Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein

详细信息    查看全文

• 作者：De Vendittis ; E ; Amatruda ; MR ; Raimo ; G ; Bocchini ; V
• 关键词：elongation factor 2 ; Sulfolobus solfataricus ; recombinant EF-2 ; thermostability
• 刊名：Biochimie
• 出版年：1997
• 出版时间：May, 1997
• 年：1997
• 卷：79
• 期：5
• 页码：303-308
• 全文大小：476 K

文摘

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-β-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2_rec) 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-2_rec did not undergo post-translational modification of His₆₀₃ into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2_rec 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-2_rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2_rec 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.