IMPROVED PROPERTIES OF FLP RECOMBINASE EVOLVED BY CYCLING MUTAGENESIS

The site-specific recombinases FLP and Cre are useful for genomic engi neering in many living systems. Manipulation of their enzymatic proper ties offers a means to improve their applicability in different host o rganisms. We chose to manipulate the thermolabilty of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for sel ection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through c ycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of scree ning at the same stringency to enrich for clones with improved fitness . An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.