کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020949 1069218 2010 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1
چکیده انگلیسی

The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. FMRP has two types of RNA binding domains, two K-homology domains and an arginine–glycine–glycine box domain, and it is proposed to act as a translation regulator of specific messenger RNA. The interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies is high. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro expression has also resulted in limited success. In addition, the in vitro and eukaryotic expression systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and long-term storage of FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing the study of the direct effects of these modifications individually and simultaneously. In order to assure that FMRP retained activity throughout the process, we used fluorescence spectroscopy to assay the binding activity of the FMRP arginine–glycine–glycine box for the semaphorin 3F mRNA and confirmed that FMRP remained active.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 74, Issue 2, December 2010, Pages 242–247
نویسندگان
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