Enzyme-pore constructs

Patent metadata

Field	Value
Publication number	US-11859247-B2
Application number	US-202117355668-A
Country	US
Kind code	B2
Filing date	Jun 23, 2021
Priority date	Jul 7, 2008
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

The invention relates to constructs comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme. The pore subunit is covalently attached to the enzyme such that both the subunit and enzyme retain their activity. The constructs can be used to generate transmembrane protein pores having a nucleic acid handling enzyme attached thereto. Such pores are particularly useful for sequencing nucleic acids. The enzyme handles the nucleic acid in such a way that the pore can detect its component nucleotides by stochastic sensing.

First claim

The invention claimed is:

1. A method of purifying a transmembrane pore comprising at least one transmembrane construct, the transmembrane construct comprising a transmembrane protein pore subunit and a nucleic acid handling enzyme, wherein the subunit is covalently attached to the enzyme, wherein the subunit retains its ability to form a pore and wherein the enzyme retains its ability to handle nucleic acids, said method comprising:
(a) providing the at least one construct and other subunits required to form the pore;
(b) oligomerising the at least one construct and other subunits on lipid vesicles;
(c) contacting the vesicles with a non-ionic surfactant; and
(d) recovering the oligomerised pore.

2. The method according to claim 1 , wherein the lipid vesicles in (b) are synthetic lipid vesicles.

3. The method according to claim 1 , wherein step (d) comprises recovering the oligomerised pore by liquid chromatography.

4. The method according to claim 1 , wherein after steps (a) to (d) the oligomerised pore is substantially pure and in a form that comprises less than 10% of other components.

5. The method according to claim 1 , wherein after steps (a) to (d) the oligomerised pore is substantially pure and in a form that comprises less than 5% of other components.

6. The method according to claim 1 , wherein after steps (a) to (d) the oligomerised pore is substantially pure and in a form that comprises less than 2% of other components.

7. The method according to claim 1 , wherein the nucleic acid handling enzyme is a polymerase, exonuclease, helicase or topoisomerase.

8. The method according to claim 1 , wherein the transmembrane protein pore subunit is from a β-barrel pore or an α-helix pore.

9. The method according to claim 1 , wherein the transmembrane protein pore subunit is from α-hemolysin.

10. The method according to claim 1 , wherein the transmembrane pore comprises 6, 7 or 8 transmembrane protein pore subunits.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Jayasinghe Lakmal
• Bayley John Hagan Pryce
• Cheley Stephen
• Mckeown Brian
• White James
• Clarke James

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12N9/127

  RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase · CPC title

• C12N9/1247

  DNA-directed RNA polymerase (2.7.7.6) · CPC title