A simple procedure for bacterial expression and purification of the fragile X protein family

Abstract

AbstractThe fragile X protein family consists of three RNA-binding proteins involved in translational regulation. Fragile X mental retardation protein (FMRP) is well-studied, as its loss leads to fragile X syndrome, a neurodevelopmental disorder which is the most prevalent form of inherited mental retardation and the primary monogenetic cause of autism. Fragile X related proteins 1 and 2 (FXR1P and FXR2P) are autosomal paralogs of FMRP that are involved in promoting muscle development and neural development, respectively. There is great interest in studying this family of proteins, yet researchers have faced much difficulty in expressing and purifying the full-length versions of these proteins in sufficient quantities. We have developed a simple, rapid, and inexpensive procedure that allows for the recombinant expression and purification of full-length human FMRP, FXR1P, and FXR2P from Escherichia coli in high yields, free of protein and nucleic acid contamination. In order to assess the proteins’ function after purification, we confirmed their binding to pseudoknot and G-quadruplex forming RNAs as well as their ability to regulate translation in vitro.