流感嗜血杆菌表面天冬氨酸酶和其突变体蛋白的表达纯化及酶活性测定

PURIFICATION AND EXPRESSION OF RECOMBINANT HAEMOPHILUS IN FLUENZAE SURFACE ASPARTATE AMMONIA LYASE AND CARBOXYL-TERMINAL LYSINE-TRUNCATED VARIANT AND DETECTION OF THEIR ENZYME ACTIVITY

目的表达纯化不可分型流感嗜血杆菌表面天冬氨酸酶(r ASP)和其敲除羧基末端赖氨酸的重组蛋白(r ASPΔK)。方法克隆了不可分型流感嗜血杆菌ATCC49247的ASP基因和ASPΔK基因,与载体连接后,转入大肠杆菌表达蛋白,并用Co2+亲和柱层析纯化蛋白;采用酶促反应测定纯化蛋白的酶活性。结果两种基因的克隆和表达质粒构建成功,目的蛋白表达量大且具有较高的酶活性。结论成功表达纯化了r ASP和r ASPΔK蛋白。

Objective To express and purify the recombinant haemophilus influenzae surface aspartate ammonia lyase（ r ASP） and its carboxyl-terminal lysine-truncated variant（ r ASPΔK）. Methods We cloned ASP and ASPΔK from Non-typeable Haemophilus influenzae ATCC49247,linked with p ASK-IBA37 vector,produced r ASP and r ASPΔK in E. coli and purified them by affinity chromatography with Co^2＋affinity resins. The enzymatic reaction was completed to determine the recombinant proteins activity. Results PCR ampifing ASP and ASPΔK and plasmid building on linking with p ASK- IBA37 vector were succusful; The purified r ASP and r ASPΔK were profuse,and found to have abundant aspartase activity. Conclusion We purified r ASP and r ASPΔK succusfully.