A Single Protein Detection Using a Biological Nanopore Formed By Perforin
Authors: Watanabe Hirokazu , Kawano Ryuji 
Published: February 2020

This paper describes the single protein detection of granzyme B (≈5 nm, GZB) using human perforin (PFN, 5.3 nm) nanopores. Nanopore sensing is a strong tool for a single molecule analysis of the translocating molecules through the nanopore. This method has already implemented extensive biochemical measurement at single molecule level, such as DNA sequencing and analysis of folding/unfolding protein behaviors. Although alpha-hemolysin (αHL, 1.4 nm) has been mainly used as the nanopore, the analytes that size is larger than αHL pore, for example the most of proteins, cannot be measured using αHL. To address this issue, we have tried to reconstitute larger sized nanopores, such as PFN, in bilayer lipid membranes (BLMs), and it has been successfully detected an apoptosis-inducing serine protease, GZB. As the result, we clarified the volume of GZB and time of translocation. Thus, we consider that PFN is a candidate as a nanopore for detecting single protein molecules.

Nanopore sensing has been used for analysis of small molecules, for example, sequencing of a single-stranded DNA and estimating the molecular weight of PEG. αHL is used in most of previous studies as biological nanopore. In this sensing, reconstituting αHL in BLMs separating two chambers filled with buffered electrolyte and applying a voltage across the BLMs, we can detect passing ions through pore as channel current. Moreover, when the comparable sized molecule translocate the pore, they cause blocking of ion passing, and we can examine volume and translocating velocity from current reduction. Although nanopore sensing is a strong tool, it has a problem that only a few type of nanopores has been used and diameter is restricted only near 1 nm. Hence, we can’t detect the larger molecule than αHL because they are enable to translocate the nanopore. Therefore, in this study, we used PFN which forms pores on infected cells in human immune system, for detection of proteins.

BLMs were prepared by “droplet contact method” using a device fabricated with microfabrication technology. Two lipid monolayers contacted and formed BLMs on the micropore of the separator. PFN was added one side of chambers (Cis side) at 1 nM, and reconstituted in BLMs. We measured channel current by a patch-clamp amplifier and calculated pore diameter from channel current. As the result, PFN formed heterogeneous sized pores. We considered PFN formed pores by oligomerization, and estimated the number of assembling monomers (n). First, we regarded the area of pore formed by n-mers as that of regular n-sided polygon. Then, we derived the formula and estimated the number of monomers. As the result, we concluded PFN formed variable-sized pores, and the most probable value of pore diameter and the number of monomers was 5.3 nm and 8-mer. Finally, we show the detection of GZB, which is serine protease and induces apoptosis of target cells. GZB applied into Cis chamber translocated through PFN and created the spike-like current blocking, and the analysis of this blocking revealed that translocation speed of GZB and volume of GZB. In this way, we analyzed behaviors of larger protein by PFN pore. We believe extensive sized PFN pore enable to analyze of targeting-sized molecule at the single molecule level.