Premium
Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus
Author(s) -
Ianniciello G.,
Masullo M.,
Gallo M.,
Arcari P.,
Bocchini V.
Publication year - 1996
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1996.tb00362.x
Subject(s) - sulfolobus solfataricus , t7 rna polymerase , eukaryotic translation elongation factor 1 alpha 1 , escherichia coli , biology , recombinant dna , elongation factor , microbiology and biotechnology , biochemistry , gene , rna , ribosome , archaea , bacteriophage
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.