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| Expression, Purification and Activity Assay of the Full-length and Truncated Human Cystathionine β-Synthase |
| NIU, Wei-ning, YANG Meng-lin, CAO Shan-shan, XU Le, QIN Chuan-guang |
| Faculty of Life Science, Northwestern Polytechnical University, Xi'an 710072, China |
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Abstract 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-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-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.
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Received: 20 May 2011
Published: 25 December 2011
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