A Method to Prepare Claudin-Modulating Recombinant Proteins.

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.