title: Reducing mutational bias in random protein libraries.
abstract: The success of protein optimization through directed molecular evolution depends to a large extent on the size and quality of the displayed library. Current low-fidelity DNA polymerases that are commonly used during random mutagenesis and recombination in vitro display strong mutational preferences, favoring the substitution of certain nucleotides over others. The result is a biased and reduced functional diversity in the library under selection. In an effort to reduce mutational bias, we combined two different low-fidelity DNA polymerases, Taq and Mutazyme, which have opposite mutational spectra. As a first step, random mutants of the Bacillus thuringiensis cry9Ca1 gene were generated by separate error-prone polymerase chain reactions (PCRs) with each of the two polymerases. Subsequent shuffling by staggered extension process (StEP) of the PCR products resulted in intermediate numbers of AT and GC substitutions, compared to the Taq or Mutazyme error-prone PCR libraries. This strategy should allow generating unbiased libraries or libraries with a specific degree of mutational bias by applying optimal mutagenesis frequencies during error-prone PCR and controlling the concentration of template in the shuffling reaction while taking into account the GC content of the target gene.
authors.affiliation: Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Ghent University, B-9000 Ghent, Belgium. Thomas.Vanhercke@bayercropscience.com
authors.forename: Thomas
authors.initials: T
authors.lastname: Vanhercke
authors.forename: Christophe
authors.initials: C
authors.lastname: Ampe
authors.forename: Luc
authors.initials: L
authors.lastname: Tirry
authors.forename: Peter
authors.initials: P
authors.lastname: Denolf
mesh_terms: D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D004731:Endotoxins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D006460:Hemolysin Proteins; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics*; D019914:Taq Polymerase / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011506:Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D004247:DNA; D019914:Taq Polymerase