Abstract
The epithelium forms tight junctions by sealing the paracellular space, and tight junctions prevent the free movement of solutes. Claudin is an important structural and functional component of tight junctions and contributes to the formation of paracellular pathways for different populations of size- and charge-selective solutes. Therefore, modulation of tight junctions is important to develop drug delivery strategies. Clostridium perfringens enterotoxin (CPE) causes food poisoning in humans and is a 35-kDa polypeptide, consisting of 319 amino acids and two functional regions. The C-terminal region of CPE (C-CPE) is not cytotoxic and binds to its receptor claudin, which in turn modulates the epithelial tight junction barrier. Thus, claudin binders, such as C-CPE, are useful tools for drug delivery targeting tight junctions. Here, we provide a protocol for the expression and purification of recombinant C-CPE proteins as claudin binders, an analysis method for C-CPE binding affinity, and a procedure for assessing the effect of modulating tight junction integrity.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kondoh M, Takahashi A, Yagi K (2012) Spiral progression in the development of absorption enhancers based on the biology of tight junctions. Adv Drug Deliv Rev 64:515–522
Hashimoto Y, Yagi K, Kondoh M (2017) Roles of the first-generation claudin binder, Clostridium perfringens enterotoxin, in the diagnosis and claudin-targeted treatment of epithelium-derived cancers. Pflugers Arch 469:45–53
Van Itallie CM, Anderson JM (2006) Claudins and epithelial paracellular transport. Annu Rev Physiol 68:403–429
Czeczulin JR, Hanna PC, McClane BA (1993) Cloning, nucleotide sequencing, and expression of the Clostridium perfringens enterotoxin gene in Escherichia coli. Infect Immun 61:3429–3439
Sonoda N, Furuse M, Sasaki H et al (1999) Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: evidence for direct involvement of claudins in tight junction barrier. J Cell Biol 147:195–204
Katahira J, Inoue N, Horiguchi Y et al (1997) Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol 136:1239–1247
Van Itallie CM, Betts L, Smedley JG et al (2008) Structure of the claudin-binding domain of Clostridium perfringens enterotoxin. J Biol Chem 283:268–274
Uchida H, Kondoh M, Hanada T et al (2010) A claudin-4 modulator enhances the mucosal absorption of a biologically active peptide. Biochem Pharmacol 79:1437–1444
Takahashi A, Saito Y, Kondoh M et al (2012) Creation and biochemical analysis of a broad-specific claudin binder. Biomaterials 33:3464–3474
Srinivasan B, Kolli AR, Esch MB et al (2015) TEER measurement techniques for in vitro barrier model systems. J Lab Autom 20:107–126
Takahashi A, Komiya E, Kakutani H et al (2008) Domain map** of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis. Biochem Pharmacol 75:1639–1648
Hashimoto Y, Shirakura K, Okada Y et al (2017) Claudin-5-binders enhance permeation of solutes across the blood-brain barrier in a mammalian model. J Pharmacol Exp Ther 363:275–283
Acknowledgements
We would like to thank Y. Fujieda for technical assistance with the experiments. This research was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19H04468, 18K19400, 18H03190, 16K13044, and 24390042); Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Numbers JP19am0101085, JP19am0101077, JP19am0101084, and JP19am0101090.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media New York
About this protocol
Cite this protocol
Tachibana, K., Kondoh, M. (2019). A Method to Prepare Claudin-Modulating Recombinant Proteins. In: Turksen, K. (eds) Epidermal Cells. Methods in Molecular Biology, vol 2109. Humana, New York, NY. https://doi.org/10.1007/7651_2019_258
Download citation
DOI: https://doi.org/10.1007/7651_2019_258
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0250-8
Online ISBN: 978-1-0716-0251-5
eBook Packages: Springer Protocols