%0 Journal Article
%T Prokaryotic expression, purification and enzymatic properties of nuclease P1<br>核酸酶P1的原核表达、纯化及酶学特性分析
%A Yanan Wang
%A Aiyun Wei
%A Meiyan Wang
%A Xiaobin Wei
%A Chao Zhang
%A Liwei Shan
%A Sanhong Fan
%A <br>王亚楠
%A 魏爱云
%A 王美艳
%A 卫晓彬
%A 张超
%A 单丽伟
%A 范三红
%J 生物工程学报
%D 2012
%I 
%X To establish a prokaryotic expression and purification protocol for nuclease P1 (NP1), we first obtained a synthetic NP1 by splicing 22 oligonucleotides with overlapping PCR. We constructed and transformed a secretory expression vector pMAL-p4X-NP1 into Escherichia coli host strains T7 Express and Origami B (DE3) separately. Then, the recombinant NP1 was purified by amylose affinity chromatography, and its activity, thermo-stability and metal-ion dependence were investigated systematically. The results indicated that the expressed fusion proteins MBP-NP1 (Maltose binding protein-NP1) existed mainly in soluble form both in host strains T7 Express and Origami B (DE3), but the specific activity of recombinant protein from Origami B(DE3) strain was higher than T7 Express strain (75.48 U/mg : 51.50 U/mg). When the MBP-tag was cleaved by protease Factor Xa, the specific activity both increased up to 258.1 U/mg and 139.2 U/mg. The thermal inactivation experiments demonstrated that the recombinant NP1 was quite stable, and it retained more than 90% of original activity after incubated for 30 min at 80 °C. Zn2+ (2.0 mmol/L) could increase enzyme activity (to 119.1%), on the contrary, the enzyme activity was reduced by 2.0 mmol/L Cu2+ (to 63.12%). This research realized?the functional expression of NP1 in the prokaryotic system for?the?first?time, and provided an alternative pathway for NP1 preparation.
%K nuclease P1
%K prokaryotic expression
%K affinity purification
%K thermo-stability<br>核酸酶P1，原核表达，亲和纯化，热稳定性
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=324F8504B2709856A136D62897F5752D&yid=99E9153A83D4CB11&vid=D3E34374A0D77D7F&iid=708DD6B15D2464E8&sid=77586D59F496BF38&eid=4108C40BBEB9A8DE&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=0