Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1
Introduction
Fragile X syndrome, the most prevalent inheritable mental retardation which affects ∼1 in 4000 males and ∼1 in 8000 females, is caused by the lack of fragile X mental retardation protein (FMRP)1 expression [1]. The absence of FMRP is the result of transcriptional inactivation of the fragile X mental retardation-1 (FMR1) gene by the unstable expansion of a cytosine–guanine–guanine (CGG) trinucleotide repeat in its 5′-untranslated region [2], [3]. Upon exceeding ∼200 CGG repeats, the cytosines of the repeats are hypermethylated leading to the transcriptional silencing of FMR1 and loss of FMRP expression. In addition to a nuclear localization signal near its N-terminus and a nuclear export signal near its C-terminus, FMRP carries two types of RNA binding domains: two K-homology domains and one arginine–glycine–glycine box (RGG box), suggesting that the protein exerts its function via RNA binding (Fig. 1) [4], [5], [6]. Despite extensive studies of the FMRP function, the mechanism by which its loss of expression leads to mental retardation is still not fully understood. FMRP has demonstrated RNA chaperone properties and has been found localized to actively translating ribosomes, leading to the postulation that it serves as a translational regulator for specific messenger RNA (mRNA) targets [7], [8], [9]. FMRP is expressed in most tissues, being found abundantly in the dendritic spines of neurons [10], [11], [12].
The FMR1 gene has 17 exons and it can undergo alternative splicing that involves the exons 12 and 14 and the choice of acceptor sites at exons 15 and 17 [4], [13], [14], [15]. These splicing patterns could potentially result in 20 FMRP isoforms, however, so far only five isoforms have been detected in various tissues [15], [16]. The largest molecular weight FMRP species corresponds to the isoform 1 (ISO1) (Fig. 1) [13].
Previous attempts towards FMRP recombinant bacterial expression, purification and long-term storage in sufficient quantities for biochemical and biophysical studies resulted in limited success, which was not greatly improved upon the subsequent utilization of eukaryotic cells or in vitro systems [4], [12], [15], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]. Following an extensive study, we have isolated the experimental conditions for expression, purification and storage of active recombinant ISO1, representing full-length FMRP, in Escherichia coli. Besides improving yield, the bacterial expression of FMRP ISO1 has the advantage that it renders the protein product devoid of posttranslational modifications, such as phosphorylation and arginine methylation. This is particularly important, as it has been proposed that phosphorylation and arginine methylation might be relevant for the regulation of FMRP function. Mammalian FMRP was found to be phosphorylated in a region N-terminal to its RGG box [31]. The initial suggestion that this modification modulates the FMRP translation regulator function came from the findings that unphosphorylated FMRP associates with actively translating polyribosomes, whereas phosphorylated FMRP associates with stalled polyribosomes [30]. Recently, two enzymes implicated in the phosphorylation and dephosphorylation of FMRP were identified as the ribosomal protein S6 kinase and the protein phosphatase 2A, respectively [32], [33]. It has been suggested that FMRP phosphorylation is coupled to translational suppression, whereas the FMRP dephosphorylation releases the target mRNAs for translation [32], [33], [34]. However, it is not clear if these phosphorylation and dephosphorylation events regulate the FMRP translational regulator function by affecting its RNA binding properties or its interactions with other protein partners. In addition, it has been proposed that the phosphorylation state of FMRP determines its inclusion with the microRNA pathway by regulating its association with Dicer and Dicer-containing complexes [35].
It has also been shown that FMRP is methylated within its RGG box domain and that its binding affinity for homoribopolymers (poly (rG), poly (rC), poly (rA) or poly (rU)) changes when the protein is produced in the presence of protein methyltransferases [19], [36], [37], [38]. Moreover, FMRP methylation reduced its ability to associate with Sc1 RNA, a G quadruplex forming synthetic RNA [19]. These results suggest that protein arginine methylation is important in defining the interactions of FMRP with its G quadruplex forming mRNA targets.
As stated previously, the expression of FMRP ISO1 using E. coli renders the protein devoid of posttranslational modifications. Thus, this method allows for the future analysis of the explicit effects of each posttranslational modification, phosphorylation and arginine methylation, individually and simultaneously. The ability to study the effects of these modifications as potential regulators of the mRNA-binding activity of FMRP is particularly important due to their locations relative to the mRNA-binding RGG box domain, as the four proposed sites of phosphorylation occur N-terminal, but in close proximity to the RGG box domain (Fig. 1B). Moreover, of the arginine methylation sites, two occur on the arginine residues of the RGG repeats within the RGG box, whereas the other two sites occur at the only two arginines found between these repeats. Hence, the probability that the posttranslational modifications of arginine methylation and phosphorylation acting as a regulatory mechanism for the mRNA-binding activity of the RGG box domain is high.
Thus, the successful purification of a significant yield of active recombinant FMRP ISO 1 expressed in E. coli, devoid of posttranslational modifications, is significant in that it will allow us to investigate to what extent FMRP posttranslational modifications, such as phosphorylation and arginine methylation, modulate its translational regulator function.
Section snippets
Expression of recombinant FMRP ISO1
The recombinant plasmid pET21a-FMRP, encoding ISO1 fused with a C-terminal 6× histidine tag, was a gift kindly provided by Dr. Bernhard Laggerbauer [17]. The plasmid was transformed into and cultured using Rosetta 2(DE3) pLysS E. coli cells (Novagen). The cells were cultured at 37 °C, 250 rpm in Luria–Bertani (LB; Fisher Scientific) media containing 200 μg/mL ampicillin (Amp; MP Biomedical) and 15 μg/mL chloramphenicol (Chl; MP Biomedical) and then mixed 1:1 with glycerol and frozen at −80 °C.
Cells
Results and discussion
Since the discovery of the FMR1 gene, the recombinant expression of FMRP in E. coli has been continuously pursued, however the results of these efforts have been only satisfactory, as we and others have experienced problems involving low levels of FMRP expression and yield, as well as protein precipitation [4], [15], [17], [18], [19], [21], [22], [24], [25], [27], [28]. In trying to circumvent these problems, other studies have employed eukaryotic cell lines and in vitro transcription and
Acknowledgments
We thank Dr. Bernhard Laggerbauer (Department of Biochemistry, Theodor Boveri Institute) for the plasmid pET21a-FMRP encoding for FMRP ISO1, and Dr. Stephanie Ceman (Department of Cell and Developmental Biology, University of Illinois, Urbana-Champaign) and Dr. Robert Denman (Department of Molecular Biology, Biochemical Molecular Neurobiology Laboratory, New York State Institute for Basic Research in Developmental Disabilities) for their helpful suggestions in the initial stages of this study.
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