Fig. 1: Schematic diagram and purification of MCA proteins.
a Schematic diagram of intact MCA1 and MCA2 and their C-terminally truncated proteins. TM, transmembrane segment. D21, Asp-21 crucial for Ca2+ influx17. b Purification of MCA2(1-173)-6H. Protein samples were subjected to SDS-PAGE followed by silver staining. Lane 1, A crude proteoliposome fraction. Lane 2, 1% DDM-solubilized sample. Lane 3, A flow-through fraction from a Ni-NTA column. Lane 4, An eluate from the same column eluted with 300 mM imidazole. Lane 5, An eluate from a PD-10 desalting column eluted with MOPS buffer described in the Methods section. One-4000th of each sample was loaded onto the respective lanes. M, Molecular weight markers. A typical result from six independent experiments is shown. c Purified MCA proteins used for reconstitution into liposomes. Each sample is an eluate from the PD-10 desalting column described above. Lane 1, MCA1(1-173)D21N-6H; Lane 2, MCA1(1-173)-6H; Lane 3, MCA2(1-173)D21N-6H; Lane 4, MCA2(1-173)-6H. A typical result from three independent experiments is shown. Note that the molecular weights of MCA1(1-173)-6H and MCA2(1-173)-6H were calculated from their amino acid sequences to be 21.7 and 21.4 kDa, respectively. For comparison, note that the molecular weights of MCA1(1-173) and MCA2(1-173) tagged with four tandem hemagglutinin repeats, which were previously used17, but not in the present study, were calculated to be 27.8 and 27.5 kDa, respectively.
