EXPRESSION IN ESCHERICHIA-COLI OF THERMOSTABLE ELONGATION-FACTOR-I-ALPHA FROM THE ARCHAEON SULFOLOBUS-SOLFATARICUS

The elongation factor 1 alpha from the archaeon Sulfolobus solfataricu s (SsEF-1 alpha) was expressed in Escherichia coli and purified. The S sEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of t he pT7-7 expression vector, under the control of the promoter of T7 RN A polymerase. Upon induction with isopropyl beta-D-thiogalactopyranosi de, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from t he E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained, The N-t erminal sequence of the first 30 amino acid residues of recSsEF-1 alph a was identical with that translated from the nucleotide sequence of t he corresponding gene, except for the initial residue, which in recSsE F-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determi ned by electrospray MS) was almost coincident with that of the natural ly occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were ide ntical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to e licit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Boc chini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.