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TPxcDNA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24460。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。
Objective：To construct a prokaryotic expression vector carrying thioredoxin peroxidase （TPx） gene of Bramchiostoma 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 SDSPAGE; the activity and structure of the protein were analyzed. Results： The recombinant plasmid pET 32a（ ＋ ）MTPx 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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2008年第7期781-786,共6页
Academic Journal of Second Military Medical University
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
thioredoxin peroxidase
Branchiostoma belcheri Tsingtaunese
prokaryotic expression
protein purification
enzymatic activity
structure
