一种合成的IgG结合结构域与水母发光蛋白的融合蛋白：在大肠杆菌细胞中的表达、纯化及其应用。

A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

作者信息

Inouye Satoshi, Sahara-Miura Yuiko

机构信息

Yokohama Research Center, JNC Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan.

出版信息

Protein Expr Purif. 2017 Sep;137:58-63. doi: 10.1016/j.pep.2017.06.017. Epub 2017 Jun 28.

DOI:10.1016/j.pep.2017.06.017

PMID:28668497

Abstract

Aequorin is a Ca-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.

摘要

水母发光蛋白是一种与钙结合的光蛋白，它是脱辅基水母发光蛋白（脱辅基 AQ）和 2-过氧腔肠素的复合物。在本研究中，由源自金黄色葡萄球菌蛋白 A 的合成 IgG 结合结构域（ZZ 结构域）和脱辅基 AQ 组成的融合蛋白（ZZ-脱辅基 AQ）被表达至大肠杆菌细胞的周质空间。ZZ-脱辅基 AQ 先通过镍螯合亲和层析，再通过 IgG 亲和层析进行高度纯化。通过与腔肠素孵育从纯化的 ZZ-脱辅基 AQ 制备 ZZ-AQ，并对其进行表征，包括其发光特性。ZZ-AQ 可用作检测 IgG 的报告分子，包被在 96 孔板上的 IgG 的可测量范围为 1 - 1000 ng/mL。