Kolb A F, Siddell S G
Institute of Virology and Immunology, University of Würzburg, Germany.
Gene. 1996 Dec 12;183(1-2):53-60. doi: 10.1016/s0378-1119(96)00470-2.
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.
噬菌体P1的Cre重组酶可催化原核细胞和真核细胞中lox重组靶位点之间的位点特异性重组，因此已成为基因研究中一种常用的工具。当使用Cre重组酶蛋白而非cre表达质粒来指导位点特异性重组时，有利于DNA序列在真核基因组中预先存在的lox位点处进行稳定的、Cre介导的整合（Baubonis和Sauer（1993年），《核酸研究》，21，2025 - 2029）。我们通过细菌生产了一种含有核定位信号的Cre重组酶，它作为与大肠杆菌麦芽糖结合蛋白（MBP）的融合蛋白，并通过一步亲和层析法纯化该蛋白。随后用蛋白酶因子Xa进行切割，可从麦芽糖结合蛋白上释放出包含核定位信号的Cre重组酶。令人惊讶的是，我们发现未切割的MBP - Cre融合蛋白的重组活性与天然Cre重组酶的活性几乎相同。这表明融合蛋白的MBP部分表现为一个独立的蛋白结构域，不干扰Cre活性，因此可作为一个独立的分子标签。此外，融合蛋白对蛋白水解降解具有很强的抗性，并且在很宽的温度范围内都具有活性。它能在体外和真核细胞中高效催化切除和整合反应。最后，我们能够证明，通过使用MBP - Cre，在一次转染实验中就可以从质粒上切除一个lox侧翼的DNA序列，并将其整合到基因组中预先存在的lox位点处。由此可以排除可能产生不良影响的载体骨架序列。本文所述的MBP - Cre融合蛋白不仅将成为体外和体内催化Cre介导的重组反应的有用工具，还将用于位点特异性重组机制的分析。
