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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Satchell KJ (2015) Multifunctional-autoprocessing repeats-in-toxin (MARTX) toxins of Vibrios. Microbiol Spectr 3:3. https://doi.org/10.1128/microbiolspec.VE-0002-2014
Gavin HE, Satchell KJ (2015) MARTX toxins as effector delivery platforms. Pathog Dis 73:ftv092
Lupardus PJ, Shen A, Bogyo M et al (2008) Small molecule-induced allosteric activation of the Vibrio cholerae RTX cysteine protease domain. Science 322:265–268
Prochazkova K, Satchell KJ (2008) Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin. J Biol Chem 283:23656–23664
Prochazkova K, Shuvalova LA, Minasov G et al (2009) Structural and molecular mechanism for autoprocessing of MARTX Toxin of Vibrio cholerae at multiple sites. J Biol Chem 284:26557–26568
Egerer M, Satchell KJ (2010) Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins. PLoS Pathog 6:e1000942
Shen A, Lupardus PJ, Albrow VE et al (2009) Mechanistic and structural insights into the proteolytic activation of Vibrio cholerae MARTX toxin. Nat Chem Biol 5:469–478
Shen A, Lupardus PJ, Morell M et al (2009) Simplified, enhanced protein purification using an inducible, autoprocessing enzyme tag. PLoS One 4:e8119
Satchell KJ (2010) Cysteine protease autoprocessing of fusion proteins US Patent No. 8,257,946 B2. Date of issue: Sep. 4, 2012
Sheahan KL, Cordero CL, Satchell KJ (2007) Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain. EMBO J 26:2552–2561
Biancucci M, Dolores JS, Wong J et al (2017) New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag. BMC Biotechnol 17:1
Eschenfeldt WH, Makowska-Grzyska M, Stols L et al (2013) New LIC vectors for production of proteins from genes containing rare codons. J Struct Funct Genomics 14:135–144
Lee PY, Costumbrado J, Hsu CY et al (2012) Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp 62:3923. https://doi.org/10.3791/3923
Makovets S (2013) Basic DNA electrophoresis in molecular cloning: a comprehensive guide for beginners. Methods Mol Biol 1054:11–43
Gibbins JM (2004) Techniques for analysis of proteins by SDS-polyacrylamide gel electrophoresis and Western blotting. Methods Mol Biol 273:139–152
Chakavarti B, Chakavarti D (2008) Electrophoretic separation of proteins. J Vis Exp 16:758. https://doi.org/10.3791/758
Gibson DG, Benders GA, Andrews-Pfannkoch C et al (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220
Gibson DG, Benders GA, Axelrod KC et al (2008) One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci U S A 105:20404–20409
Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345
Kwon K, Pieper R, Shallom S et al (2007) A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins. Protein Expr Purif 55:368–378
Samuelson JC (2011) Recent developments in difficult protein expression: a guide to E. coli strains, promoters, and relevant host mutations. Methods Mol Biol 705:195–209
Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421
Lin W, Fullner KJ, Clayton R et al (1999) Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci U S A 96:1071–1076
Dolores J, Satchell KJ (2013) Analysis of Vibrio cholerae genome sequences reveals unique rtxA variants in environmental strains and an rtxA-null mutation in recent altered El Tor isolates. mBio 4:e00624
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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: Miller, G. (eds) Inositol Phosphates. Methods in Molecular Biology, vol 2091. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0167-9_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0167-9_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0166-2
Online ISBN: 978-1-0716-0167-9
eBook Packages: Springer Protocols