KANSL1蛋白的真核表达纯化及功能验证

Eukaryotic expression and purification of KANSL1 protein and its functional verification

目的利用昆虫杆状病毒表达系统(Bac-to-Bac)表达KANSL1蛋白并进行纯化,同时验证该蛋白在体外对微管蛋白聚合的影响。方法构建真核表达载体pFast-Bac-HTB-mCherry-KANSL1,转化大肠杆菌DH10Bac感受态细胞获得重组穿梭质粒(Bacmid),用脂质体转染介导Bacmid进入草地贪夜蛾细胞(Sf9 cell),以产生可表达目的基因的重组杆状病毒。随后用此重组杆状病毒感染并扩增Sf9细胞使其大量表达His-mCherry-KANSL1融合蛋白。通过镍柱亲和层析对表达的His-mCherry-KANSL1融合蛋白进行初步纯化,再经快速蛋白液相色谱(FPLC)进一步纯化,利用Western印迹、考马斯亮蓝染色鉴定目的蛋白的表达纯化效果,最后通过体外微管聚合实验分析纯化的KANSL1蛋白对微管蛋白聚合的影响。结果质粒测序显示,pFast-Bac-HTB-mCherry-KANSL1真核表达质粒构建成功;考马斯亮蓝染色、Western印迹表明纯化得到正确的His-mCherry-KANSL1蛋白,体外微管聚合实验显示KANSL1蛋白显著促进微管蛋白的聚合。结论成功构建pFast-Bac-HTB-mCherry-KANSL1真核表达质粒并在真核表达系统中表达和纯化,重组KANSL1蛋白具有促进微管聚合的作用,为后续KANSL1蛋白功能研究奠定了基础。

Objective To express and purify KANSL1 protein with the Bac-To-Bac baculovirus expression system and verify the role of purified KANSL1 protein in microtube assembly.Methods The eukaryotic expression vector pFast-BacHTB-mCherry-KANSL1 was constructed and transformed into E.coli DH10 Bac to obtain the recombinant shuttle vector named Bacmid-KANSL1,which was transfected into Sf9 cells with CellfectinⅡReagent,thus producing the recombinant baculovirus that could express the target proteins.The recombinant baculovirus was used to infect Sf9 cells to express HismCherry-KANSL1 fusion protein.Ni-NTA agarose was used to preliminarily purify the expressed His-mCherry-KANSL1 fusion protein,while fast protein liquid chromatography(FPLC)was used to further purify protein.Coomassie blue staining and Western blot were used to identify the expression of the target gene protein.Finally,the role of purified KANSL1 protein in microtube assembly was verified by in vitro tubulin polymerization assay.Results Sequencing analysis indicated that the eukaryotic expression vector pFast-Bac-HTB-mCherry-KANSL1 was successfully constructed.The specific band was shown by Coomassie blue staining and Western blot,which indicated that KANSL1 protein was expressed in Sf9 cells and highly purified His-mCherry-KANSL1 proteins were obtained.In vitro tubulin polymerization assays showed that KANSL1 stimulated microtube assembly.Conclusion The eukaryotic expression vector pFast-Bac-HTB-mCherryKANSL1 is successfully constructed and expressed effectively in the eukaryotic expression system,and KANSL1 fusion protein promotes microtube assembly,which can shed light on the function of KANSL1.