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结核分枝杆菌H37Rv高丝氨酸激酶基因的克隆、表达及酶学性质分析 被引量:1

Expression,Purification and Characterization of Homoserine Kinase from Mycobacterium tuberculosis H37Rv
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摘要 采用PCR方法克隆到结核分枝杆菌H37Rv的高丝氨酸激酶基因thrB,将其连接到pET-28a(+)表达载体中,在大肠杆菌E.coliBL21(DE3)中经丙基硫代半乳糖苷(IPTG)诱导得到高效表达.用Ni.NTA His.Bind亲和层析柱对表达的活性重组蛋白进行了分离纯化,并对其酶学性质进行了研究.结果表明:重组结核分枝杆菌高丝氨酸激酶能以L-高丝氨酸和ATP为底物催化L-高丝氨酸生成O-磷酰-L-高丝氨酸,该酶的比活力为2.946 U/mg,对底物L-高丝氨酸和ATP的米氏常数分别为2.303 1 mmol/L和2.342 9 mmol/L. The thrB gene encoding homoserine kinase (HSK) was amplified by PCR from genomic DNA of Mycobacterium tuberculosis H37Rv. Its purified product was cloned into pET-28a ( + ) vector to construct recombinant expression plasmid pET-28a-HSK. MtHSK was highly expressed with induction of IPTG after recombinant plasmid was transformed into competent cells of E. coli BL 21 (DE3). Purified fusion protein was obtained with one-step Ni-NTA affinity chromatography. Under optimal conditions, the enzymatic properties of MtHSK were studied. Purified MtHSK can catalyze L-homoserine to O-phospbo-L-homoserine. The specific activity of MtHSK was 2. 946 U/rag. The kinetic constants were determined: Km for L-homoserine and ATP was found to be 2. 303 1 mmol/L and 2. 342 9 mmol/L respectively.
出处 《复旦学报(自然科学版)》 CAS CSCD 北大核心 2008年第3期295-300,共6页 Journal of Fudan University:Natural Science
基金 国家自然科学基金资助项目(30670109) 科技部"973"计划资助项目(2005CB523102)
关键词 结核分枝杆菌 高丝氨酸激酶 L-高丝氨酸 ATP NADH Mycobacterium tuberculosis homoserine kinase L-Homoserine ATP NADH
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