人P选择素lectin基因片段的克隆及表达

Cloning and Expression of Lectin of Human P-selectin

应用PCR技术扩增P选择素lectin基因 ,克隆入pET42b (+)载体 ,测序验证后在大肠杆菌BL2 1中表达了lectinC端融合 6×His蛋白 ,表达产物经Ni2 + NTAsuperflow亲和柱纯化 ,纯度可达 90 %以上 ,得率为 0 9mg/ 10 0ml,其表达量达到总菌体蛋白的 15 %。SDS PAGE和Western印迹试验显示 ,表达产物相对分子质量为 130 0 0。

PCR was applied to amplify lectin gene of P-selectin and cloned into pET42b(+). The sequenced vector was expressed in E.coli with high level fusion protein tailed with six additional histidine residues at its C-terminus,and the histidine-tagged lectin was purified with Ni 2+ -NTAsuperflow affinity chromatography, It's purifity reached 90%, 100 ml induction culture had 0.9 mg lectin-(His) 6 fusion proteins, reaching 15% of the total bacteria protein. SDS-PAGE and Western blot showed that the molecular weight of expressed lectin was 13 000.