Tiam1截短体融合蛋白的原核表达与纯化

Prokaryotic expression and purification of Tiam1 truncated proteins

目的 Tiam1是一种鸟嘌呤核苷酸转换因子,与肿瘤的侵袭和转移密切相关。文中构建Tiam1基因的截短体原核表达重组质粒,在大肠埃希菌中诱导蛋白表达,并对GST融合蛋白进行纯化和鉴定。方法采用PCR方法扩增Tiam1的C685、C751、C1199基因片段,将目的基因片段插入p GEX-4T-1质粒载体后转化大肠埃希菌BL21(DE3)感受态细胞,用IPTG诱导蛋白表达。利用谷胱甘肽琼脂糖珠进行分离纯化,再采用Western blot检测目的蛋白表达情况。结果构建了3个Tiam1基因的截短体重组质粒,IPTG诱导显示,GST-Tiam1 C685、GST-Tiam1 C751、GST-Tiam1 C1199主要以可溶性蛋白形式在细胞裂解液上清中表达,表达产物的相对分子量分别为100 000、108 000、157 000。结论成功构建可以表达生物活性蛋白的GST-Tiam1 C685、GST-Tiam1 C751、GST-Tiam1 C1199融合蛋白原核表达载体,为进一步研究Tiam1蛋白奠定了基础。

objective Tiaml, a member of guanine nucleotide exchange factors, plays an important role in tumor invasion and metastasis. This study aimed to construct Tiaml truncated recombinant plasmids and induce the expression of GST-tagged human Tiaml fusion proteins in Escherichia coli （ E. coli） , followed by purification and identification of the GST-Tiaml fusion protein. Methods The cDNA fragments of Tiaml C685, C751 and Cl 199 were amplified by PCR and cloned into the pGEX-4T-l vector. After verified by DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 （ DE3） competent cells, and the expression of the fusion protein was induced by IPTG. The GST-tagged human Tiaml fusion proteins were purified with glutathione-agarose resin and indentified by Western blot. Results Three Tiaml truncated recombinant plasmids were constructed successfully. The recombinant fusion proteins GST-Tiaml C685,GST-Tiaml C751, and GST-Tiaml Cl 199 were expressed mainly in the form of soluble proteins in the cell lysate supernatant with expected relative molecu-lar weight of 100, 108,and 157 kD. Conclusion The recombi-nant plasmids expressing the bioactive fusion proteins were construc-ted successfully, which has prepared the ground for the subsequent studies of the Tiaml protein.