An effective family shuffling method using single-stranded DNA

Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fr agments into parental gene sequences, because such a process prevents the f ormation of chimeric sequences. In order to improve the efficiency of hybri d 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. Wh en these ssDNAs were digested by DNase I and reassembled, chimeric genes we re obtained at a rate of 14%, which was much higher than the rate of less t han 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. (C) 2000 Pu blished by Elsevier Science B.V. All rights reserved.