叶绿体分裂蛋白PDV1胞质侧结构域可溶性表达条件的研究及纯化

目的:探索叶绿体分裂蛋白PLASTID DIVISION1(PDV1)胞质侧结构域的高效可溶性表达条件,并得到高纯度目的蛋白。方法:通过改变表达载体种类、基因片段大小、诱导剂浓度、诱导温度的方法,以及运用分子伴侣的协助,实现目的蛋白高效可溶性表达。通过镍柱亲和层析和分子筛层析纯化目的蛋白。结果:(1)带His标签的目的蛋白大部分以包涵体形式存在于沉淀中;(2)截掉疏水区域并与增溶标签GST或NusA融合表达,再通过改变诱导表达条件,可以实现PDV1胞质侧结构域的可溶性表达;(3)比较目的蛋白可溶性表达量,选择高效可溶性表达体系,并在该条件下纯化得到高纯度目的蛋白。结论:PDV1胞质侧结构域的高效可溶性表达及纯化,为进一步研究该蛋白的结构及其在叶绿体分裂过程中的作用奠定了一定基础。

Optimization of Conditions for Soluble Expression and Purification of the Cytosolic Region of Arabidopsis thaliana PDV1

Plastid Division1(PDV1),an outer envelope membrane protein of chloroplasts,with its larger N-terminal portion facing the cytosol and its smaller C-terminal portion existing in the inter-membrane space,plays a key role in chloroplast division.To improve the soluble expression level of Arabidopsis thaliana PDV1,and get large-scale purified proteins,types of the expression vector,different regions of the gene,concentrations of IPTG,temperatures of induction were optimized.Additionally,the co-expression system of recombinant plastids and five molecular chaperone vectors(pG-KJE8、pGro7、pKJE7、pGTf2 and pTf16)were constructed respectively.The results are described as follows:(1) The target proteins expressed from the recombinant plastids,which were constructed in pET22b and pET28b with the N-terminal 1-206,1-180,38-206 and 38-180 amino acid residues of PDV1,formed into inclusion bodies,even with different expression conditions.(2) The soluble protein level was increased significantly when the N-terminal 180 amino acid residues of PDV1 was cloned into the expression vector pGEX6p-1,induced with 0.2 mmol/L IPTG and grown at 16℃ overnight.However,the proteins fused with GST expressed from pGEX6p-1-PDV1-206 formed into inclusion bodies.The soluble protein level was higher when a region of the N-terminal 180 amino acid residues was expressed with the expression vector pET44a.(3) Highly pure soluble NusA-PDV1-180 fusion protein was obtained through Ni-NTA and gel filtration chromatography.The soluble proteins provide a potential value for the further mechanistic study of the structure and function of PDV1 in chloroplast division.