GENOMIC TARGETING WITH AN MBP-CRE FUSION PROTEIN

The Cre recombinase of bacteriophage P1 catalyses site-specific recomb ination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic resear ch. Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombina se protein rather than a cre-expression plasmid is used to direct site -specific recombination (Baubonis and Sauer (1993) Nucleic Acids Res., 21, 2025-2029). We bacterially produced a Cre recombinase containing a nuclear localisation signal as a fusion protein with the E. coli mal tose binding protein (MBP) and purified the protein by one step affini ty chromatography. Subsequent cleavage with the protease factor Xa rel eases the Cre recombinase including the nuclear localisation signal fr om the maltose binding protein. Surprisingly, we found that the recomb ination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase. This suggests that th e MBP portion of the fusion protein behaves as a separate protein doma in which does not interfere with Cre activity and can thus be used as an independent molecular tag. Additionally, the fusion protein is very resistant to proteolytic degradation and active over a wide range of temperatures. It efficiently catalyses excision and integration reacti ons in vitro and in eukaryotic cells. Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DN A sequence from a plasmid and integrate it into a pre-existing lox sit e in the genome in one transfection experiment. Vector backbone sequen ces which might have undesirable effects can thereby be excluded. The MBP-Cre fusion protein described here will be a useful tool not only f or the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific re combination.