海洋假单胞杆菌褐藻胶裂解酶基因在大肠杆菌中的高效表达和活性检测

利用PCR方法从假单胞杆菌基因组DNA扩增胞外褐藻胶裂解酶的全基因和缺损片段,构建表达质粒pQE30-aly32和pQE30-aly165,并在大肠杆菌M15中成功表达。表达的重组蛋白大部分存在于包涵体中,经洗涤,变性和复性,镍柱亲和层析纯化,获得了电泳纯的Aly32和Aly165。酶活性检测发现,仅Aly165具有较强活性,108 u/mg,该酶对poly(G)和poly(M)都有活性,对poly(M)更敏感。对表达体系进行优化,确定IPTG的最佳浓度为0.6mmol/L,最佳诱导菌液密度OD600为0.5~0.6,最佳表达时间为3h,28℃低温诱导可以提高重组蛋白的可溶性,可溶性蛋白相对表达量达到25%。

High Expression of Alginate Lyase from Pseudoalteromonas elyakovii IAM 14594 in Escherichia coli and Detection of Enzyme Activity

The full length and N-terminal deleted Aly gene were amplified from the genomic DNA of Pseudoalteromonas sp.by using PCR and inserted into pQE30 vector to construct the expression plasmid called pQE-aly32 and pQE-aly165.The expression plasmids were transfected into M15 and the Aly protein was found to be highly expressed as inclusion bodies.The inclusion bodies were isolated and subjected to denaturzing and renaturation,followed by Ni-NTA agarose affinity chromatography to purify the expressed Aly.The specific activity of purified Aly165 on both polyM and polyG block,especially on polyM,was close to that of native Aly.The expression system of the Aly165 in E.coli was optimized,showing that the most effective concentration of IPTG was 0.6 mmol/L,the OD600 of bacteria density of E.coli ranged from about 0.5 to 0.6 and the best expression time was 3 h.The soluble Aly165 protein which was expressed at 28 ℃was 25%.