Protein Engineering Protocols - Mycobacteriology research center

11Degenerate Oligonucleotide Gene ShufflingPeter L. Bergquist and Moreland D. GibbsSummaryImprovement of the biochemical characteristics of enzymes has been aided by misincorporationmutagenesis and DNA shuffling. Shuffling techniques can be used on a collection of mutantsof the same gene, or related families of genes can be shuffled to produce mutants encodingchimeric gene products. One difficulty with current shuffling procedures is the predominance ofunshuffled (“parental”) molecules in the pool of mutants. We describe a procedure for gene shufflingusing degenerate primers that allows control of the relative levels of recombination betweenthe genes that are shuffled and reduces the regeneration of unshuffled parental genes. This procedurehas the advantage of avoiding the use of endonucleases for gene fragmentation beforeshuffling and allows the use of random mutagenesis of selected segments of the gene as part ofthe procedure. We illustrate the use of the technique with a diverse family of β-xylanase genesthat possess widely different G and C contents.Key Words: Polymerase chain reaction; primer extension; degenerate primers; in vitro evolution;gene shuffling; complementary degenerate-end primers.1. IntroductionUntil recently, the most popular methods of creating combinatorial librarieswere recursive strategies that sought to evolve sequences by the addition of pointmutations. For in vitro evolution, inclusion of recombinant polymerase chainreaction (PCR; gene shuffling) offers practical and theoretical advantages oversimple recursive mutagenesis methods (1–3). It will rapidly fine-tune the mutationalload in several parts of the protein by recombining point mutations andwild-type sequences. Family shuffling is usually achieved by fragmentation of thegenes to be shuffled, followed by PCR. It relies on homologous recombinationduring the PCR reassembly step. Most methods require relatively high levels ofsequence similarity between the genes to be shuffled, because “crossover points”seem to occur in these regions.From: Methods in Molecular Biology, vol. 352: Protein Engineering ProtocolsEdited by: K. M. Arndt and K. M. Müller © Humana Press Inc., Totowa, NJ191