机构：[1]Department of Neurobiology，Beijing Institute of Geriatrics，Xuanwu Hospital of Capital University of Medical Sciences，Beijing 100053老年医学科首都医科大学宣武医院
Objective To clone the cDNA of rat alpha-Syn gene, investigate its prokaryotic expression and produce purified recombinant rat alpha-Syn protein. Methods Rat alpha-Syn cDNA was amplified from the rat brain total RNA by RT-PCR and was cloned into pGEX-4T-1, a prokaryotie expressing vector. The recombinant plasmid containing rat alpha-Syn gene was transformed into E. Coli BL21 to express a fusion protein with rat alpha-Syn protein tagged by glutathione-S-transferase (GST). The fusion protein was then cleaved by thrombin during passing through the GST-agarose 4B column to release the recombinant rat alpha-Syn protein. The recombinant rat alpha-Syn protein was further purified using Superdex S200 gel filtration.Results DNA sequencing confirmed that the cloned cDNA contained 420 base pairs encoding 140 amino acids, which was identical to the reported amino acid sequence of rat alpha-Syn. After transformation, the recombinant plasmid pGEX-raSyn expressed a soluble protein that was inducible by IPTG. The purified recombinant protein was shown to be single band on SDS-PAGE, with a molecular size of around 18000, which was identical to the reported molecular size of rat alpha-Syn.Western blot analysis demonstrated that the recombinant protein was recognized by specific antibody against alpha-Syn. Conclusion The rat alpha-Syn gene was successfully expressed in prokaryotic expression system and highly purified rat alpha-Syn recombinant protein was produced.
基金：
This work was suppomed by Key
Project of National Natural Science Foundation of China
(30430280)：National NatureJ Science Fonndalion of China
(3027I437．30270482)：Natural Science Foundation of
Beiijng(7022011)
语种：
外文
PubmedID：
中科院(CAS)分区：
出版当年[2005]版：
无
最新[2023]版：
大类|2 区医学
小类|3 区神经科学
第一作者：
第一作者机构：[1]Department of Neurobiology，Beijing Institute of Geriatrics，Xuanwu Hospital of Capital University of Medical Sciences，Beijing 100053
通讯作者：
通讯机构：[1]Department of Neurobiology，Beijing Institute of Geriatrics，Xuanwu Hospital of Capital University of Medical Sciences，Beijing 100053
推荐引用方式(GB/T 7714)：
Xin LI,Yao-Hua LI,Jun-Yan HAN,et al.cDNA cloning, prokaryotic expression and purification of rat alpha-synuclein.[J].Neuroscience bulletin.2006,22(1):29-33.
APA：
Xin LI,Yao-Hua LI,Jun-Yan HAN,Shun YU&Biao CHEN.(2006).cDNA cloning, prokaryotic expression and purification of rat alpha-synuclein..Neuroscience bulletin,22,(1)
MLA：
Xin LI,et al."cDNA cloning, prokaryotic expression and purification of rat alpha-synuclein.".Neuroscience bulletin 22..1(2006):29-33
