Alpha-hemolysin variants
Described herein are engineered alpha-hemolysin subunits having mutated oligomerization domains for assembling into heptameric nanopores in lipid bilayers.
1. A hetero-oligomeric α-hemolysin (αHL) heptamer, comprising at least one preceding and at least one following subunit, each subunit comprising at least one αHL monomer and/or at least one polypeptide comprising concatenated αHL monomers,
wherein the at least one αHL monomer and/or the at least one polypeptide comprising concatenated αHL monomers comprises a mutation corresponding to H35L of SEQ ID NO:3; and
wherein the heptamer comprises exactly 7 αHL monomers, each αHL monomer comprising a first oligomerization domain and a second oligomerization domain, the first oligomerization domain of each αHL monomer being linked to the second oligomerization domain of a preceding αHL monomer and the second oligomerization domain of each αHL monomer being linked to the first oligomerization domain of a following αHL monomer.
2. The hetero-oligomeric αHL heptamer of claim 1 , wherein at least one αHL monomer further comprises a time-to-thread (TTT) substitution at a position corresponding to one or both of position 12 and position 17 of SEQ ID NO: 3.
3. An isolated polypeptide comprising one or more alpha-hemolysin monomers, wherein at least one of the alpha-hemolysin monomers comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 and an H35L substitution.
4. The isolated polypeptide of claim 3 , wherein one or more of the alpha-hemolysin monomers further comprise a time-to-thread (TTT) substitution at a position corresponding to position 12 and/or position 17 of SEQ ID NO: 3.
5. The isolated polypeptide of claim 4 , wherein the TTT substitution is selected from the group consisting of T12K, T12R, N17K, and N17R.
6. The isolated polypeptide of claim 3 having one alpha-hemolysin monomer.
7. The isolated polypeptide of claim 3 having at least two alpha-hemolysin monomers, wherein each monomer of the polypeptide is separated from each adjacent monomer in the polypeptide by a flexible linker.
8. The isolated polypeptide of claim 7 having from two to seven alpha-hemolysin monomers.
9. The isolated polypeptide of claim 3 , wherein at least one of the one or more alpha-hemolysin monomers further comprises an attachment component.
10. The isolated polypeptide of claim 9 , wherein the attachment component comprises a SpyTag or SpyCatcher peptide.
11. The isolated polypeptide of claim 9 , wherein the attachment component joins the alpha-hemolysin monomer to a DNA polymerase, an RNA polymerase, or a reverse transcriptase.
12. A heptameric pore complex comprising seven alpha-hemolysin monomers, wherein at least one of the alpha-hemolysin monomers comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 and an H35L substitution.
13. The heptameric pore complex of claim 12 , further comprising a polymerase linked to one of the alpha-hemolysin monomers.
14. The heptameric pore complex of claim 13 , wherein the polymerase is covalently linked to the alpha-hemolysin monomer.
15. The heptameric pore complex of claim 12 , wherein one or more of the alpha-hemolysin monomers further comprises a time-to-thread (TTT) substitution at a position corresponding to one or both of position 12 and position 17 of SEQ ID NO: 3.
16. The heptameric pore complex of claim 15 , wherein the TTT substitution or substitutions is/are selected from the group consisting of T12K, T12R, N17K, and N17R.
17. The heptameric pore complex of claim 12 , wherein each alpha-hemolysin monomer of the heptameric pore complex is disposed within a separate polypeptide from the other alpha-hemolysin monomers of the heptameric pore complex.
18. The heptameric pore complex of claim 12 , wherein from two to seven of the alpha-hemolysin monomers of the heptameric pore complex are disposed within a single polypeptide.
19. The isolated polypeptide of claim 12 , wherein at least one of the one or more alpha-hemolysin monomers further comprises an attachment component.
20. The isolated polypeptide of claim 19 , wherein the attachment component comprises a SpyTag or SpyCatcher peptide.
21. The isolated polypeptide of claim 19 , wherein the attachment component joins the alpha-hemolysin monomer to a DNA polymerase, an RNA polymerase, or a reverse transcriptase.
22. A method of making a heptameric pore complex, the method comprising heating a plurality of polypeptides of claim 3 in the presence of a lipid at a temperature greater than 25° C. for a sufficient period of time for the polypeptides to self-aggregate into alpha-hemolysin heptamers.
23. The method of claim 22 , wherein the temperature at which the polypeptides are heated is 30° C. or higher.
24. The method of claim 23 , wherein the temperature at which the polypeptides are heated is from 30° C. to 50° C.
25. A chip for nucleic acid sequencing, said chip comprising a heptameric pore complex of claim 12 disposed in a membrane adjacent to or in proximity to an electrode.