hGSTP1-1及其半胱氨酸突变体的原核表达载体构建、诱导表达、纯化和部分性质研究和两种EGFP-hsTRAIL融合蛋白表达载体的构建、融合蛋白纯化、生物学活性分析

【作者】 沈佳胤；

【导师】 殷志敏；

【作者基本信息】 南京师范大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 人胎盘型谷胱甘肽S-转移酶（GST P1-1、GST-π、GST-pi）是人体内GSTs的一种重要的活性亚型，因其首先在人体胎盘（placenta）中被克隆而得名。GST P1-1在多种肿瘤组织细胞和转化的细胞株中丰度极高，且与肿瘤细胞的耐药性密切相关。对GST P1-1的早期研究集中于分子空间结构及其多态性和催化活性与上述人类疾病的关系，是目前所有人类GSTs中研究最为广泛和深入的一种亚型。本实验构建了E. coli重组表达载体pET-28a-His₆-hGST P1-1、pET-28a-His₆-hGST P1-1^C47A／C101A、pET-28a-His₆-hGST P1-1^{C14A／C47A／C101A}、pET-28a-His₆-hGST P1-1^{C14A／C47A／C101A／C169A}、pET-28a-His₆-hGST P1-1^Y7F和pET-28a-His₆-rEK-hGST P1-1。并在E. coli BL21（DE3）中实现了高效诱导表达，靶蛋白约占菌体总蛋白的30-50％。并初步建立了His₆-hGST P1-1蛋白纯化工艺，利用该工艺路线我们可以从1 L细菌诱导物中最终获得50-60 mg纯度大于93％的His₆-hGST P1-1蛋白，转移酶活性及对底物GSH和CDNB亲和常数K_m与已报道的重组hGST P1-1和从人胎盘纯化的hGST P1-1相似，为进一步研究His₆-hGST P1-1的性质奠定了坚实的基础。我们的实验结果表明，hGST P1-1半胱氨酸的突变并未使酶的催化活性完全丧失，但在一定程度上抑制了hGST P1-1对底物GSH和CDNB亲和力，因此半胱氨酸并不是hGST P1-1发挥转移酶活性所必需的，但的确影响了酶的催化效率。另外，热稳定性研究推测Cys169在维持酶的热稳定性方面发挥重要作用。我们的研究揭示，hGST P1-1在氧化应激时单体间通过Cys47和Cys101间形成二硫键，之后进一步使Cys14和Cys1169间形成单体间二硫键。肿瘤坏死因子相关的凋亡诱导配体（TNF-related apoptosis-inducing ligand, TRAIL／Ap02L）属于肿瘤坏死因子超基因家族成员。TRAIL及其受体在诱导肿瘤细胞凋亡的同时能保护正常细胞而免于细胞毒副反应，是一种具有开发潜力的新的抗肿瘤药物。本实验室从MegaMan^TM人转录组文库(MegaMan^TM Human Transcriptome Library)中扩增得到了表达人肿瘤坏死因子相关的凋亡诱导配体（hTRAIL）胞外可溶性功能区（114位氨基酸残基至281位氨基酸残基，命名为sTR114和95位氨基酸残基至281位氨基酸残基，命名为sTR95）的cDNA序列，通过重叠延伸PCR将TR95s和TR114s的cDNA序列分别与增强型绿色荧光蛋白（EGFP）基因融合，并将此融合基因克隆至pET-28a表达载体中。融合蛋白（EGFP-sTR95和EGFP-sTR114）诱导表达后通过IDA-Ni2⁺亲和层析柱纯化得到的EGFP-sTR95和EGFP-sTR114这两种融合蛋白在体外诱导多种肿瘤细胞系凋亡的活性。由于并融合蛋白保留了EGFP的荧光活性，可用于检测细胞表面表达TRAIL受体的各种细胞系。更多还原

【Abstract】 The bacterial expression and purification of human glutathione S-transferase P1-1（hGST P1-1）, as a hexahistidine-tagged polypeptide, was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two （C47A/C101A）, three （C14A/C47A/C101A） or all four （C14A/C47A/ C101A/C169A） cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis （SDS-PAGE） under nonreducing conditions, hexahistidine-tagged hGST P1-1 （His₆-hGST P1-1） treated with 1 mM H₂O₂ showed at least three extra bands, in addition to the native His₆-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed. The extracellular portion （amino acids 95-281 or 114-281） of the human TNF-related apoptosis-inducing ligand （sTRAIL） was genetically linked to the C-terminus of the fluoresce-enhanced green fluorescent protein variant （EGFP） to generate two versions of EGFP-sTRAIL fusion protein, designated EGFP-sTR95 and EGFP-sTR114 respectively. The two versions of EGFP-sTRAIL fusion protein both induce extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. In addition, the two versions of fusion protein retain similar fluorescence spectra to those of EGFP, and have shown the specific binding to TRAIL receptor positive-cells, thus the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion protein represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.更多还原