Enzyme-pore constructs
AssigneeOxford Nanopore Technologies PLC
InventorsLakmal Jayasinghe, John Hagan Pryce Bayley, Stephen Cheley, Brian McKeown, James White, James Clarke
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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.
Claims13
-42. (canceled)
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 the 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.
A method of purifying a heteroheptameric pore comprising seven transmembrane protein pore subunits wherein each subunit retains its ability to form a pore, said method comprising:
- (a) providing the seven subunits required to form the pore;
- (b) oligomerising the subunits on lipid vesicles;
- (c) contacting the vesicles with a non-ionic surfactant;
- and
- (d) recovering the oligomerised pore.
Dependencies are inferred from the claim text.