| RESEARCH PAPERS |
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| Optimization of Conditions for Soluble Expression and Purification of the Cytosolic Region of Arabidopsis thaliana PDV1 |
| HAN Cui-xiao, LI Feng, YAN Xiao-jin, FENG Duo, LI Qian-qian, GAO Hong-bo, GAO Wei |
| College of Biological Sciences and Biotechnology, Science, Beijing Forestry University, Beijing 100083, China |
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Abstract 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.
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Received: 20 February 2012
Published: 25 June 2012
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