cDNA cloning, prokaryotic expression and purification of rat alpha-synuclein.
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.
Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital of Capital University of Medical Sciences, Beijing 100053, China E-mail: liyaohua1962@ yahoo.com.cn.
Xin
X
Li
Yao-Hua
YH
Li
Jun-Yan
JY
Han
Shun
S
Yu
Biao
B
Chen