荞麦胰蛋白酶抑制剂的原核表达及纯化

根据文献报道的荞麦胰蛋白酶抑制剂的氨基酸序列及本研究室先前已获得的部分基因序列设计引物,经过RT-PCR扩增,获得荞麦胰蛋白酶抑制剂编码区基因全序列.将该基因克隆到原核表达载体pQE-31中,并转化至大肠杆菌M15,经IPTG诱导表达获得可溶性目的蛋白,其表达量约占菌体总蛋白的25%.该目的蛋白经Ni2 -NTA柱亲和纯化,SDS-PAGE分析显示,在大约9kD处出现明显的目的条带,与预计蛋白分子量大小一致.Western blot鉴定证实,目的蛋白N端带有6个组氨酸标签.活性测定表明,目的蛋白具有专一性的胰蛋白酶抑制剂活性,抑制活性约为77U/mg纯化蛋白.本实验为进一步研究荞麦胰蛋白酶抑制剂结构与功能的关系奠定了基础.

Prokaryotic Expression and Purification of Buckwheat Trypsin Inhibitor

Based on the reported amino acid sequence of buckwheat trypsin inhibitor(BTI) and the partial gene sequence obtained by our lab,special primers were designed,buckwheat trypsin inhibitor gene was amplified through RT-PCR.The BTI gene was cloned into the expression vector pQE-31,and expressed in E.coli M15(pREP4) host cells.Recombinant BTI induced by IPTG was analyzed by Tris-Tricine-SDS-PAGE.The result showed that most of recombinant protein found was soluble,accounted for 25% of the total bacterial lysate.After purified through HiTrap Chelating HP column and analyzed by SDS-PAGE,the approximate molecular weight of a fusion protein was 9 kD and the purity of the target protein reached above 95%.The six histidine residues in the fusion protein were confirmed by Western blotting.The recombinant protein was similar to the native trypsin inhibitors in buckwheat seeds.The inhibitory activity of recombinant protein was about 77 U/mg.This work will be useful for further studying on the molecular mechanism and the potential applications of buckwheat trypsin inhibitor.