关键词: α-突触核蛋白, 纯化

Abstract: Objective To investigate bacteria expression of rat α-Synuclein cDNA,and prepare the recombinant protein.Methods Total RNA were isolated from Wistar Rat brain.A cDNA fragment encoding the α-Synuclein was amplified by RT-PCR,and was subcloned into plasmid pGEX-4T-1.The recombinant plasmid DNA pGEX-raSYN was expressed with bacteria BL21(DE3),and Glutathione S transferase(GST) tagged protein was purified by column chromatography.Recombinant rat α-Synuclein was freed by thrombin cleavage,and confirmed with SDS-PAGE and western blot analysis.Results Amplified cDNA fragment was composed of 420 bp and encodes 140 amino acids.The fusion protein GST-raSYN was expressed in the bacteria,with IPTG present in the condition medium.The recombinant rat α-Synuclein was purified to homogeneity from soluble fraction of the bacteria.The molecular weight of the purified protein was calculated to be approximately 524 000 on superdex S-200HR and to be 18 000 on SDS-PAGE in the presence of b-mercaptoethanol.Western blot analysis demonstrates that the purified recombinant protein was reactive to the anti α-Synuclein monoclonal or polyclonal antibodes.Conclusion Rat α-Synuclein cDNA was effectively expressed with bacteria,and pure recombinant rat α-Synuclein was prepared,which may be samples to studies physiological function of the protein.

Key words: Parkinson disease, α-Synuclein, purification, rat