麦芽糖结合蛋白-微管驱动蛋白11删除型融合基因表达载体的构建及其蛋白表达

Construction and Prokaryotic Expression of Maltose Binding Protein-Kinesin Superfamily Protein 11 Truncated Fusion Gene

目的:构建删除型微管驱动蛋白(KIF)11与麦芽糖结合蛋白(MBP)的融合蛋白的原核表达载体,并纯化得到高纯度的MBP-删除型KIF11融合蛋白。方法:PCR法从全长的KIF11基因组中扩增KIF11的相关删除型基因并连接到原核表达载体pMal中,此载体包含一个表达MBP的基因,删除型KIF11片段插入后与其融合,筛选和测序鉴定阳性克隆。将重组质粒转化到Rosseta大肠杆菌中,经异丙基硫代-β-D-半乳糖苷诱导表达和亲和层析分离纯化表达产物,对纯化的蛋白进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)和蛋白质免疫印迹杂交(Western blot)鉴定。结果:成功扩增删除型KIF11基因,构建MBP-KIF11-head,-stalk和-tail重组质粒并在Rosseta中诱导表达出MBP-KIF11-head,-stalk,-tail融合蛋白。经SDS-PAGE和Western blot法验证了高纯度纯化的融合蛋白。结论:建立了高效稳定的MBP-KIF11表达体系,为进一步研究KIF11与mRNA调控蛋白锌指结合蛋白1相互作用的区域打下基础。

Objective： To recombine truncated Kinesin superfamily protein （KIF） 11 gene in E.coli cells, and to purify the different fusion proteins of maltose binding protein （MBP）-KIF11. Methods： KIF11 cDNA was amplified by PCR. The KIF11 eDNA was inserted into the plasmid pMal. There was a MBP gene in pMal. By screening and sequenceing the recombinant plasmid, the positive constructs were transformed into the Rosseta host cells and induced by isopropyl-13-d-thiogalactoside. The recombinant products were purified in affinity chromatography and identified in sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Westem blot methods. Results： The truncated K/Fll gene was obtained successfully. The recombinant plasmid MBP-KIF 11-head, -stalk, -tail were constructed respectively. By SDS-PAGE and Westem blot methods, the fusion proteins MBP-KIFll-head, -stalk, -tail were purified successfully. Conclusion： The efficient prokaryotic expression system of MBP-KIF 11 is established. The purified MBP-KIF 11 protein provides to further study of the interaction between KIF 11 and mRNA regulation protein ZBP1.