摘要
叶绿体是植物光合作用的重要场所。PLASTID DIVISION2(PDV2)是叶绿体外膜上调控叶绿体分裂的关键蛋白之一。PDV2的N末端较大伸向细胞质,C末端较小伸向膜间隙,探索叶绿体分裂蛋白PDV2-213(N末端213氨基酸残基)胞质侧结构域可溶性表达获得高纯度的蛋白质,为叶绿体分裂过程中其结构与功能的研究提供依据。通过pET表达载体的构建,优化原核表达条件,实现PDV2-213蛋白的可溶性表达;运用镍柱亲和层析和分子排阻层析的方法,对目的蛋白进行分离纯化。本研究可溶性表达了PDV2-213胞质侧结构域蛋白质,通过表达条件和镍柱亲和层析的优化,降低杂蛋白对PDV2-213蛋白质纯化过程的影响,获得高纯度的蛋白质。PDV2-213胞质侧结构域的高效可溶性表达和纯化,为叶绿体分裂过程中其结构与功能的研究提供依据。
Chloroplast is a major place for plant photosynthesis. PLASTID DIVISION2 （PDV2） is one of the key proteins that regulates the division of chloroplast on chloroplast outer membrane. The larger N-terminal of PDV2 protein faces the cytosol and its smaller C-terminal reaches the inter-membrance space. The obtain of highly purified protein through the exploration of the soluble expression of cytoplasmic domain of chloroplast mitotic protein PDV2-213 （N-terminal 213 amino acid residues） could provide the basis for the study of the structure and fun- ction of chloroplast in its division process. The pET expression vector was constructed and the prokaryotic expression conditions were optimized to obtain the soluble expression of PDV2-213. In the study, the Ni-affinity chromatography and size-exclusive chromatography method were adopted to separate and purify the target protein. In addition, cytoplasmic domain protein PDV2-213 was solubly expressed in this study. The optimization of expression conditions and Ni-affinity chromatography reduced the influence of impure proteins on the purification process of PDV2-213 protein and obtained highly purified protein. High soluble expression and purification of cytoplasmic domain of PDV2-213 provided reference for the further study of the structure and function of PDV2 in the division process of chloroplast.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2017年第3期1028-1034,共7页
Genomics and Applied Biology
基金
中央高校基本科研基金(2015ZCQ-LY-02)
国家自然科学基金(31070651)共同资助
作者简介
通讯作者，w_gao@bjfu．edu．cn
