Expression and Purification of CytK Nanopores.

Nanopores are emerging as powerful tools for peptide and protein sensing and sequencing. Cytotoxin K (CytK), a highly stable β-barrel pore, shows strong potential for both applications. The incorporation of an acidic-aromatic recognition site significantly enhances its ability to resolve different peptides. Other CytK variants with enhanced electroosmotic flow enable the measurement of unfolded native proteins. Together, these properties position CytK as an effective tunable nanopore for label-free detection of peptides and for analyzing native full-length proteins under denaturing conditions. This chapter provides a step-by-step protocol for the expression and purification of CytK nanopores.