血红素标记蛋白的亲和纯化。

Affinity purification of heme-tagged proteins.

作者信息

Asher Wesley B, Bren Kara L

机构信息

Division of Molecular Therapeutics Department of Psychiatry, Columbia University, NYSPI Kolb Annex, 3rd Floor 1051 Riverside Drive, Unit 25, New York, NY, 10032, USA,

出版信息

Methods Mol Biol. 2014;1177:17-33. doi: 10.1007/978-1-4939-1034-2_2.

DOI:10.1007/978-1-4939-1034-2_2

PMID:24943311

Abstract

Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay.

摘要

蛋白质亲和纯化技术被广泛用于分离纯的目标蛋白质，以进行生化和结构表征。在此，我们描述了一种基于亲和的大肠杆菌表达蛋白纯化方案，该方案利用共价修饰有血红素c的肽标签（称为血红素标签）与固定在琼脂糖树脂上的L-组氨酸之间的配位作用。这种方法提供了一种肉眼可见的亲和纯化标签，便于追踪蛋白质。此外，我们还描述了使用血红素染色程序在SDS-PAGE凝胶中特异性检测血红素标记蛋白质的方法，以及使用吡啶血色原测定法对蛋白质进行定量的方法。