重组酶Cre基因在大肠杆菌中的高效表达及其一步纯化和活性检测

Recombinase Cre's overexpression in Escherichia coli BL21(DE3) and its one-step purification and activity assay

摘要: 为了实现DNA重组酶Cre在体外的应用,以pET-30a高效表达载体为基础构建了pTE30-Cre质粒.将此质粒导入大肠杆菌BL21(DE3)中进行IPTG诱导的Cre基因的高效表达.对表达出的Cre重组酶蛋白进行镍离子鳌合法一步纯化,并以带有两个同向loxP位点的质粒为底物,对纯化出的Cre酶进行活性检测.该实验为Cre酶的体外应用奠定了基础.

Abstract: In order to realize the application of Cre recombinase in vitro,plasmid pTE30-Cre was constructed based on a pET-30a vector.This plasmid was transferred into Escherichia coli BL21(DE3).The Cre gene was overexpressed and then induced by IPTG.The Cre recombinase protein was easily purified using a Ni⁺ affinity column and its activity was tested in vitro by providing a plasmid which contained two of the same directional loxP sites.