尖吻蝮蛇类凝血酶在大肠杆菌中的表达及其优化

Prokaryotic expression and optimization of a thrombin-like enzyme from deinagkistrodon acutus in E. coli

将尖吻蝮蛇类凝血酶基因克隆到载体pET32a(+)上,在大肠杆菌Rosetta2(DE3)中表达N端融合了硫氧还原蛋白的类凝血酶.通过SDS-PAGE检测融合蛋白条带,纤维蛋白原凝结实验检测酶学活性.确定表达菌株后,对其诱导条件进行优化,在30℃诱导3 h,诱导剂IPTG浓度为0.5 mmol·L-1条件下类凝血酶表达水平最高.

In this article, the gene of snake venom thrombin - like enzyme from deinagkistrodon acutus, acutobin, was cloned into expression vector pET32a（ ＋ ）. The enzyme fused with thioredoxin as its N - terminal part was expressed in E. coli Rosetta2 （ DE3 ） under the control of 30 ℃ with 0.5 mmol · L ^- 1 IPTG for 3 hours. SDS - PAGE was used to confirm the purity of target protein. A higher bioactivity of purified products was shown by fibrinogen - clotting analysis.