Site-directed mutant libraries for isolating minimal mutations yielding functional changes.
Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity.
Department of Chemistry, University of California, Davis, CA 95616, USA.
Dong Hee
DH
Chung
Department of Chemistry, University of California, Davis, CA 95616, USA.
Sarah C
SC
Potter
Department of Chemistry, University of California, Davis, CA 95616, USA.
Ammon C
AC
Tanomrat
Department of Physics, University of California, Davis, CA 95616, USA.
Krishnakumar M
KM
Ravikumar
Department of Chemistry, University of California, Davis, CA 95616, USA.
Michael D
MD
Toney
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D000637:Transaminases / Q000737:chemistry / Q000235:genetics
D029968:Escherichia coli Proteins; D000637:Transaminases
mutant library; protein engineering; reaction specificity; site-directed mutagenesis