期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 99, 期 -, 页码 64-69出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2014.04.001
关键词
Self-cleavage; Protein expression; Human rhinovirus 3C protease (HRV3C); Escherichia coli (E. coli)
类别
资金
- National High-Tech Research and Development Program of China [2011AA10A212]
- Special Fund for Agro-scientific Research in the Public Interest [201203056]
- State Key Laboratory of Veterinary Biotechnology [SKLVBP201316]
- National Natural Science Foundation of China [31302065]
- China Postdoctoral Science Foundation [2013M540172]
Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications. (C) 2014 Elsevier Inc. All rights reserved.
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