Construction, expression, and purification of the wild-type recombinant
human beta-cardiac myosin sS1 and the hypertrophic cardiac myopathy H251N
mutant49 (link) are described
in detail elsewhere49 (link)-51 (link). Briefly, a truncated version
of MYH7 (residues 1–808), corresponding to sS1, with a C-terminal
enhanced green fluorescent protein (eGFP), was co-expressed with myosin light
chain 3 (MYL3), encoding human ventricular essential light chain (ELC), and
containing an N-terminal FLAG tag (DYKDDDDK) and tobacco etch virus (TEV)
protease site in mouse myoblast C2C12 cells using the AdEasy Vector System
(Obiogene Inc.). The myosin heavy chain with its associated FLAG-tagged ELC was
first purified from clarified lysate with anti-FLAG resin (Sigma). After
cleaving off the FLAG tag with TEV protease, the human beta-cardiac sS1 was
further purified using anion exchange chromatography on a 1-mL HiTrap Q HP
column (GE Healthcare). Peak fractions were eluted with column buffer (10 mM
imidazole, pH 7.5, ~200 mM NaCl, 4 mM MgCl2, 1 mM DTT, 2 mM ATP, and 10%
sucrose) and concentrated by centrifugation in Amicon Ultra-0.5 10-kDa cutoff
spin filters (EMD Millipore) before being used in assays or stored at −80
°C.
human beta-cardiac myosin sS1 and the hypertrophic cardiac myopathy H251N
mutant49 (link) are described
in detail elsewhere49 (link)-51 (link). Briefly, a truncated version
of MYH7 (residues 1–808), corresponding to sS1, with a C-terminal
enhanced green fluorescent protein (eGFP), was co-expressed with myosin light
chain 3 (MYL3), encoding human ventricular essential light chain (ELC), and
containing an N-terminal FLAG tag (DYKDDDDK) and tobacco etch virus (TEV)
protease site in mouse myoblast C2C12 cells using the AdEasy Vector System
(Obiogene Inc.). The myosin heavy chain with its associated FLAG-tagged ELC was
first purified from clarified lysate with anti-FLAG resin (Sigma). After
cleaving off the FLAG tag with TEV protease, the human beta-cardiac sS1 was
further purified using anion exchange chromatography on a 1-mL HiTrap Q HP
column (GE Healthcare). Peak fractions were eluted with column buffer (10 mM
imidazole, pH 7.5, ~200 mM NaCl, 4 mM MgCl2, 1 mM DTT, 2 mM ATP, and 10%
sucrose) and concentrated by centrifugation in Amicon Ultra-0.5 10-kDa cutoff
spin filters (EMD Millipore) before being used in assays or stored at −80
°C.
