Purification of Recombinant Wild Type and Mutant Ryanodine Receptors Expressed in HEK293 Cells.

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer , 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee , 1994 ) and later adapted for cryo-EM ( Samsó , 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra , 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer , 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.