%0 Journal Article
%T Prokaryotic Expression of DNA Recombinase FLP and Its Purification with Enzymatic Activity<br>DNA重组酶FLP原核表达与多步法纯化及其活性检测
%A GAI Ying
%A WANG Wen_Qi
%A LU Hai
%A IANG Xiang_Ning
%A <br>盖颖
%A 王文棋
%A 陆海
%A 蒋湘宁
%J 生物工程学报
%D 2007
%I 
%X DNA recombinase FLP gene exists on the 2 micro 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 0.5-1.0 mL 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.
%K recombinase FLP
%K overexpression
%K ammonium sulfate precipitation
%K micro-column Ni~ 2
%K -affinity purification
%K activity assay<br>重组酶FLP
%K 高效表达
%K 硫酸铵沉淀
%K 亲合层析
%K 交换活性
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=E46711854C9D46F4&yid=A732AF04DDA03BB3&vid=EA389574707BDED3&iid=E158A972A605785F&sid=94655B9881133A28&eid=B3AAD7DC3C912B50&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=19