# US 11,078,530 B2 · Enzyme-pore constructs > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,530 B2 - Document ID: US11078530B2 - Status: Granted - Kind: B2 - Application number: 16/129,693 - Filed: 2018-09-12 - Published: 2019-01-03 - Granted: 2021-08-03 - Earliest claimed: 2008-07-07 - Assignees: Oxford Nanopore Technologies PLC - Applicants: Oxford Nanopore Technologies Ltd. - Inventors: Lakmal Jayasinghe; John Hagan Pryce Bayley; Stephen Cheley; Brian McKeown; James White; James Anthony Clarke - Web page: https://patentgrep.com/patent/US11078530B2 ## CPC - C12Q 1/6869: Methods for sequencing - C07K 14/31: from Staphylococcus (G) - C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)} - C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase} - C12N 9/127: {RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase} - C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase} - C12N 9/16: acting on ester bonds (3.1) - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - C12N 9/52: derived from bacteria {or Archaea} - C12N 9/90: Isomerases (5.) - C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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. ## Claims ### Claim 1 A method of attaching an enzyme to a transmembrane protein pore subunit comprising: (a) providing a preparation of a first fusion protein, wherein each of the first fusion proteins comprise (i) an enzyme and (ii) a first polypeptide linker, wherein one end of the first polypeptide linker is attached to the carboxyl or amino terminus of the enzyme, and wherein the first polypeptide linker comprises an internal reactive lysine residue; (b) providing a preparation of a second fusion protein, wherein each of the second fusion proteins comprise (i) a transmembrane protein pore subunit and (ii) a second polypeptide linker, wherein one end of the second polypeptide linker is attached to the carboxyl or amino terminus of the transmembrane protein pore subunit and wherein the second polypeptide linker comprises an internal reactive amino acid group having a carboxyl group that is specific for the reactive lysine in (a), and an affinity purification tag; (c) performing a coupling reaction between the two fusion proteins of (a) and (b) above under suitable conditions such that the internal reactive lysine residue of the first linker covalently attaches to the internal reactive amino acid group of the second linker, thereby generating a population of enzyme-attached transmembrane protein pore subunits; and (d) separating the preparation of enzyme-attached transmembrane protein pore subunits from any unreacted first fusion protein and second fusion protein. ### Claim 2 (depends on 1) A method according to claim 1 wherein the first polypeptide linker is attached to the carboxyl terminus of the enzyme. ### Claim 3 (depends on 1) A method according to claim 1 wherein the second polypeptide linker is attached to the carboxyl terminus of the transmembrane protein pore subunit. ### Claim 4 (depends on 1) A method according to claim 1 wherein the first polypeptide linker is attached to the carboxyl terminus of the enzyme and the second polypeptide linker is attached to the carboxyl terminus of the transmembrane protein pore subunit. ### Claim 5 (depends on 1) A method according to claim 1 wherein the enzyme is a nucleic acid handling enzyme. ### Claim 6 (depends on 1) A method according to claim 1 wherein the enzyme is a polymerase, exonuclease, helicase or topoisomerase. ### Claim 7 (depends on 1) A method according to claim 1 wherein the enzyme is capable of catalyzing the synthesis of a polymer. ### Claim 8 (depends on 1) A method according to claim 1 wherein the enzyme is a DNA polymerase or is an RNA polymerase. ### Claim 9 (depends on 1) A method according to claim 1 wherein the transmembrane protein pore subunit is from a β-barrel pore or an α-helix pore. ### Claim 10 (depends on 1) A method according to claim 1 wherein the transmembrane protein pore subunit is from α-hemolysin. ### Claim 11 (depends on 5) A method according to claim 5, wherein the enzyme retains the ability to handle nucleic acids after being attached to the transmembrane protein pore subunit. ### Claim 12 (depends on 7) A method according to claim 7, wherein the enzyme retains the ability to catalyze synthesis of a polymer after being attached to the transmembrane protein pore subunit. ### Claim 13 (depends on 1) A method according to claim 1, wherein the transmembrane protein pore subunit retains its ability to form a pore after being attached to the enzyme. ### Claim 14 (depends on 1) The method of claim 1 further comprising, after step (d): (e) combining the preparation of enzyme-attached transmembrane protein pore subunits with one or more wild-type transmembrane protein pore subunits to produce heteroligomeric transmembrane protein pores having a desired stoichiometry of enzyme-attached transmembrane protein pore subunits to wild-type subunits. ### Claim 15 (depends on 14) The method according to claim 14, wherein the heteroligomeric transmembrane protein pores comprise an α-hemolysin transmembrane protein pore subunit. ### Claim 16 (depends on 14) The method according to claim 14, wherein the enzyme attached to the heteroligomeric transmembrane protein pores is an exonuclease or a polymerase enzyme. ### Claim 17 (depends on 14) The method according to claim 14, wherein the one or more wild-type transmembrane protein pore subunits are 5, 6, 7, or 8 wild-type transmembrane protein pore subunits. ### Claim 18 (depends on 14) The method of claim 14, wherein the enzyme forms part of the cis side of the heteroligomeric transmembrane protein pores. ### Claim 19 A method of attaching an enzyme to a transmembrane protein pore subunit comprising: (a) providing a preparation of a first fusion protein, wherein each of the first fusion proteins comprise (i) transmembrane protein pore subunit and (ii) a first polypeptide linker, wherein one end of the first polypeptide linker is attached to the carboxyl or amino terminus of the transmembrane protein pore subunit, and wherein the first polypeptide linker comprises an internal reactive lysine residue; (b) providing a preparation of a second fusion protein, wherein each of the second fusion proteins comprise (i) an enzyme and (ii) a second polypeptide linker, wherein one end of the second polypeptide linker is attached to the carboxyl or amino terminus of the enzyme and wherein the second polypeptide linker comprises an internal reactive amino acid group having a carboxyl group that is specific for the internal reactive lysine in (a), and an affinity purification tag; (c) performing a coupling reaction between the two fusion proteins of (a) and (b) above under suitable conditions such that the internal reactive lysine residue of the first linker covalently attaches to the internal reactive amino acid group of the second linker, thereby generating a population of enzyme-attached transmembrane protein pore subunits; and (d) separating the preparation of enzyme-attached transmembrane protein pore subunits from any unreacted first fusion protein and second fusion protein. ### Claim 20 (depends on 19) The method of claim 19 further comprising, after step (d): (e) combining the preparation of enzyme-attached transmembrane protein pore subunits with one or more wild-type transmembrane protein pore subunits to produce heteroligomeric transmembrane protein pores having a desired stoichiometry of enzyme-attached transmembrane protein pore subunits to wild-type subunits.