The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named Se1B. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNA(Sec), In addition, Se1B 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 Se1B 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 Se1B with guanine nucleotides, SECIS elements, and selenocysteyl-tRNA(Sec). 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 Se1B and the mechanisms of selenocysteine incorporation. (C) 2003 Elsevier Science (USA). All rights reserved.