Purification of Lethocerus Troponin C Domains

Full-length Lethocerus F2TnC and the two isolated N and C lobes, spanning residues 1–88 and 88–158, respectively, were produced by overexpression in Escherichia coli. The purification procedure was the same as the ones described previously (25 (link)). In short, all constructs were cloned in a pET24d (M11) expression vector (Novagen) containing an N-terminal hexahistidine tag followed by a tobacco etch virus protease cleavage site. The overexpressed proteins were purified by affinity using nickel-nitrilotriacetic acid resin (Qiagen) in two steps separated by an overnight tag cleavage step using in-house produced tobacco etch virus protease. The proteins were then passed through a size exclusion Superdex75 column in 20 mm Tris-HCl buffer at pH 6.8 with 100 mm KCl to remove any high molecular weight contaminants. Proteins were then concentrated on Vivaspin to 0.8 mm samples. Calcium depletion was achieved by a Chelex100 resin (Sigma) and adding 250 mm EDTA to the solution. The holo samples were obtained by adding a 5-fold molar excess of CaCl₂.