Purification and biochemical characterization of the 19-kDa signal recognition particle RNA-Binding protein expressed as a hexahistidine-tagged polypeptide in Escherichia coli

KA Henry, C Zwieb, HM Fried

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

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 SRP 19, we expressed in Escherichia coli a human SRP19 cDNA with an amino-terminal addition of six histidine residues. Expression at 25°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 K(d) 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.

Original languageAmerican English
Pages (from-to)15-26
Number of pages12
JournalProtein Expression and Purification
Volume9
Issue number1
DOIs
StatePublished - Feb 1997

Keywords

  • Amino Acid Sequence
  • Binding, Competitive
  • Cross-Linking Reagents
  • Escherichia coli/genetics
  • Histidine
  • Humans
  • Molecular Sequence Data
  • Oligopeptides/genetics
  • Peptides/genetics
  • Protein Binding
  • RNA-Binding Proteins/genetics
  • Recombinant Fusion Proteins/isolation & purification
  • Signal Recognition Particle/genetics
  • Ultraviolet Rays

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