Biopolym. Cell. 2014; 30(2):96-106.
http://dx.doi.org/10.7124/bc.000887
Structure and Function of Biopolymers
Structural dissection of human translation elongation factor 1Bγ (eEF1Bγ): expression of full-length protein and its truncated forms
1Trosiuk T. V., 1, 2Liudkovska V. V., 1Shalak V. F., 1Negrutskii B. S., 1El’skaya A. V.
  1. State Key Laboratory of Molecular and Cellular Biology
    Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Institute of Genetics and Biotechnology, Warsaw University
    5a, Pawinskiego, 02-106, Warsaw, Poland

Abstract

Aim. To gain more insights into properties of the human translation elongation factor eEF1Bγ and its interaction with partners we intended to produce the full-length protein and its truncated forms. Methods. cDNAs encoding truncated forms of eEF1Bγ 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-eEF1Bγ were optimized. Several truncated forms of eEF1Bγ were also expressed and purified to homogeneity. Two short variants of C-terminal domain comprising amino acids 263–437 or 228–437 were obtained in monomeric state. Two short variants of N-terminal domain comprising amino acids 1–33 or 1–230, fused with glutathione S-transferase, were obtained and estimated to be dimers by gel filtration. The mutants of N-terminal domain comprising amino acids 1–93 or 1–165, fused with maltose binding protein, were obtained as soluble high molecular weight aggregates only. Conclusions. The purified recombinant His-eEF1Bγ 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.
Keywords: eEF1Bγ, protein structural domains, expression of recombinant proteins, protein purification
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Supplementary data

References

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