中文摘要：

将人胱硫醚β-合酶（CBS）基因克隆至质粒pGEX-4T-1中,获得的重组质粒pGEX-4T-1-CBS转入大肠杆菌E.coli Rosetta（DE3）菌株,构建了高效表达CBS的重组菌E.coli Rosetta（pGEX4T-1-CBS）。重组菌在0.1mmol/L的IPTG于30℃诱导16h,可溶性CBS表达量达到28mg/L培养基。将重组菌破碎后上清液经GSTrap Fast Flow亲和层析一步纯化得到CBS融合蛋白,在凝血酶柱上切割缓冲液中加入3%甘油和0.1%CHAPS可以有效抑制酶切后CBS聚沉,酶活性回收率为54.8%,蛋白质产率为15.2mg/L培养基,纯度达到95%,单位酶活为143U/mg,终浓度为1mmol/L的S-腺苷甲硫氨酸（AdoMet）可使CBS单位酶活提高5.1倍,达到735U/mg。同时构建了表达CBS1-413（删除了CBS羧基端调控域138个氨基酸残基）的重组菌E.coli Rosetta（pETDuet-1-CBS1-413）,经过一步HisTrap Fast Flow亲和层析,酶活性回收率为74.3%,蛋白质产率为12.8mg/L培养基,纯度达到95%,单位酶活为965U/mg;还表达和纯化了胱硫醚β-裂解酶（CBL）,并在此基础上建立了一种新的CBL偶联的CBS酶活性测定方法。

英文摘要：

The human cystathionine β-synthase （CBS） gene was ligated into vector pGEX-4T-1. The recombinant pGEX-4T-1-CBS was transformed into E. coli Rosetta （DE3）, and the recombinant E. coli Rosetta （pGEX4T-1-CBS） strain which highly express CBS gene was constructed. After the recombinant E. coli was grown at 37℃ to an A60o of 0. 4 -0. 6, induced with IPTG at a final concentration of 0. lmmol/L for 16h at 30℃. The productivity of the soluble CBS reached 28mg/L. The supernatant of the disrupted cells by sonication was directly loaded on GSTrap Fast Flow, and the GST-CBS fusion protein was absorbed on the column. The GST- tagged CBS fusion protein bound to the column was treated with thrombin（ 10 unit of thrombin/mg protein） at 22℃ for 12-16h in the presence of 3% glycerol and 0. 1% CHAPS. An easy one-step protocol to purify recombinant human CBS and in 54. 8% overall yield had been used. From a 1L culture, some 15.2 mg of purified CBS protein at 95% purity as judged by SDS-PAGE could be abtained, and the purified enzyme had a specific activity of 143 unit/rag protein. S-adenosylmethionine（AdoMet） activates the CBS enzyme by as much as 5.1-fold in the presence of 1 mmol/L AdoMet with a specific activity of 735 unit/mg protein. Additionally, the recombinant E. eoli Rosetta （pETDuet-l-CBS1.413 ） strain which highly express truncated CBS （CBS1.413 ） gene was constructed. Using a HisTrap Fast Flow affinity chromatography, the purity of recombinant CBS1-413 lacking the C- terminal regulatory domain reached 95% by one-step purification with the specific activity of 965 unit/mg. The productivity of the soluble CBS reached 12. 8mg/L and in 74.3% overall yield. In addition, The expression and purification of recombinant cystathionine β-1yase （CBL） in E. coli were described. The present study established a novel method, which relies on CBL as coupling enzyme, for detemination of CBS activity based on the color reaction between pyruvate and 2,4-dinitrophenylhydrazine.