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Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum

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Abstract

Gene encoding a novel translation initiation factor PeIF5B from Pisum sativum with sequence similarity to eIF5B from H. sapiens, D. melanogaster, S. cerevisiae as well as archaeal aIF5B from M. thermoautotrophicum was earlier reported by us. We now describe the expression and purification of 96 kDa recombinant PeIF5B (rPeIF5B) protein. Using fluorescence and circular dichroism spectra analyses, we show that Mg2+ binding does not lead to any change in PeIF5B aromatic amino acid micro-environment, whereas GTP binding induces significant changes in the local environment of the aromatic amino acids. However, the protein undergoes changes in secondary structure upon metal ion and nucleotide binding. Charged initiator tRNA binding to PeIF5B is found to be cofactor dependent. PeIF5B binds to GTP in vitro as evident from autoradiography. Based on homology modeling of the catalytic domain of PeIF5B, we could confirm the conformational changes in PeIF5B following ligand binding.

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Acknowledgements

S.R. and M.S. thank the CSIR and ICMR, respectively, for Senior Research Fellowship. S.E.H. is a JC Bose National Fellow.

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Authors and Affiliations

  1. Department of Biochemistry, University of Hyderabad, Prof. C. R. Rao Road, Hyderabad, 500 046, India

    Sheeba Rasheedi & Seyed E. Hasnain

  2. Laboratory of Molecular and Cellular Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, 500 001, India

    Sheeba Rasheedi

  3. Molecular Biology Unit, National Institute of Nutrition, Hyderabad, 500 007, India

    Madhuri Suragani, Soghra K. Haq & Nasreen Z. Ehtesham

  4. Institute of Life Sciences, University of Hyderabad Campus, Prof. C. R. Rao Road, Hyderabad, 500 046, India

    Sachchidanand, Seyed E. Hasnain & Nasreen Z. Ehtesham

  5. Biomedical Informatics Center, National Institute of Nutrition, Hyderabad, 500 007, India

    Rajesh Bhardwaj & Nasreen Z. Ehtesham

  6. Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, 560 064, India

    Seyed E. Hasnain

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  1. Sheeba Rasheedi
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  2. Madhuri Suragani
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Correspondence to Nasreen Z. Ehtesham.

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11010_2010_526_MOESM1_ESM.ppt

The following supplementary material is available: Fig. S1 Ramachandran plots for free (a), inactive (b) and active (c) forms of PeIF5B vs. 1g7r, 1g7 s and 1g7t forms of aIF5B from M. thermoautotrophicum, respectively. (PPT 911 kb)

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Rasheedi, S., Suragani, M., Haq, S.K. et al. Expression, purification and ligand binding properties of the recombinant translation initiation factor (PeIF5B) from Pisum sativum . Mol Cell Biochem 344, 33–41 (2010). https://doi.org/10.1007/s11010-010-0526-2

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  • DOI: https://doi.org/10.1007/s11010-010-0526-2

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