Nivala, J. et al. Nat. Biotechnol. 31, 247–250 (2013).

Nanopore-based DNA sequencing is well on its way to becoming a practical technique. Such an approach could also be adapted to sequence proteins. Before a bench-top protein sequencer can be made, however, several methodological challenges must be overcome. In new work, Nivala et al. address two of these challenges: how to unfold the protein to allow it to go through the pore, and how to ensure that it goes through the pore processively and unidirectionally. The researchers created a system using the α-hemolysin pore with the unfoldase ClpX positioned on the trans side of the pore. They designed a polyanionic tag (for the protein of interest) that is captured and threaded through the pore when a voltage is applied; ClpX then recognizes a targeting motif on the C terminus of the tag and, fueled by ATP hydrolysis, unfolds and pulls the protein of interest through the pore.