Recombinant bacterial expression and purification of human fragile X mental retardation protein isoform 1

Abstract

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.

Figure 1

(A) Schematic of full length FMRP showing the nuclear localization signal (NLS), K-homology domains (KH1, KH2), nuclear export signal (NES), region of phosphorylation (P), and the RGG box (RGG) domain. (B) Sequence of the phosphorylation region and the RGG box domain of full-length ISO1. The sequence shown represents splice site acceptor 15a of FMR1 mRNA. The RGG repeats of the RGG box domain are underlined; the phosphorylation sites are indicated by a double dagger (‡); the arginine methylation sites, located within the RGG box domain, are indicated by an arrow (↓). Also shown is the sequence of the synthetic RGG box peptide used in the previous studies of binding with the S3F-sh_8AP mRNA [42]. Adapted from [28].

Figure 2

Tris-glycine SDS-PAGE (10%) analysis of FMRP ISO1 purity (A) after the Ni-NTA affinity chromatography method developed herein and (B) prior to the optimization of the purification protocol. FMRP ISO1 is not expressed strongly enough in the presence of IPTG to be visibly distinguished from other protein bands in the cleared crude supernatant hence that sample is not shown. (A) Lane 1: EZ-Run protein marker (Fisher Scientific). Lanes 2-8: eluted pure FMRP ISO1 fractions 4-10, respectively. (B) Lane 1: EZ-Run protein marker. Lanes 2-5: eluted impure FMRP ISO1 fractions 5-8, respectively.

Figure 2

Tris-glycine SDS-PAGE (10%) analysis of FMRP ISO1 purity (A) after the Ni-NTA affinity chromatography method developed herein and (B) prior to the optimization of the purification protocol. FMRP ISO1 is not expressed strongly enough in the presence of IPTG to be visibly distinguished from other protein bands in the cleared crude supernatant hence that sample is not shown. (A) Lane 1: EZ-Run protein marker (Fisher Scientific). Lanes 2-8: eluted pure FMRP ISO1 fractions 4-10, respectively. (B) Lane 1: EZ-Run protein marker. Lanes 2-5: eluted impure FMRP ISO1 fractions 5-8, respectively.

Figure 3

Tris-glycine SDS-PAGE (10%) analysis of FMRP ISO1 after the purification, concentration and dialysis processes and ready for binding analysis with G quadruplex mRNAs. Lane 1: EZ-Run protein marker. Lane 2: dialyzed FMRP ISO1.

Figure 4

Proposed G quadruplex structure of S3F-sh mRNA [42]. The adenine at position 8 replaced with fluorescent 2-aminopurine tag, to form S3F-sh_8AP, is shown in red.

Figure 5

Fluorescence spectroscopy assay where FMRP ISO1 (black squares) is titrated into a fixed concentration of S3F-sh_8AP mRNA resulting in a K_d of 104 ± 11 nM and R² value of 0.995. The K_d value was obtained by curve-fitting (red curve) the titration data points with equation 1. As a negative control, BSA (blue triangles) was similarly titrated and showed no significant binding to S3F-sh_8AP mRNA.