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US 11,307,192 B2Granted
Method for producing a hetero-oligomeric pore comprising two different monomers in a specific stiochiometric ratio
AssigneeOxford Nanopore Technologies PLC
InventorsLakmal Jayasinghe, John Joseph Kilgour, Neil Roger Wood
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Abstract
The invention relates to a novel method of producing hetero-oligomeric pores. The invention also relates to hetero-oligomeric pores produced using the method and polynucleotide characterisation using the hetero-oligomeric pores.
Claims20
A method for producing a hetero-oligomeric pore comprising two different monomers in a specific stoichiometric ratio, comprising:
- (a) transfecting or transforming a cell with the first different monomer in a first vector, wherein expression of the first monomer is under the control of a first inducible promoter;
- (b) transfecting or transforming the cell with the second different monomer in a second vector, wherein expression of the second monomer is under the control of a second inducible promoter that is not the same as the first inducible promoter;
- and
- (c) controlling the concentration of a first inducing molecule relative to a second inducing molecule in the cell such that the first and second inducible promoters are simultaneously induced to produce the hetero-oligomeric pore comprising the first and second different monomers in the specific stoichiometric ratio,
- wherein the specific stoichiometric ratio is not 1:1.
2 more elements
A method for producing a hetero-oligomeric pore comprising two different monomers in a specific stoichiometric ratio, comprising:
- (a) transfecting or transforming a cell with the first different monomer in a first vector, wherein expression of the first monomer is under the control of a first inducible promoter;
- (b) transfecting or transforming the cell with the second different monomer in a second vector, wherein expression of the second monomer is under the control of a second inducible promoter that is not the same as the first inducible promoter, wherein the second different monomer is genetically fused to a peptide or polypeptide tag which reduces its ability to oligomerise with itself or reduces its expression compared with its expression in the absence of the tag;
- and
- (c) simultaneously inducing the first and second inducible promoters such that the cell produces the hetero-oligomeric pore comprising the first and second different monomers in the specific stoichiometric ratio,
- wherein the specific stoichiometric ratio is not 1:1.
2 more elements
Dependencies are inferred from the claim text.