Eukaryotic translation elongation factor 1Ba (eEF1Ba) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Ba functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The Cterminal domain of eEF1Ba is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bc (eEF1Bc) to form a tight complex. However, eEF1Bc has been shown to enhance the catalytic activity of eEF1Ba attributed to the C-terminal domain of eEF1Ba. This suggests that the N-terminal domain of eEF1Ba may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Ba and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Ba, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Ba. A similar effect on the catalytic activity of eEF1Ba was observed after its interaction with eEF1Bc. We suggest that the non-catalytic N-terminal domain of eEF1Ba may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bc and eEF1Ba N-terminal domains abolishes this inhibitory effect.
To gain more insights into properties of the human translation elongation factor eEF1Bg and its interaction with partners we intended to produce the full-length protein and its truncated forms. Methods. cDNAs encoding truncated forms of eEF1Bg were generated by PCR amplification with respective primers and cloned into vectors providing polyhistidine, glutathione S-transferase or maltose binding protein tags. The recombinant proteins were expressed in Escherichia coli and purified by affinity chromatography. An aggregation state of the proteins was analyzed by analytical gel filtration. Results. The expression, purification and storage conditions for the full-length recombinant His-eEF1Bg were optimized. Several truncated forms of eEF1Bg were also expressed and purified to homogeneity. Two short variants of C-terminal domain comprising amino acids 264-437 or 229-437 were obtained in monomeric state. Two short variants of N-terminal domain comprising amino acids 1-33 or 1-228, fused with glutathione S-transferase, were obtained and estimated to be dimers by gel filtration. The mutants of N-terminal domain comprising amino acids 1-92 or 1-163, fused with maltose binding protein, were obtained as soluble high molecular weight aggregates only. Conclusions. The purified recombinant His-eEF1Bg and several truncated forms of the protein were obtained and characterized. These protein variants will be used for further studies on the protein-protein interaction.
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