Enzyme-pore constructs

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.