%0 Journal Article
%T Fusion expression of fibrinolytic enzyme gene PPFE-I from endophytic Paenibacillus polymyxa in Escherichia coli and activity analysis<br>内生多粘类芽胞杆菌纤溶酶基因PPFE-I在大肠杆菌中融合表达及活性分析
%A 吕凤霞
%A 陆兆新
%A 别小妹
%A 林谦
%A 张充
%A 曹林
%A 郭瑶
%A 汤彦翀
%J 生物工程学报
%D 2010
%I 
%X With the genomic DNA of strain EJS-3 as the template, we amplified the gene of fibrinolytic enzyme from Paenibacillus polymyxa (PPFE-I) by PCR. We purified the PCR product and ligated it into pMD19-T. After DNA sequencing, we cloned the PPFE-I gene into expression vector pET-DsbA and transformed it into Escherichia coli BL21(DE3). Upon induction of IPTG, we found that the activity of recombinant fibrinolytic enzyme fused with DsbA expressed in Escherichia coli was 228 IU/mL. SDS-PAGE analysis showed that the recombinant enzyme was soluble and accounted for about 18.4% of total cell protein. Western blotting demonstrated that the recombinant protein was DsbA-PPFE-I. We purified the recombinant enzyme by Ni affinity chromatography, thrombin digestion and sephadex G-100 gel-filtration, and identified the molecular weight of purified product to be 66.3 kDa with MALDI-TOF mass spectrometry. The purified enzyme exhibited distinct fibrinolytic activity on fibrin plate.
%K endophytes
%K paenibacillus polymyxa
%K fibrinolytic enzyme
%K gene cloning
%K fusion expression<br>内生菌，多粘类芽胞杆菌，纤溶酶，基因克隆，融合表达
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=96D2F75FCFD47738336CC19D80419AF3&yid=140ECF96957D60B2&vid=96C778EE049EE47D&iid=5D311CA918CA9A03&sid=A91C28383511878F&eid=9F83C44826B8A7D6&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=0