Expression of Flp Protein in a Baculovirus/Insect Cell System for Biotechnological Applications.

Abstract

The Saccharomyces cerevisiae Flp protein is a site-specific recombinase that recognizes and binds to the Flp recognition target (FRT) site, a specific sequence comprised of at least two inverted repeats separated by a spacer. Binding of four monomers of Flp is required to mediate recombination between two FRT sites. Because of its site-specific cleavage characteristics, Flp has been established as a genome engineering tool. Amongst others, Flp is used to direct insertion of genes of interest into eukaryotic cells based on single and double FRT sites. A Flp-encoding plasmid is thereby typically cotransfected with an FRT-harboring donor plasmid. Moreover, Flp can be used to excise DNA sequences that are flanked by FRT sites. Therefore, the aim of this study was to determine whether Flp protein and its step-arrest mutant, FlpH305L, recombinantly expressed in insect cells, can be used for biotechnological applications. Using a baculovirus system, the proteins were expressed as C-terminally 3 × FLAG-tagged proteins and were purified by anti-FLAG affinity selection. As demonstrated by electrophoretic mobility shift assays (EMSAs), purified Flp and FlpH305L bind to FRT-containing DNA. Furthermore, using a cell assay, purified Flp was shown to be active in recombination and to mediate efficient insertion of a donor plasmid into the genome of target cells. Thus, these proteins can be used for applications such as DNA-binding assays, in vitro recombination, or genome engineering.