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

Bauerová-Hlinková, Vladena, Hostinová, Eva, Gašperík, Juraj, Beck, Konrad, Borko, Lubomír, Lai, F. Anthony, Zahradníková, Alexandra and Ševčík, Jozef 2010. Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2. Protein Expression and Purification 71 (1) , pp. 33-41. 10.1016/j.pep.2009.12.014

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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 RyR21–606·His6, RyR2391–606·His6, RyR2409–606·His6, Trx·RyR2384–606·His6, Trx·RyR2391-606·His6 and Trx·RyR2409–606·His6. The folding of RyR21–606·His6 was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR21–606·His6, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His6·Tag antibody indicated that RyR21–606·His6 is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Dentistry
Subjects: Q Science > QH Natural history > QH301 Biology
R Medicine > R Medicine (General)
Uncontrolled Keywords: Human cardiac ryanodine receptor 2 (RyR2); Calcium release channel; Recombinant expression; Bioinformatics prediction; Proteolytic digestion
Publisher: Elsevier
ISSN: 1046-5928
Date of First Compliant Deposit: 9 August 2017
Last Modified: 09 Aug 2017 11:16
URI: http://orca-mwe.cf.ac.uk/id/eprint/15770

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