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

Fusion expression of fibrinolytic enzyme gene PPFE-I from endophytic Paenibacillus polymyxa in Escherichia coli and activity analysis

摘要:

以内生多粘类芽胞杆菌EJS-3基因组DNA为模板，PCR扩增PPFE-I基因，并克隆到pMD19-T载体上，构建克隆载体pMD-PPFE-I，经测序正确后，将PPFE-I基因克隆至表达载体pET-DsbA上构建重组表达质粒pET-DsbA/PPFE-I，将其转化至E. coli BL21(DE3)，在IPTG诱导下实现了融合蛋白DsbA-PPFE-I的表达，表达产物酶活性达228 IU/mL。表达产物用SDS-PAGE和Western blotting进行鉴定。SDS-PAGE电泳检测表明融合蛋白主要以可溶形式表达，占菌体总蛋白的18.4％。Western blotting结果表明在相应分子量处有一条特异性条带，证实该蛋白为DsbA-PPFE-I融合蛋白。表达产物通过Ni亲和柱、凝血酶酶切及Sephadex G-100等步骤进行分离纯化，并用 MALDI-TOF 质谱对重组酶进行了鉴定。纯化后的表达产物在纤维蛋白平板上表现出明显的纤溶活性。

Abstract:

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.