cre基因在大肠杆菌中的表达及表达蛋白活性的检测

Expression of cre Gene in Escherichia coli and Bioassay Its Expression Product

Cre重组酶来自噬菌体P1,可以识别特异的loxP位点的DNA序列 ,并进行专一性的剪切和拼接。利用PCR技术将cre基因克隆至原核表达载体pET 2 9a ,在大肠杆菌BL2 1(DE3)得到了高效表达。采用DEAE 5 2柱层析的方法对表达蛋白进行了纯化。体外生物学活性检测表明 ,表达蛋白对含有同向loxP位点的质粒有切割活性。

The Cre recombinase from bacteriophage P1 can recognize specific DNA sequences, cleave DNA at specific target sites, and then ligate it to the cleaved DNA of a second site. In this study, cre gene was cloned into the pGEM-T Easy vector via PCR procedure. Then the cre gene was inserted into an expression vector pET-29a and expressed in E.coli BL21(DE3). A 38kD soluble protein was expressed and named CRE. CRE was purified by DEAE-52 chromatography. Bioassay of the partially purified product showed that CRE can cleave the plasmid pGLGFP which contains two loxP sites with the same direction.