title: Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli.
abstract: The signal recognition particle (SRP) is a ribonucleoprotein complex that mediates translocation of proteins into the endoplasmic reticulum. Protein SRP19 is an essential structural component of SRP and is believed to promote assembly of the particle. In order to have a convenient source for the purification of milligram amounts of SRP19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25 degrees C eliminated formation of insoluble SRP19 and resulted in accumulation of soluble hexahistidine-SRP19 to 68% of total cell protein after 24 h. Metal chelation chromatography yielded 40 mg of hexahistidine-SRP19 per liter of culture, with a purity slightly greater than 97%. To examine protein function, the RNA-binding properties of the purified protein were determined by RNA electromobility shift assays. The histidine-tagged SRP19 bound specifically to a 150-nucleotide RNA derived from SRP RNA, with an apparent Kd of 1 nM, and bound, with greatly reduced affinity, to a mutagenized form of the SRP RNA derivative that contained an altered helix 6 tetranucleotide loop. The purified protein was also photochemically crosslinked to the 150-nucleotide SRP RNA fragment, providing the means to potentially identify portions of hexahistidine-SRP19 which are in close proximity to the RNA molecule.
authors.affiliation: Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, 27599, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Henry
authors.forename: C
authors.initials: C
authors.lastname: Zwieb
authors.forename: H M
authors.initials: HM
authors.lastname: Fried
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D010455:Peptides / Q000235:genetics; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D018271:Signal Recognition Particle / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D009842:Oligopeptides; D010455:Peptides; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D018271:Signal Recognition Particle; C033223:polyhistidine; D006639:Histidine