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 a rchaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterolog ous product did not increase upon addition:of isopropyl-beta-thiogalac topyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2 ) was about 3 mg from 60 g of transformed wet cells. Recombinant and n aturally occurring SsEF-2 showed identical electrophoretic mobility, i mmunological properties and the N-terminal amino acid sequence; both w ere lacking the initial methionine. Differently from SsEF-2, SsEF-2(re c) did not undergo post-translational modification of His(603) into di phthamide, 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(P he) synthesis, to bind CDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2(rec) was endowed with the s ame heat stability as SsEF-2. Altogether these findings proved that Ss EF-2(rec) was functionally active as SsEF-2. The used expression syste m could allow to produce mutated forms of SsEF-2 obtained by mutagenes is of the corresponding gene.