用于在大肠杆菌中过表达和纯化带标签的外膜蛋白、周质蛋白和分泌蛋白的载体套件。

A Vector Suite for the Overexpression and Purification of Tagged Outer Membrane, Periplasmic, and Secreted Proteins in E. coli.

作者信息

Casasanta Michael A, Slade Daniel J

机构信息

Department of Biochemistry, Virginia Polytechnic Institute, State University, Blacksburg, VA, USA.

出版信息

Methods Mol Biol. 2019;1960:123-138. doi: 10.1007/978-1-4939-9167-9_11.

DOI:10.1007/978-1-4939-9167-9_11

PMID:30798527

Abstract

Outer membrane and secreted proteins in Gram-negative bacteria constitute a high percentage of virulence factors that are critical in disease initiation and progression. Despite their importance, it is often difficult to study these proteins due to challenges with expression and purification. Here we present a suite of vectors for the inducible expression of N-terminally 6His-tagged outer membrane, periplasmic, and secreted proteins in E. coli and show this system to be capable of producing milligram quantities of pure protein for downstream functional and structural analysis. This system can not only be used to purify recombinant virulence factors for structural and functional studies but can also be used to create gain-of-function E. coli for use in phenotypic screens, and examples of each are provided herein.

摘要

革兰氏阴性菌的外膜蛋白和分泌蛋白构成了很大比例的毒力因子，这些因子在疾病的发生和发展中起关键作用。尽管它们很重要，但由于表达和纯化方面的挑战，研究这些蛋白质往往很困难。在此，我们展示了一套载体，用于在大肠杆菌中诱导表达N端带有6His标签的外膜蛋白、周质蛋白和分泌蛋白，并表明该系统能够产生毫克量的纯蛋白，用于下游的功能和结构分析。该系统不仅可用于纯化重组毒力因子以进行结构和功能研究，还可用于创建功能获得型大肠杆菌以用于表型筛选，本文提供了这两方面的示例。