人源磷脂酶PLA2异源可溶表达纯化及酶学分析  

Prokaryotic Expression,Purification and Enzymatic Characterization of Recombinant Human Phospholipase A2

作　　者：马文君 滕琳 田顺利 郭校燕 王培培 郑春阳 卫宏远[1] MA Wenjun;TENG Lin;TIAN Shunli;GUO Xiaoyan;WANG Peipei;ZHENG Chunyang;WEI Hongyuan(Tianjin University,Tianjin 300350 China;Robustnique Corporation Ltd.,Tianjin 300384,China;Health and Medical Department,General Hospital,Tianjin Medical University,Tianjin 300052,China)

机构地区：[1]天津大学,天津300350 [2]天津强微特生物科技有限公司,天津300384 [3]天津医科大学总医院保健医疗部,天津300052

出　　处：《食品工业科技》2021年第2期70-75,82,共7页Science and Technology of Food Industry

摘　　要：为实现人源磷脂酶A2的原核异源可溶表达,并对其进行纯化和初步的酶学性质分析。通过检索NCBI确定人源PLA2(PLA2G10),并将其构建于载体pET28a+上,并以麦芽糖结合蛋白(MBP)为促溶标签,成功构建载体pET28a-MBP-PLA2,转化E.coli BL21(DE3)后,经PTG低温诱导表达,SDS-PAGE鉴定后,确认其在上清中大量表达。根据蛋白的性质建立了一整套蛋白纯化工艺,包括Q柱洗脱,硫酸铵盐析,Phenyl柱纯化,Amylose柱纯化,分离纯化获得重组酶,SDS-PAGE测定该酶纯度大于90%。以卵磷脂为底物,酸碱滴定法测定其比活为127.04 U/mg,纯化得率48.8%,纯化倍数为10.3倍。酶学性质研究表明,该酶的分子量为56.5 kDa,最适反应温度为40℃,最适反应pH为8.0,是钙离子依赖酶,对PC亲和能力最强,Km值为12.2 mmol/L,V(max)为0.19 mmol/L/min。本试验建立的磷脂酶A2的异源表达、纯化体系,为其进一步的理论和工业研究奠定了基础。The prokaryotic expression and purification of human phospholipase A2 was realized,purification and the enzymatic characterization was analyzed.Human PLA 2(PLA2G10)was identified by searching NCBI,and constructed on the vector pET28a+,and maltose binding protein(MBP)was used as the solubilizing label.The vector pET28a-MBP-PLA was successfully constructed.After purification BL21(DE3)was transformed,it was induced to express at low temperature,and identified by SDS-PAGE,and confirmed to be massively expressed in the supernatant.According to the properties of the protein,a set of protein purification process was established,including Q-column elution,ammol/Lonium sulfate salting out,phynyl column purification,and xylose column purification.The purification of the recombinant enzyme was more than 90%by SDS-PAGE.The specific activity of lecithin was 127.04 U/mg by acid-base titration.The purification yield was 48.8%,and the purification multiple was 10.3 times.The study of enzymatic properties showed that the enzyme had a molecular weight of 56.5 kDa,the optimum reaction temperature was 40℃,the optimum reaction pH was 8.0.It was a calcium dependent enzyme with the strongest affinity to PC,K m value of 12.2 mmol/L,V max of 0.19 mmol/L/min.In this study,the heterologous expression and purification system of phospholipase A 2 was established,which laid a foundation for further theoretical and industrial research.

关 键 词：人磷脂酶A2 载体构建 异源可溶表达 蛋白纯化 活性测定 

分 类 号：TS201.2[轻工技术与工程—食品科学]