کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10843377 | 1069231 | 2010 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
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چکیده انگلیسی
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 â¼23% and â¼29%, 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 â¼40 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 71, Issue 1, May 2010, Pages 33-41
Journal: Protein Expression and Purification - Volume 71, Issue 1, May 2010, Pages 33-41
نویسندگان
Vladena Bauerová-Hlinková, Eva Hostinová, Juraj GaÅ¡perÃk, Konrad Beck, ĽubomÃr Borko, F. Anthony Lai, Alexandra ZahradnÃková, Jozef Å evÄÃk,