title: An effective family shuffling method using single-stranded DNA.
abstract: Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fragments into parental gene sequences, because such a process prevents the formation of chimeric sequences. In order to improve the efficiency of hybrid formation in family shuffling, single-stranded DNAs (ssDNAs) were used as templates. The ssDNAs of two catechol 2,3-dioxygenase genes, nahH and xylE, were prepared, the xylE strand being complementary to the nahH strand. When these ssDNAs were digested by DNase I and reassembled, chimeric genes were obtained at a rate of 14%, which was much higher than the rate of less than 1% obtained by shuffling with double-stranded DNAs. Chimeric catechol 2,3-dioxygenases that were more thermally stable than the parental enzymes, XylE and NahH, were obtained by this ssDNA-based DNA shuffling.
authors.affiliation: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
authors.forename: K
authors.initials: K
authors.lastname: Ohnishi
authors.forename: S
authors.initials: S
authors.lastname: Harayama
mesh_terms: D050557:Catechol 2,3-Dioxygenase; D004247:DNA / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D049308:Dioxygenases; D004795:Enzyme Stability; D005818:Genetic Engineering / Q000379:methods*; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D004277:DNA, Single-Stranded; D011993:Recombinant Fusion Proteins; D004247:DNA; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase