Jurynec, M.J., Xia, R., Mackrill, J.J., Gunther, D., Crawford, T., Flanigan, K.M., Abramson, J.J., Howard, M.T and Grunwald, D.J.
Notes: The authors show that SepN, a selenoprotein of unknown function, and ryanodine receptor (RyR) intracellular calcium release channel are both required for normal muscle development in zebrafish. Furthermore SepN and RyR interact, and SepN is required for full activity of the RyR channel. As part of their study, the authors expressed SepN as a polyhistidine-tagged (8X His) protein in TNT® Coupled Reticulocyte Lysate System and purified it using the MagZ™ Protein Purification System. The TNT® reaction was modified to optimize selenocysteine incorporation efficiency; the reaction contained 80% rabbit reticulocyte lysate (RRL), 1mM methionine, 0.4mM spermidine, 0.01µg/ml SepN-8X-His DNA and 300nM of the C-terminus of Secis Binding Protein 2 , which is required for efficient incorporation of selenocysteine in RRL-based reactions. (3898)