Control of subunit stoichiometry in single-chain msp nanopores

A method for preparing a purified population of single chain Mycobacterium smegmatis porin A (MspAs) comprising:

• (a) expressing in E. coli α polypeptide comprising a single chain MspA, wherein the polypeptide comprises, in the following order,
• (i) a first affinity tag, wherein the affinity tag is a polyhistidine tag;
• (ii) a first amino acid linker;
• (iii) a single chain MspA, wherein the single chain MspA comprises at least a first MspA monomer sequence and a second MspA monomer sequence;
• wherein the first and second monomer sequence are linked by a second amino acid linker;
• (iv) a third amino acid linker;
• and
• (v) a second affinity tag;
• (b) recovering inclusion bodies that express the single chain MspAs from the E. coli under denaturing conditions;
• (c) using Ni-affinity chromatography to obtain one or more fractions comprising single chain MspAs from the inclusion bodies under denaturing conditions;
• (d) optionally separating the single chain MspAs in the one or more fractions using size exclusion chromatography to obtain a desired fraction comprising MspAs;
• (e) purifying the MspAs from the one or more fractions of step (c) or the desired fraction of step (d) using second affinity tag purification under denaturing conditions;
• (f) refolding the purified MspAs of step (e) in a refolding buffer, wherein the refolding buffer comprises about 50 mM to about 200 mM L-Arginine, about 800 mM to about 1000 mM urea, about 0.5% to about 1.0% OPOE, wherein the buffer has a pH of about 7.5 to about 8.5;
• (g) concentrating the refolded MspAs using size exclusion chromatography to obtain a purified population of single chain MspAs.