Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus.
The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified. The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the 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-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.
Ianniciello
G
G
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Masullo
M
M
Gallo
M
M
Arcari
P
P
Bocchini
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016944:Sulfolobus; D013696:Temperature
D001426:Bacterial Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins