Lipid-free anchoring of thermophilic bacteriophage G20C portal adapter into solid-state nanopores
AssigneesNortheastern University, The University of York
InventorsMeni Wanunu, Alfred Antson, Sandra Greive, Benjamin Cressiot
Life of this patent
Estimating the term…
Abstract
Hybrid nanopores, comprising a protein pore supported within a solid-state membrane, which combine the robust nature of solid-state membranes with the easily tunable and precise engineering of protein nanopores. In an embodiment, a lipid-free hybrid nanopore comprises a water soluble and stable, modified portal protein of the Thermus thermophilus bacteriophage G20c, electrokinetically inserted into a larger nanopore in a solid-state membrane. The hybrid pore is stable and easy to fabricate, and exhibits low peripheral leakage, allowing sensing and discrimination among different types of biomolecules.
Claims15
A protein variant, the protein variant comprising a modification of an amino acid sequence comprising SEQ ID NO:
- 1, the modification comprising one or more of:
- (i) a modification of an upper internal surface residue or a lower internal surface residue of the protein variant, the modification comprising an alteration of an electrostatic surface potential of an upper internal surface or a lower internal surface of SEQ ID NO:
- 1;
- (ii) a modification comprising an insertion of a cysteine residue into SEQ ID NO:
- 1 or a replacement of an amino acid residue of SEQ ID NO:
- 1 with a cysteine residue;
- (iii) a modification of a tunnel loop residue of the protein variant, the modification comprising an expansion of a narrowest constriction of a tunnel loop of SEQ ID NO:
- 1, a restriction of the narrowest constriction of the tunnel loop of SEQ ID NO:
- 1, or a removal of the narrowest constriction of the tunnel loop of SEQ ID NO:
- 1;
- (iv) a modification of a tunnel loop residue of the protein variant, the modification resulting in an alteration of an electrostatic charge property of a tunnel loop of SEQ ID NO:
- 1;
- (v) a modification which alters an external charge of the protein variant;
- (vi) a modification which promotes binding of the protein variant to a solid-state matrix;
- (vii) a modification which extends an N-terminus or a C-terminus of SEQ ID NO:
- 1 or an N-terminus or a C-terminus of a cleaved portion of SEQ ID NO:
- 1;
- and
- (viii) a deletion of an amino acid residue of at least one of a C-terminal region and an N-terminal region of SEQ ID NO:
- 1.
A protein variant, the protein variant comprising a modification of an amino acid sequence comprising SEQ ID NO:
- 1, the modification comprising one of:
- SEQ ID NO:
- 27;
- SEQ ID NO:
- 11;
- SEQ ID NO:
- 37;
- SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 21, SEQ ID NO:
- 23, SEQ ID NO:
- 25, SEQ ID NO:
- 19, SEQ ID NO:
- 9, SEQ ID NO:
- 13, SEQ ID NO:
- 29, SEQ ID NO:
- 31, SEQ ID NO:
- 35, SEQ ID NO:
- 33, SEQ ID NO:
- 3, SEQ ID NO:
- 5, and SEQ ID NO:
- 7.
A protein variant, the protein variant comprising a modification of an amino acid sequence comprising SEQ ID NO:
- 1, the modification comprising one of:
- SEQ ID NO:
- 27;
- SEQ ID NO:
- 37;
- SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 23, SEQ ID NO:
- 25, SEQ ID NO:
- 19, SEQ ID NO:
- 9, SEQ ID NO:
- 13, SEQ ID NO:
- 29, SEQ ID NO:
- 31, SEQ ID NO:
- 35, SEQ ID NO:
- 33, SEQ ID NO:
- 3, SEQ ID NO:
- 5, and SEQ ID NO:
- 7.
A protein variant encoded by a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- (i) a modification in a portion of the nucleic acid sequence that encodes an upper internal surface residue or a lower internal surface residue of the protein variant encoded by the nucleic acid sequence, the modification producing an alteration of an electrostatic surface potential of an upper internal surface or a lower internal surface of the protein variant encoded by the nucleic acid sequence;
- (ii) a modification comprising an insertion of a cysteine residue into the protein variant encoded by the nucleic acid sequence or a replacement of an amino acid residue of the protein variant encoded by the nucleic acid sequence with a cysteine residue;
- (iii) a modification to produce a modification of a tunnel loop residue of the protein variant encoded by the nucleic acid sequence, the modification in the tunnel loop residue comprising an expansion of a narrowest constriction of a tunnel loop of the protein variant encoded by the nucleic acid sequence, a restriction of the narrowest constriction of the tunnel loop of the protein variant encoded by the nucleic acid sequence, or a removal of the narrowest constriction of the tunnel loop of the protein variant encoded by the nucleic acid sequence;
- (iv) a modification to produce a modification of a tunnel loop residue of the protein variant encoded by the nucleic acid sequence, the modification resulting in an alteration of an electrostatic charge property of a tunnel loop of the protein variant encoded by the nucleic acid sequence;
- (v) a modification which alters an external charge of the protein variant encoded by the nucleic acid sequence;
- (vi) a modification which promotes binding of the protein variant encoded by the nucleic acid sequence to a solid-state matrix;
- (vii) a modification which extends an N-terminus or a C-terminus of the protein variant encoded by the nucleic acid sequence or an N-terminus or a C-terminus of a cleaved portion of the protein variant encoded by the nucleic acid sequence;
- and
- (viii) a deletion of an amino acid residue of at least one of a C-terminal region and an N-terminal region of the protein variant encoded by the nucleic acid sequence.
A protein variant encoded by a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 12;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 22, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
A protein variant encoded by a modification of a nucleic acid sequence of SEQ ID NO:
- 2, the modification comprising one or more of:
- SEQ ID NO:
- 28;
- SEQ ID NO:
- 38;
- SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 24, SEQ ID NO:
- 26, SEQ ID NO:
- 20, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 30, SEQ ID NO:
- 32, SEQ ID NO:
- 36, SEQ ID NO:
- 34, SEQ ID NO:
- 4, SEQ ID NO:
- 6, and SEQ ID NO:
- 8.
Dependencies are inferred from the claim text.