Producing Chimeric Genes by CLERY: In Vitro and In Vivo Recombination

Directed evolution usually starts from the analysis of variant genes. Among methods used to produce such diversity, recombination between members of a gene family is a frequent approach (1 ). Building a library of mosaic genes can been achieved by fragmentation of parental genes and subsequent recombination by PCR without primers (2 ,3 ). Cloning of the newly generated fragments need a ligation step followed by transformation of the universal cloning host: Escherichia coli . Here we describe a method combining a PCR-dependent reassembly of fragmented full expression vectors using optimized temperature cycles and an in vivo recombination and self-cloning in yeast. Cloning performed in yeast avoid the usual bias that could be introduced by ligation and propagation in E. coli , particularly any toxicity or counter-selection that would selectively apply to clones in the library. The method is illustrated by the construction of a combinatorial library between the human CYP1A1 and the CYP1A2 cDNA, which share 74% nucleotide sequence identity. Formation of at least 86% of mosaic genes was observed.