| 摘要: |
| 目的：将编码青岛文昌鱼硫氧还蛋白过氧化物酶(TPx)的基因克隆于原核表达载体,并通过表达获得重组的TPx蛋白。方法：将已获得的TPx cDNA片段克隆于原核表达载体pET-32a(+)M,转化大肠杆菌BL21(DE3),获得重组表达质粒pET-32a(+)M-TPx。37℃下经IPTG诱导,该融合蛋白在大肠杆菌BL21(DE3)中表达。获得的重组TPx蛋白再经过纯化,用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析重组蛋白。并对重组蛋白进行活性鉴定和结构分析。 结果：构建的重组质粒pET-32a(+)M-TPx在大肠杆菌中高效表达,经纯化后的重组TPx蛋白纯度可达90%以上,其单体相对分子质量为24 460。重组TPx蛋白是以同源二聚体和单体混合的形式存在,在二硫苏糖醇(DTT)存在时具有还原H2O2活性和对超螺旋DNA或对硫醇基敏感蛋白的保护作用。 结论：本实验在大肠杆菌表达系统中高效表达了重组TPx蛋白,并且证实其活性与其高级结构紧密相关。 |
| 关键词: 硫氧还蛋白过氧化物酶 青岛文昌鱼 原核表达 蛋白质纯化 酶活性 结构 |
| DOI：10.3724/SP.J.1008.2008.00781 |
| 投稿时间：2007-11-30修订日期：2008-03-03 |
| 基金项目: |
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| Expression,purification and activity characterization of a thioredoxin peroxidase from Branchiostoma belcheri Tsingtaunese |
| 廖剑 |
| () |
| Abstract: |
| Objective：To construct a prokaryotic expression vector carrying thioredoxin peroxidase (TPx) gene of Branchiostoma belcheri Tsingtaunese and express it in E.coli.Methods: The cDNA fragments encoding TPx were obtained from Branchiostoma belcheri Tsingtaunese and were cloned into the expression vector pET-32a (+)M; the product was used to transform BL21(DE3) cells and expression of TPx protein was induced by IPTG.The recombinant TPx was expressed as a histidine fusion protein in E.coli and was purified with Ni chromatography and SP cation exchange chromatography.The expression and purification of TPx were analyzed by SDS-PAGE; the activity and structure of the protein were analyzed.Results: The recombinant plasmid pET-32a(+)M-TPx was highly expressed in E.coli.The purity of the protein was over 90% after purification; the molecular weight of the protein monomer was 24 460.The recombinant TPx protein existed as a mixture of both dimer and monomer.The recombinant TPx had a significant thiol-dependent peroxidase activity in the presence of dithiothreitol (DTT),and it could protect plasmid DNA and thiol-protein from damages caused by metal-catalyzed oxidation (MCO).Conclusion: The recombinant TPx protein has been successfully expressed in E.coli; its activity is closely related to the advanced structures. |
| Key words: thioredoxin peroxidase Branchiostoma belcheri Tsingtaunese prokaryotic expression protein purification |
