Genomic targeting with an MBP-Cre fusion protein.
The Cre recombinase of bacteriophage P1 catalyses site-specific recombination between lox-recombination target sites both in prokaryotic and eukaryotic cells and has thus become a popular tool in genetic research. Stable, Cre-mediated integration of DNA sequences at pre-existing lox sites in the eukaryotic genome is facilitated when a Cre recombinase 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 maltose binding protein (MBP) and purified the protein by one step affinity chromatography. Subsequent cleavage with the protease factor Xa releases the Cre recombinase including the nuclear localisation signal from the maltose binding protein. Surprisingly, we found that the recombination activity of the uncleaved MBP-Cre fusion protein is virtually identical to that of the native Cre recombinase. This suggests that the MBP portion of the fusion protein behaves as a separate protein domain 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 reactions in vitro and in eukaryotic cells. Finally, we could show that, by using MBP-Cre, it is possible to concomitantly excise a lox-flanked DNA sequence from a plasmid and integrate it into a pre-existing lox site in the genome in one transfection experiment. Vector backbone sequences which might have undesirable effects can thereby be excluded. The MBP-Cre fusion protein described here will be a useful tool not only for the catalysis of Cre-mediated recombination reactions in vitro and in vivo but also for the analysis of the mechanism of site-specific recombination.
Kolb
A F
AF
Institute of Virology and Immunology, University of Würzburg, Germany.
Siddell
S G
SG
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D017101:Bacteriophage P1 / Q000201:enzymology* / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D002415:Cats; D002460:Cell Line; D002467:Cell Nucleus; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015951:Factor Xa; D005347:Fibroblasts; D018390:Gene Targeting / Q000379:methods*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D013696:Temperature; D014162:Transfection; D014764:Viral Proteins
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C461599:maltose transport system, E coli; C045073:Cre recombinase; D019426:Integrases; D015951:Factor Xa