Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

Abstract

We report the domain analysis of the N-terminal region (residues 1-759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR2(1-606)xHis(6), RyR2(391-606)xHis(6), RyR2(409-606)xHis(6), Trx.RyR2(384-606)xHis(6), TrxxRyR2(391-606)xHis(6) and Trx.RyR2(409-606)xHis(6). The folding of RyR2(1-606)xHis(6) was analyzed by circular dichroism spectroscopy resulting in alpha-helix and beta-sheet content of approximately 23% and approximately 29%, respectively, at temperatures up to 35 degrees C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR2(1-606)xHis(6), resulted in the appearance of two specific subfragments of approximately 40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His(6).Tag antibody indicated that RyR2(1-606)xHis(6) is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.