带有六组氨酸标签的麦芽糖结合蛋白作为在大肠杆菌中生产可溶性重组蛋白的融合伴侣。

Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli.

作者信息

Austin Brian P, Nallamsetty Sreedevi, Waugh David S

机构信息

Macromolecular Crystallography Laboratory, Center for CancerResearch, National Cancer Institute at Frederick, Frederick, MD, USA.

出版信息

Methods Mol Biol. 2009;498:157-72. doi: 10.1007/978-1-59745-196-3_11.

DOI:10.1007/978-1-59745-196-3_11

PMID:18988025

Abstract

Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way to circumvent this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) has emerged as one of the most effective solubilizing agents. In this chapter, we describe how to construct combinatorially-tagged His(6)MBP fusion proteins by recombinational cloning and how to evaluate their yield and solubility. We also describe a procedure to determine how efficiently a His(6)MBP fusion protein is cleaved by tobacco etch virus (TEV) protease in E. coli and a method to assess the solubility of the target protein after it has been separated from His(6)MBP.

摘要

重组蛋白在大肠杆菌中的不溶性是其用于结构和功能研究生产的主要障碍。解决这个问题的一种方法是将易于聚集的蛋白与高度可溶的伴侣蛋白融合。大肠杆菌麦芽糖结合蛋白（MBP）已成为最有效的增溶剂之一。在本章中，我们描述了如何通过重组克隆构建组合标记的His(6)MBP融合蛋白，以及如何评估它们的产量和溶解性。我们还描述了一种确定His(6)MBP融合蛋白在大肠杆菌中被烟草蚀纹病毒（TEV）蛋白酶切割效率的方法，以及一种在目标蛋白与His(6)MBP分离后评估其溶解性的方法。