Nanopore sensor for enzyme-mediated protein translocation

Abstract

Described herein is a device and method for translocating a protein through a nanopore and monitoring electronic changes caused by different amino acids in the protein. The device comprises a nanopore in a membrane, an amplifier for providing a voltage between the cis side and trans side of the membrane, and an NTP driven unfoldase which processed the protein to be trans-located. The exemplified unfoldase is the ClpX unfoldase from E. coli.

1 .- 30 . (canceled) 
     
     
         31 . A method of determining one or more characteristics of a protein, comprising the steps of:
 (a) providing a device for translocating a protein through a nanopore, comprising:
 (i) a nanopore in a membrane separating a fluidic chamber into a first side and a second side; and 
 (ii) a circuit for providing a voltage between the first side and the second side and for monitoring ionic current flowing through the nanopore; 
   (b) adding to the first side of the fluidic chamber a protein translocase;   (c) monitoring ionic current changes during translocation of a protein by the protein translocase through the nanopore in a direction toward the first side of the fluidic chamber; and   (d) determining one or more characteristics of the protein based on the ionic current changes.   
     
     
         32 . The method of  claim 31 , wherein the nanopore is a pore protein. 
     
     
         33 . The method of  claim 32 , wherein the nanopore is α-hemolysin. 
     
     
         34 . The method of  claim 31 , wherein the protein translocase is attached to the nanopore on the first side of the fluidic chamber. 
     
     
         35 . The method of  claim 31 , wherein the protein translocase is a ring-shaped NTP driven unfoldase. 
     
     
         36 . The method of  claim 35 , wherein the ring-shaped NTP driven unfoldase is an ATPases Associated with diverse cellular Activities (AAA+) enzyme. 
     
     
         37 . The method of  claim 36 , wherein the AAA+ enzyme is ClpX. 
     
     
         38 . The method of  claim 31 , wherein the circuit comprises a patch clamp amplifier applying a constant voltage between the first side of the fluidic chamber and the second side of the fluidic chamber. 
     
     
         39 . The method of  claim 31 , wherein the protein is in a non-denatured state. 
     
     
         40 . The method of  claim 31 , wherein the nanopore is  Mycobacteria smegmatis  porin A (MspA). 
     
     
         41 . The method of  claim 31 , wherein the one or more characteristics is the identity of the protein. 
     
     
         42 . The method of  claim 31 , wherein the one or more characteristics is a sequence of the protein. 
     
     
         43 . The method of  claim 31 , wherein the protein comprises an exogenous sequence. 
     
     
         44 . The method of  claim 43 , wherein the exogenous sequence comprises a targeting domain for the protein translocase. 
     
     
         45 . The method of  claim 31 , wherein the nanopore is a solid-state pore. 
     
     
         46 . A device for determining one or more characteristics of a protein, the device comprising:
 a nanopore in a membrane separating a fluidic chamber into a first side and a second side; and   a circuit for providing a voltage between the first side and the second side and for monitoring ionic current flowing through the nanopore; and   instructions that cause the device to:
 monitor ionic current changes during translocation of a protein by a protein translocase on the first side of the fluidic chamber through the nanopore in a direction toward the first side of the fluidic chamber; and 
 determine one or more characteristics of the protein based on the ionic current changes. 
   
     
     
         47 . The device of  claim 46 , wherein the one or more characteristics is an identity of the protein. 
     
     
         48 . The device of  claim 46 , wherein the one or more characteristics is a sequence of the protein. 
     
     
         49 . The device of  claim 46 , wherein the nanopore is a pore protein. 
     
     
         50 . The device of  claim 46 , wherein the nanopore is a solid-state pore.