Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein.
The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells. Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine. Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2. The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.
de Vendittis
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E
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy.
Amatruda
M R
MR
Raimo
G
G
Bocchini
V
V
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006361:Heating; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016944:Sulfolobus / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins