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

为实现人源肿瘤抑素Tumstatin在大肠杆菌中的可溶性表达,构建了5种不同促溶性融合标签与tumstatin基因融合表达的重组大肠杆菌BL21(DE3),利用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)诱导重组菌进行摇瓶发酵,利用SDS-PAGE和Western-Blot分析并筛选出能可溶性表达Tumstatin蛋白的重组菌。构建的重组菌中BL21/pGEX-6p-1-Tum(含谷胱甘肽巯基转移酶标签—GST标签)、BL21/pET28a-MBP-Tum(含麦芽糖结合蛋白标签—MBP标签)、BL21/pET28a-Tum(无标签)都是以包涵体的形式表达融合蛋白Tumstatin,只有重组菌BL21/pET28a-XXA-Tum(含抗冻蛋白的反向蛋白—XXA标签)能以可溶性的形式表达融合蛋白且目的蛋白占菌体总蛋白45%以上。利用亲和层析对目的蛋白进行纯化并进行Western-Blot分析,得到分子质量为50.9 kDa的目的条带,与理论分子质量一致。进一步对重组菌BL21/pET28a-XXA-Tum的诱导剂添加浓度、诱导剂添加时间、诱导温度、诱导时间诱导条件进行优化。最终选择Tumstatin融合蛋白诱导条件为:诱导剂浓度0.1 mmol/L、2 h添加诱导剂、诱导温度16℃、诱导时间36 h。人源肿瘤抑素在大肠杆菌中可溶性表达为大量制备可溶性人源肿瘤抑素奠定了基础,可为功能性多肽在大肠杆菌中的可溶性表达提供借鉴。

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-thiogalactopyranoside(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-1-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 theoretical 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 provide a reference for the soluble expression of functional polypeptides in E.coli.