Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.