囊素三肽的基因克隆和表达

Cloning and expression of the bursin gene in E.coli

以合成的BS1、BS2为引物扩增囊素三肽(BS)8串联片段(BS8),克隆至pBAD/Thio-TOPO载体,用pBAD/TOPO○RThio大肠杆菌表达系统进行表达,含BS8融合蛋白的分子量大小约为19kD,BS8融合蛋白主要以可溶形式存在。用蛋白质纯化树脂50%Ni-NTA纯化融合蛋白,高纯度的融合蛋白经透析除咪唑、浓缩后,再经胰蛋白酶水解获得大量BS单体。高压液相色谱分析表明,经过DEAE阴离子交换层析,获得纯化的BS单体。

The tandem 8 repeats BS8 of BS gene was amplified with PCR and inserted into the prokaryotic expression vehicle pBAD/TOPO to obtain the recombinant plasmid pBAD-BSS. The recombinant plasmid was transformed into E. coli TOP10 and expressed. SDS-PAGE analysis and densitometry test showed an expressed protein with a molecular weight of approximately 19kD after inducted with L-arabinose for 4 hours at 37℃. Following induction and massive expression of the fusion protein, the soluble fraction was purified with 50% Ni-NTA resin affinity chromatography, which was proven by SDS-PAGE to be a method that can achieve high purity of fusion protein. After cleavage of the fusion protein by trypsin at 37℃ ,BS was obtained by ion exchange methods and proved by HPLC analysis.