食品与发酵工业2024，Vol.50Issue(12)：1-8,8.DOI:10.13995/j.cnki.11-1802/ts.037565
人工蛋白XXA促进人源肿瘤抑素Tumstatin可溶性表达
Artificial protein XXA promotes soluble expression of human-derived Tumstatin
摘要
Abstract
To realize the soluble expression of human-derived Tumstatin in Escherichia coli.Five recombinant E.coli(BL21(DE3))expressed by fusion of different solubility-promoting fusion tags and tumstatin genes were constructed,and isopropyl-beta-D-thiogalactopyr-anoside(IPTG)was used to induce the recombinant bacteria to express the target protein.SDS-PAGE and Western-Blot were employed to analyze and screen for the recombinant bacteria that could solubility express Tumstatin.The results indicated that BL21/pGEX-6p-l-Tum(containing glutathione mercaptotransferase tag-GST tag),BL21/pET28a-MBP-Tum(containing maltose-binding protein tag-MBP tag),BL21/pET28a-Tum(no tag)all expressed the fusion protein Tumstatin in the form of inclusion bodies.Only the recombinant bacteria BL21/pET28a-XXA-Tum(containing the reverse protein of antifreeze protein-XXA tag)expressed fusion proteins in a soluble form,and the target protein accounts for more than 45％of the total protein of the bacteria.The target protein was purified by affinity chromatography and analyzed by Western-Blot.The band of interest with a molecular weight of 50.9 kDa was obtained,which was consistent with the theo-retical molecular weight.Furthermore,the inducer concentration,addition time,induction temperature,and induction time of recombinant bacteria BL21/pET28a-XXA-Tum were optimized.Finally,the induction conditions of tumstatin fusion protein were selected as follows:Inducer concentration 0.1 mmol/L,inducer added at 2 h,induction temperature 16 ℃,induction time 36 h.The soluble expression of human Tumstatin in E.coli laid the foundation for the preparation of soluble human-derived Tumstatin in a large scale,which could pro-vide a reference for the soluble expression of functional polypeptides in E.coli.关键词
人源肿瘤抑素/大肠杆菌/可溶性表达/融合标签/抗冻蛋白的反向蛋白Key words
human-derived Tumstatin/Escherichia coli/soluble expression/fusion tags/reverse protein of antifreeze protein引用本文复制引用
敖龙,张梁,朱玲玉,庞欣,辛瑜,郭忠鹏,朱瑞,李默影,顾正华,郭自涛..人工蛋白XXA促进人源肿瘤抑素Tumstatin可溶性表达[J].食品与发酵工业,2024,50(12):1-8,8.基金项目
国家重点研发计划(2021YFC2100300) （2021YFC2100300）
