γ-突触核蛋白的克隆、表达及纯化

目的对γ-突触核蛋白(synuclein)基因进行克隆,并对其表达及纯化条件进行优化。方法根据GenBank中γ-突触核蛋白的基因序列,设计PCR引物,从人脑cDNA文库中扩增γ-突触核蛋白的编码DNA序列,将其克隆至pGEX-6p-1载体中,构建重组质粒,将测序正确的重组质粒转化入大肠杆菌BL21(DE3)中,用IPTG诱导表达;对培养温度、IPTG的用量、诱导时间等条件进行了优化;用SDS-PAGE和质谱分析表达的目标蛋白;用亲和层析、离子交换层析、凝胶过滤纯化目标蛋白。结果γ-突触核蛋白在大肠杆菌中高效、可溶性表达,经质谱鉴定及SDS-PAGE分析,表达产物为γ-突触核蛋白。经过纯化,得到纯度高达98%的γ-突触核蛋白。结论成功地在大肠杆菌中高效表达了γ-突触核蛋白,并建立了纯化工艺,得到高纯度的重组蛋白,为进一步研究γ-突触核蛋白的晶体结构、生物学活性及功能,探讨其在肿瘤中的发病机制奠定了基础。

Cloning, Expression and Purification of Human Synuclein Gamma

Objective To study cloning, prokaryotic expression and purification of human synuclein gamma. Methods A DNA fragment encoding human synuclein gamma was amplified by polymerase chain reaction from a human cDNA library and was cloned into vector pGEX-6p-1. The construct carrying the coding DNA sequence of synuclein gamma fused with GST was transformed into E.coli BL21(DE3), and the fusion protein of synuclein gamma and GST was expressed by induction with IPTG. The product was purified by affinity chromatography, ion exchange chromatography and gel filtration. Results Human synuclein gamma was successfully expressed in E.coli in soluble form. The product was identified to be human synuclein gamma by SDS-PAGE and mass spectrography. After purification, the purity of the recombinant protein was more than 98%. Conclusion An effective purification protocol was established. Recombinant synuclein gamma with a purity more than 98% was obtained, which provided a basis for the further studies of the crystal structure, biological activities and functions of synuclein gamma.