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

Expression,Purification and Characterization of Homoserine Kinase from Mycobacterium tuberculosis H37Rv

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.