人胱硫醚β合成酶原核表达纯化及鉴定

Expression,Purification and Characterization of Recombinant Human CBS in Escherichia coli

目的:构建人胱硫醚β合成酶(human cystathionineβ-synthase,hCBS)基因原核表达载体,在E.coli BL21(DE3)中表达,并进行纯化和酶活性检测。方法:以胰腺细胞cDNA文库为模板,采用聚合酶链式反应(PCR)扩增hCBS基因蛋白编码区的全序列,克隆入原核表达载体pET32a(+),构建重组质粒pET32a(+)-hCBS。经限制性内切酶双酶切及DNA序列分析鉴定目的基因后与人CBS基因(基因bank号:BT007154.1)完全一致,转入E.coli BL21(DE3)中,由IPTG诱导表达融合蛋白。结果:经SDS-PAGE、Western blot分析,证明诱导表达的蛋白为重组人CBS(rhCBS)。再由Ni-NTA树脂亲和层析,并脱盐冷冻干燥后获得重组rhCBS(约19 mg/L培养物),并测得其比活力约为57 kU/g。结论:成功地表达纯化出具有功能活性的重组蛋白rhCBS,为进一步研究该酶的相互作用蛋白以及其在生物学和临床科学的作用奠定了基础。

Objective：To construct the human cystathionine β-synthase express vector,and to investigate the mechanism and pharmaceutical function.Methods：The gene encoding hCBS was amplified by RT-PCR from human pancreatic cell cDNA library,then was inserted into the expression vector pET32a（＋） to construct the pET32a（＋）-CBS.The recombinant human CBS（rhCBS） was expressed in Escherichia coli（E.coli）.By restriction enzyme digestion and DNA sequencing analysis,the right recombinant vector was transformed into E.coli BL21（DE3）,and rhCBS was expressed by IPTG induction and purified by Ni-NTA affinity chromatography,determined by SDS-PAGE and Western blotting analysis.Results：The gene inserted into pET32a（＋） was exactly as the same as hCBS gene.A total of 19 mg of high purity（over 98%） rhCBS was obtained from 1 L culture.Conclusions：The rhCBS with functional activity was successfully expressed,and laid the foundations for further study of its interacted protein and its roles in biology and clinical science research.