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手掌参中赤霉酸诱导的富含半胱氨酸蛋白的表达、纯化及鉴定
【作者】 刘渊；
【导师】 冯娟；
【作者基本信息】 电子科技大学 ， 生物物理学， 2009， 硕士
【摘要】 在前期筛选手掌参cDNA文库获得编码赤酶酸诱导的富含半胱氨酸蛋白的基因gcgasa基础上,研究基因本身的信号肽对gcgasa基因原核表达的影响。方法:设计带有EcoR I和Hind III酶切位点的引物,分别对gcgasa基因编码区全长(gcgasa)及信号肽缺失的cDNA片段(Δgcgasa)进行PCR扩增,将目标片段克隆入原核表达载体pET-32(a),构建重组质粒pET-32(a)/gcgasa、pET-32(a)/Δgcgasa。经测序鉴定后,转化大肠杆菌BL21(DE3),经IPTG诱导表达融合蛋白,采用SDS-PAGE及Western-blotting鉴定外源蛋白的表达,并通过电镜超薄切片技术检测包涵体在细胞内的定位。对于水溶性蛋白,进一步采用亲合层析手段进行初步纯化。结果:测序鉴定外源基因成功插入表达载体。SDS-PAGE及Western-blotting结果表明:含有信号肽的外源基因在大肠杆菌中以不可溶包涵体形式存在,而信号肽缺失的片段主要以可溶形式表达。细胞超薄切片的透射电镜结果显示:pET-32(a)/gcgasa中的包涵体主要出现在周质空间,而pET-32(a)/Δgcgasa重组质粒表达蛋白主要定位在细胞质中。可溶性产物经Ni-NTA柱及凝胶过滤后可获得纯度较高的蛋白。结论:信号肽缺失的Δgcgasa能在大肠杆菌中高效表达,其表达产物可溶,并取得了一定纯度和浓度的目标蛋白,这为进一步研究GASA的结构和功能奠定了基础。
【Abstract】 The gcgasa gene encoding GA-induced cystein-rich protein was screened from the cDNA library from young shoot of Gymanadenia Conopsea R.Br. Based on this previous work, we investigated the effect of signal peptide on the prokaryotic expression.Methods:The primers bearing restriction enzyme site of EcoR I and Hind III were designed and were utilized to amplify the full-length of ORF and the signal peptide -truncated fragment. The target fragments were then cloned into pET-32(a) to produce the recombinant plasmids pET-32(a)/gcgasa, pET-32(a)/Δgcgasa. After they were identified by DNA sequencing, the recombinants were transformed into E.coli BL21(DE3) to express the fusion protein with the induction of IPTG. SDS-PAGE and Western blotting were utilized to identify the heterogenous protein. Following the solubility analysis, the expressed soluble protein was further purified by Ni-NTA column and gel filtration chromatography. In addition, inclusion body was also detected by using Transmission Electron Micrograph of ultrathin section.Results: DNA sequencing shows that the recombinant plasmids have been successfully constructed. SDS-PAGE and Western blotting analysis revealed that the presence of signal peptide gave rise to the formation of inclusion body, however, the absence of signal peptide greatly enhanced the solubility of the heterogenous protein. In the latter case, further purification was achieved using Ni-NTA column and gel filtration chromatography. Transmission electron micrograph of ultrathin section gave the location of IB in the periplasmic space.Conculsion: The gene fragementΔgcgasa having no signal peptide could be expressed in E. coli system with high efficiency to produce the soluble protein. This study will be very fundamental and essential for further investigation on the structure and function of gcgasa.