Direct cloning method for expression of recombinant proteins with an inositol hexakisphosphate inducible self-cleaving tag

Keehwan Kwon, Marco Biancucci, Patrick J. Woida, Karla J.F. Satchell*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovate to develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTXVc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages163-179
Number of pages17
DOIs
StatePublished - Jan 1 2020

Publication series

NameMethods in Molecular Biology
Volume2091
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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Keywords

  • Cloning
  • CPD
  • Gibson assembly
  • IMAC
  • Inositol hexakisphosphate
  • InsP
  • IP6
  • Protein expression
  • Protein purification
  • SDS-PAGE
  • Self-cleavage fusion tag

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Kwon, K., Biancucci, M., Woida, P. J., & Satchell, K. J. F. (2020). Direct cloning method for expression of recombinant proteins with an inositol hexakisphosphate inducible self-cleaving tag. In Methods in Molecular Biology (pp. 163-179). (Methods in Molecular Biology; Vol. 2091). Humana Press Inc.. https://doi.org/10.1007/978-1-0716-0167-9_15