@inbook{7c3d988eb4544c7e98dce8e7d8fca43d,
title = "Direct cloning method for expression of recombinant proteins with an inositol hexakisphosphate inducible self-cleaving tag",
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.",
keywords = "CPD, Cloning, Gibson assembly, IMAC, IP6, Inositol hexakisphosphate, InsP, Protein expression, Protein purification, SDS-PAGE, Self-cleavage fusion tag",
author = "Keehwan Kwon and Marco Biancucci and Woida, {Patrick J.} and Satchell, {Karla J.F.}",
note = "Funding Information: This work funded in whole or in part with Federal funds from the NIAID, NIH, DHHS, under Contracts Nos. HHSN272201200026C and HHSN272201700060C. Additional funding from the Northwestern Medicine Catalyst Fund (to K.S.), NIAID Institutional NRSA Training Grant T32AI007476 (to P. W.) and PanCan/FNCLR KRas Fellowship (to M.B). Conflict of interest: K.J.F.S. holds patents on the use of CPD for autoprocessing of fusion proteins. Publisher Copyright: {\textcopyright} Springer Science+Business Media, LLC, part of Springer Nature 2020.",
year = "2020",
doi = "10.1007/978-1-0716-0167-9_15",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "163--179",
booktitle = "Methods in Molecular Biology",
}