been investigated as a method for obtaining enzymes with altered catalytic properties. A library of staphylococcal nuclease mutants displayed on phage was created by error-prone PCR mutagenesis and selected for binding to thymidine- or guanosine-containing substrate analogs. After discarding non-binders, the binding mutants were then subjected to further mutagenesis and selection rounds. After four mutagenesis and selection cycles, the catalytic properties of some of the resulting nucleases were studied and one nuclease with nine accumulated mutations was found to have a two-fold reduction in kcat for DNA hydrolysis, but a two-fold increase in kcat/Km for hydrolysis of a thymidine containing small molecule substrate. The possibility of this technique for in vitro evolution of enzyme properties is discussed.
Light
J
J
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Lerner
R A
RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D008836:Micrococcal Nuclease