title: Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.
abstract: The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, β and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the β subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) μM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03μmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa. Electronic address: Ikechukwu.Achilonu@wits.ac.za.
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Thendo P
authors.initials: TP
authors.lastname: Siganunu
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D001665:Binding Sites; D004137:Dinitrochlorobenzene / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D020648:Peptide Elongation Factor 1 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C042630:1-chloro-2,4-dinitrobenzene-glutathione conjugate; D004137:Dinitrochlorobenzene; D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione
keywords: ANS binding; GSH-CDNB conjugation assay; GST-like N-terminus domain; Human eukaryotic elongation factor 1 gamma; Quaternary structure