Rapid, High Efficience Purification of Myofilament Proteins using Tobacco Etch Virus Protease

Abstract

As more and more cardiomyopathy causing mutations are identified, functional studies investigating contractility and myofilament mechanics with mutated proteins become more crucial. Several methods are available, but most rely on exchanging recombinant protein into permeabilized heart cells or myofilament preparation. However, success requires large amounts of highly purified myofilament proteins, a process which is usually different for each individual protein. Here, we describe one single method that can be used for purifying myofilament protein, including troponin T, I and C, as well as myosin light chain 2, which has also been adapted to other recombinant proteins. We fuse a HIS-tag and a modified tobacco etch virus (TEV) protease cleavage site, ENLYFQ/M, to the N- terminal of our protein construct. The flexibility of P1’ residue of the TEV protease cleavage site allows substitution of the native glycine to a methionine, enabling production of unmodified forms of protein after purification. We then express recombinant protein constructs in BL21 (DE3) E.coli competent cells which are lysed in buffer containing 6 M Urea. Clarified lysate is then applied to a HisTrap nickel affinity column. Following a low concentration of imidazole wash, the protein is then eluted with high imidazole, and the elution buffer is replaced by a high salt protease digestion buffer using a desalting column. Protease digestion is performed at 4°C overnight, and any undigested protein and the protease itself are removed by a second run on a HisTrap, allowing the purified recombinant protein to be collected. A final desalting step allows us to store the protein in a variety of solutions according to experimental needs. Overall, this method significantly increases yield and accelerates purification process from almost a week to only two days.