一种表达可通过固定化金属离子亲和层析快速纯化的蛋白质的原核载体的构建与应用。

Construction and application of a prokaryotic vector which expresses the protein that can be quickly purified by IMAC.

作者信息

Li F, Gong H, Li Y, Zhang Z, Hou Y

机构信息

National Laboratory of Molecular Virology and Genetic Engineering, CAPM, Beijing, China.

出版信息

Chin J Biotechnol. 1997;13(1):37-42.

PMID:9376505

Abstract

A vector was constructed by inserting a pair of complementary oligo nucleotides encoding 6 histidine residues into the polylinker's upstream of the prokaryotic high expression vector pBV220. The resultant vector is named pBV222. Proteins expressed by this vector will have a 6-histidine tail as an affinity handle fused to their N-terminus and can be quickly purified by one-step immobilized metal affinity chromatography (IMAC). This plasmid was verified by restriction mapping and DNA sequencing. When GM-CSF and IL-2 cDNA were closed into pBV222, expressed proteins in the inclusion body showed the predicted molecular weight and biological activity. The expressed bacteria were dissolved in 6 mol/L guanidine.HCl and the supernatant was loaded directly to IMAC. IL-2 and GM-CSF fusion proteins were eluted by the pH gradient, and over 90% purity was achieved.

摘要

通过将一对编码6个组氨酸残基的互补寡核苷酸插入原核高表达载体pBV220多克隆位点的上游，构建了一个载体。所得载体命名为pBV222。该载体表达的蛋白质将在其N端融合有一个6-组氨酸尾作为亲和标签，可通过一步固定金属亲和色谱（IMAC）快速纯化。通过限制性酶切图谱和DNA测序对该质粒进行了验证。当将GM-CSF和IL-2 cDNA克隆到pBV222中时，包涵体中表达的蛋白质显示出预测的分子量和生物学活性。将表达的细菌溶解于6mol/L盐酸胍中，上清液直接上样到IMAC柱。通过pH梯度洗脱IL-2和GM-CSF融合蛋白，纯度达到90%以上。