Purification and characterization of hexahistidine-tagged elongation factor SelB.
The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNASec. In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion. In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented. After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet. Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNASec. In addition, the fusion protein is fully functional in mediating UGA read-through in vivo. It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation.
Thanbichler
Martin
M
Department of Biology I, University of Munich, Maria-Ward-Str 1a, 80638 Munich, Germany. thanbichler@stanford.edu
Böck
August
A
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D006639:Histidine / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D008024:Ligands; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000302:isolation & purification; D015854:Up-Regulation
D001426:Bacterial Proteins; D008024:Ligands; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; C061878:SelB protein, Bacteria; D006639:Histidine