Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1.
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.
Evans
Timothy L
TL
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA 15282, USA.
Mihailescu
Mihaela-Rita
MR
D004926:Escherichia coli / Q000235:genetics; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence
D051860:Fragile X Messenger Ribonucleoprotein 1; D020033:Protein Isoforms; D011994:Recombinant Proteins