Expression and Purification of Chimera Proteins

Chimera proteins, CaM(1TnC), CaM(2TnC), CaM(3TnC), and CaM(4TnC), were obtained as a generous gift from Dr. George [39 (link)]. CaM and the chimera proteins were expressed and purified as described previously [14 (link)]. In general, bacterial cells carrying the plasmid containing gene inserts of CaM(1TnC), CaM(2TnC), and CaM(4TnC) were induced for protein expression by heat shock at 42°C at an OD₆₀₀ = ~1.0. The culture was continuously incubated for an additional 4–6 hrs at 42°C. CaM(3TnC) was sub-cloned to a pLW-His₆ vector and was expressed as described previously for NOX5 [40 (link)]. All proteins except CaM(3TnC) were purified through a phenyl-sepharose column [41 (link)]. CaM(3TnC) was purified through a Ni-NTA column by elution with 40 mM imidazole. The purified proteins were stored at −80°C until further use. Protein purity was checked on SDS-PAGE electrophoresis and its purity was estimated to be > 95% based on density profiles measured using UN-SCAN-IT software (Silk Scientific, Inc., Utah). The concentrations of CaM and chimera proteins were determined as reported previously [20 (link)]. Dansylation of CaM and chimeras was performed in the presence of 2 mM Ca²⁺ as described previously [30 (link)].