DNA重组酶FLP原核表达与多步法纯化及其活性检测

Prokaryotic Expression of DNA Recombinase FLP and Its Purification with Enzymatic Activity

DNA重组酶FLP存在于酵母2μ质粒上，能识别34bp的FRT位点，并根据2个FRT位点的相对方向完成位点间DNA序列的交换、重组、删除与逆转，在现代分子生物学理论研究与基因工程技术开发中具有广泛应用。构建了在原核大肠杆菌中高效表达FLP重组酶的表达载体pQE32-flpe并建立起相应的原核高效表达体系，在原核细菌大肠杆菌M15菌株中实现FLP酶蛋白的高效表达，同时建立了相应的纯化方法。纯化时先用硫酸铵沉淀法富集FLP酶蛋白，经透析脱盐后再用镍离子鳌合微柱（0．5—1．0mL）亲合层析梯度洗脱的方法获得纯化的FIJP酶蛋白。通过构建含有2个方向相同的FRT序列位点的质粒pUC18-FRT-gfp-FRT和含有1个FRT位点的表达载体pET30a-FRT，并分别以其为底物来检测FLP重组酶的删除、交换与重组功能的活性。结果表明，该方法不仅能有效表达FLP酶蛋白，并能行之有效地纯化FLP酶蛋白，以及检测纯化的FLP酶蛋白对DNA序列的删除、重组与交换功能。该方法简单易行并能获得有活性的FLP酶蛋白，为深入研究其机理以及研发相应的DNA重组技术提供重要参考。

DNA recombinase FLP gene exists on the 2μ plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of 34bp and function the sequences for exchange, recombination, deletion and reversion between the two orientated FRT sites. These functions are highly recognized by molecular biologists and biotechnology engineers for theoretic and applicable technology studies. This work constructed a prokaryotic over-expressed vector harboring FLP gene nominated as pQE30-flpe and established its over-expression culture system in which recombinase FLP could be efficiently expressed in E. coli strain M15. Purification procedures for high purity and active FLP are established through combination of ammonium sulfate precipitation with a 0.5 -1.0mL micro-column technique of Ni affinity chromatography with gradient elution. To verify the recombinase activity of purified FLP, substrate vectors, sequence donor vector （pUC18-FRT-gfp-FRT） and sequence accepting vector （pET30a-FRT） are constructed with various number, orientation of FRTs harboring the GFP gene for the expression of visible assay of the functions of recombination, exchange and deletion. Results showed that the system not only over expressed recombinase FLP in prokaryotic E. coli, but also efficiently purified the enzyme with a higher activity of the function of recombination, exchange and deletion. The system and the method are easily implemented and feasibly manipulated for theoretic study and biotechnology application.