碱性蛋白酶抑制剂lupI-MSSS原核表达载体的构建及其蛋白表达、纯化  

Construction of prokaryotic expression vector for expression and purification of inhibitor lupI-MSSS

作　　者：王昆[1,2] 梁朋娟 李尚勇[1] 王伟[1] 孙晶晶[1] 刘均忠[1] 孙谧[1] 郝建华[1] 

机构地区：[1]农业部极地渔业开发重点实验室,海洋国家实验室海洋药物与生物制品功能实验室,中国水产科学研究院黄海水产研究所,山东青岛266071 [2]上海海洋大学食品学院,上海201306

出　　处：《食品与发酵工业》2017年第7期20-26,共7页Food and Fermentation Industries

基　　金：国家自然科学基金(41376175);国家实验室-鳌山科技计划(2016ASKJ14)

摘　　要：低温碱性金属蛋白酶MP是从菌株YS-80-122中提取纯化的,属于沙雷氏蛋白酶家族,与该家族报道的其他酶的结构类似,在MP基因下游有一抑制剂基因lup I,该抑制剂可以完全抑制蛋白酶MP的活性。在野生型抑制剂基因的基础上,通过定点突变将Lup I的N端增加Met-Ser-Ser-Ser,命名为Lup I-MSSS,通过构建p ET28alup I-MSSS原核表达载体,转化大肠杆菌BL21(DE3)并诱导表达,SDS-PAGE凝胶电泳结果表明该蛋白的大小约11 kDa,与预测的蛋白分子量一致。对影响蛋白诱导表达的诱导剂添加时间、诱导温度、异丙基硫代半乳糖苷(IPTG)浓度、诱导时间4个因素进行优化,并经初步优化得到了其诱导表达的条件为:接种量2%,诱导剂添加时间3 h,诱导剂IPTG终浓度0.5 mmol/L,诱导温度为37℃,诱导时间为8 h。表达的蛋白依次通过超滤,Superdex 200凝胶过滤层析,Q-Sepharose离子交换层析进行纯化,结果显示目的蛋白纯化倍数为20,比活力为15 720U/mg,活性回收率达60.7%,纯化后的lup I通过高效液相色谱法进行纯度分析,其纯度可达99%以上。The alkaline metalloproteases MP is extracted from the strain YS-80-122 and belongs to the family of serralysin. Similarly to the other enzymes of this family reported before,there is an inhibitor gene lupI downstream of the MP gene and LupI can completely inhibit the activity of MP. On the basis of the wild-type inhibitor gene, four ami- no acids were added to the N-terminus of LupI, named LupI-MSSS. This work aims to construct a prokaryotic expres- sion vector for LupI-MSSS, the recombinant plasmid was transformed to BL21 （DE3）, and the expression was in- duced by IPTG. The recombination LupI-MSSS was separated using SDS-PAGE and the size of expressed LupI-MSSS was consistent with the prediction. Single factor experiment were used to optimize the fermentation condition and the optimal conditions were ：the inoculation amount is 2 % , IPTG as the induced agent was added at 3 h after inocula- ting, and it final concentration is 0.5 mmol/L, the recombined E. coli need inducing 8 hours at 37 ℃. Consecutive steps were used to achieve the purified protein as follows ： ultrafiltration, Q Sepharose ion exchange, Superdex 200 gel filtration, and the purity of LupI-MSSS is up to 99%

关 键 词：蛋白酶抑制剂 原核表达 蛋白纯化 

分 类 号：Q55[生物学—生物化学] Q78