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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 | |
| 引用本文： | 王虹军,刘瑞卿,金瑞良,曹健,徐胜凤,王洪海.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[J].复旦学报(自然科学版),2008,47(3). |
| 作者姓名： | 王虹军 刘瑞卿 金瑞良 曹健 徐胜凤 王洪海 |
| 作者单位： | 1. 复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海,200433;河南工业大学生物工程学院,郑州,450052 2. 复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海,200433 3. 河南工业大学生物工程学院,郑州,450052 |
| 基金项目： | 国家自然科学基金，国家重点基础研究发展计划(973计划) |
| 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.coli BL21(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. |
| 关 键 词： | 结核分枝杆菌 高丝氨酸激酶 L-高丝氨酸 |
Expression, Purification and Characterization of Homoserine Kinase from Mycobacterium tuberculosis H37Rv |
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| WANG Hong-jun,LIU Rui-qing,JIN Rui-liang,CAO Jian,XU Sheng-feng,WANG Hong-hai.Expression, Purification and Characterization of Homoserine Kinase from Mycobacterium tuberculosis H37Rv[J].Journal of Fudan University(Natural Science),2008,47(3). | |
| Authors: | WANG Hong-jun LIU Rui-qing JIN Rui-liang CAO Jian XU Sheng-feng WANG Hong-hai |
| Abstract: | 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-phosphoL-homoserine.The specific activity of MtHSK was 2.946 U/mg.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. |
| Keywords: | ATP NADH |
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