人胱硫醚β-合酶及其截短型片段的表达、纯化和活性测定

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CBS_1-413(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-CBS_1-413),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。

Expression, Purification and Activity Assay of the Full-length and Truncated Human Cystathionine β-Synthase

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 A₆₀₀ of 0.4~0.6, induced with IPTG at a final concentration of 0.1mmol/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/mg 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.coli Rosetta (pETDuet-1-CBS_1-413) strain which highly express truncated CBS(CBS_1-413) gene was constructed. Using a HisTrap Fast Flow affinity chromatography, the purity of recombinant CBS_1-413 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-lyase (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.