布氏锥虫自噬蛋白ATG12的表达、纯化及二级结构预测  

Expression,purification and secondary structure prediction of autophagy protein ATG12 in Trypanosoma brucei

作　　者：王瑞 崔冲 郑伟[1] 涂晓明[3] WANG Rui;CUI Chong;ZHENG Wei;TU Xiao-ming(School of Basic Medical Sciences,Henan University of Science and Technology,Luoyang 471023,China;Luoyang Ship Material Research Institute,Luoyang Sunrui Special Equioment Co.Ltd.,Luoyang 471000,China;Hefei National Laboratory for Physical Sciences at Microscale,University of Science and Technology of China,Hefei 230026,China)

机构地区：[1]河南科技大学基础医学院,河南洛阳471023 [2]洛阳船舶材料研究所双瑞特装有限公司,河南洛阳471000 [3]中国科学技术大学合肥微尺度实验室,安徽合肥230026

出　　处：《生物技术》2022年第2期146-152,共7页Biotechnology

基　　金：河南科技大学博士启动基金项目(4020/13480038);国家自然科学基金面上项目(31270780)。

摘　　要：[目的]表达纯化布氏锥虫自噬蛋白TbATG12,并对其二级结构进行预测。[方法]克隆TbATG12编码序列,采用酶切连接方法构建表达载体TbATG12(pET-28a),转化进入BL21(DE3)表达菌株诱导蛋白表达,利用Ni^(2+)-NAT亲和层析及分子筛层析方法纯化蛋白。采用核磁手段,采集一维和二维谱图,利用JPred 4预测其二级结构。[结果]16℃、0.5 mmol/L IPTG诱导20 h,TbATG12以可溶蛋白形式存在。纯化后蛋白纯度可达95%。TbATG12谱峰分布均匀,强度较强,二级结构预测其含有3个α-螺旋和5个β-折叠,为后续解析TbATG12溶液结构提供指导。[结论]克隆、表达纯化得到纯度>95%可溶TbATG12蛋白,二级结构预测和比对发现其结构在进化上很保守。[Objective]To express and purify autophagy protein TbATG12 of Trypanosoma brucei,and predict its secondary structure.[Method] The coding sequence of TbATG12 was cloned.The expression vector TbATG12(pET-28 a) was constructed by restriction enzyme digestion and ligation,and transformed into BL21(DE3) expression strain to induce protein expression.The protein is purified by Ni^(2+)-NAT affinity chromatography and further purified by gel-filtration.One-dimensional and two-dimensional spectra of TbATG12 were collected and its secondary structure was also predicted by JPred 4.[Result] TbATG12(pET-28 a) was expressed as soluble protein at 16 ℃ with 0.5 mmol/L IPTG induction for 20 hours.The purity of the protein could be about 95%.Spectral analysis showed that the spectral peaks of TbATG12 were uniformly distributed and strong.The secondary structure of TbATG12 contains three α-helices and five β-sheets,which provided points for the further analysis of TbATG12 solution structure.[Conclusion] The soluble TbATG12 protein with a purity of >95% was obtained by cloning,expression and purification and the protein structure is conserved in evolution by the secondary structure prediction and comparison.

关 键 词：布氏锥虫 TbATG12 自噬 蛋白表达纯化 二级结构 保守结构 

分 类 号：Q786[生物学—分子生物学]