人源内皮抑素在大肠杆菌中的可溶性表达  

Soluble expression of human endostatin in Escherichia coli

作　　者：张吉 吴志勇 朱玲玉 辛瑜 顾正华[1,2] 石贵阳 张梁[1,2] ZHANG Ji;WU Zhiyong;ZHU Lingyu;XIN Yu;GU Zhenghua;SHI Guiyang;ZHANG Liang(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2022年第16期1-10,共10页Food and Fermentation Industries

基　　金：国家重点研发计划(2021YFC2100300)。

摘　　要：为高效制备人源内皮抑素(human endostatin,hEDN),解决其在大肠杆菌中的可溶性表达。该研究在大肠杆菌BL21(DE3)中分别构建了hedn单基因、融合标签共表达和融合表达系统,将重组菌在异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导下进行摇瓶发酵,通过SDS-PAGE分析及串联飞行时间质谱(matrix-assisted laser desorption/ionization time of flight/ionization time of flight,MALDI-TOF/TOF)鉴定hEDN的表达情况。结果表明,在构建的重组菌中只有含有谷胱甘肽巯基转移酶(glutathione S-transferase,GST)促溶标签的重组菌株BL21(DE3)/pGEX-6p-1-hedn能成功实现hEDN融合蛋白的可溶性表达。进一步地,通过改变诱导温度、诱导时间、诱导剂IPTG浓度及加入诱导剂IPTG时间来优化hEDN融合蛋白的表达条件,最终确定hEDN融合蛋白的最佳发酵条件为20℃,8~12 h加入0.3 mmol/L IPTG诱导36 h。通过亲和层析(PrePack GSH Purose 4 Fast Flow预装柱)初步纯化得到hEDN融合蛋白,经重组PreScission蛋白酶(PreScission protease,PPase)酶切后得到分子质量约为22 kDa的目的条带,与hEDN理论分子质量相一致。该研究为人源多肽的可溶性表达与纯化提供了研究思路,同时对微生物发酵制备功能性多肽具有一定的借鉴意义。Human endostatin(hEDN),a bioactive polypeptide composed of 184 amino acids,is located in C-terminal of human col-lagen type XVⅢ.The molecular weight of hEDN is about 22 kDa.The three histidine residues at the N-terminal of hEDN and aspartic acid at position of 76 are four Zn^(2+) binding sites,which can bind to Zn^(2+) to play an important role in its anti-angiogenic activity.At pres-ent,the clinical application of hEDN is still facing challenges such as low solubility,poor stability,high price,and large doses.There-fore,exploring the efficient methods for the preparation of hEDN is of great significance because it is beneficial to expand its application in the field of medicine.With the development of genetic engineering technology,biological expression systems have been widely used in the expression and preparation of recombinant proteins or polypeptides.Recently,a variety of expression systems were developed to the ex-pression of hEDN,such as mammalian cells,Pichia pastoris and Escherichia coli.The E.coli expression system has a lot of advantages,such as fast growth,clear genetic background,high expression level and low cost.For these reasons,E.coli expression system is one of the more suitable systems for the production of recombinant protein or polypeptide.However,the expression product of the gene hedn in E.coli is mostly insoluble inclusion bodies,which was formed due to protein misfolding.At the same time,the processing steps of inclu-sion bodies are usually complex and it is hard to refold the inclusion bodies successfully.Therefore,some fusion tags were selected to pro-mote the soluble expression of hEDN in E.coli,such as GST tag,Trx A tag and MBP tag.This study aims to investigate the conditions of soluble expression of hEDN in E.coli.Furthermore,we tried to complete the large-scale preparation of hEDN using E.coli as a work-horse.The various expression systems including single gene,multiple genes,and fusion tags were constructed in E.coli BL21(DE3)by using multiple molecular biological techniques,re

关 键 词：人源内皮抑素 可溶性表达 融合标签 谷胱甘肽巯基转移酶 多肽 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]