Salsolinol 合成酶是一种催化多巴胺和乙醛生成Salsolinol 的酶,与帕金森病发病机制密切相关。研究发现Salsolinol 合成酶与泛素的氨基酸序列高度相似,只有4 个氨基酸位点有差异。本研究以泛素基因为模板,采用聚合酶链式反应技术对4 个位点进行定点突变,将突变基因片段克隆到载体pET30a-GST 上,构建pET30a-GST-Sal synthase 重组载体,转化BL21 后,IPTG 诱导重组菌表达融合蛋白,经亲和层析柱纯化。结果表明,实现目的位点的定点突变,获得Sal 合成酶基因,成功构建了GST-Sal synthase 原核表达质粒,在大肠杆菌中表达纯化后得到较高纯度的GST-Sal synthase 融合蛋白。
The Parkinson's disease (PD) is a common progressive neurodegenerative disorder. The main pathological characteristics of the PD are the selective loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies. The MPTP (1-methyl- 4-phenyl-1, 2, 3, 6 -tetrahydropyridine) is a kind of much studied exogenous neurotoxins, known to cause parkinsonism in humans with selective neurotoxicity to dopamine neurons in the substantial nigra. The induction of the parkinsonism in humans by the MPTP suggests that endogenous or xenobiotic neurotoxins may elicit the Parkinson's disease. The salsolinol, a very similar kind of the MPTP, is a type of products from the condensation of the aldehydes and dopamine (DA) and has neurotoxicity. The reaction of the DA and the acetaldehyde to obtain the salsolinol is an enzyme reaction. The salsolinol synthase is an enzyme for synthesizing the salsolinol from dopamine and acetaldehyde. It is closely related to the pathogenesis of the Parkinson's disease (PD). The previous research shows that the Sal synthase sees a high amino acid similarity to the ubiquitin, with only difference of four amino acids between Sal synthase and ubiqutin. The finding reveals that the enzyme may play an important role in the cell process. The salsolinol synthase gene is obtained through the site-directed mutagenesis with the ubiquitin gene by the long primer polymerase chain reaction (PCR). The mutation gene is cloned into the prokaryotic GST fusion protein expression plasmid pET30a-GST, to form the pET30a- GST-Sal synthase plasmid. The recombinant plasmid is transformed to E.coli BL2l and the expression of the GST-Sal synthase fusion protein is induced by the IPTG. The protein is purified through the affinity chromatography methods. It is shown that the four site mutations are fully consistent with the expected results, and a prokaryotic system expressing the GST-Sal synthase fusion protein is successfully constructed. The GST-Sal synthase fusion protein with high purity could be obtained after it is high efficiently expressed in the E. coli BL21 and purified with the affinity chromatography.
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