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Production and purification of the phosphoprotein of Nipah virus in Escherichia coli for use in diagnostic assays

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Abstract

Nipah Virus (NiV) is an emerging zoonotic paramyxovirus that can be fatal in humans and various types of animals. The phospho (P) protein of NiV plays an important role in RNA synthesis, replication, and genome synthesis. In this study, the NiV P gene was cloned into a pTrcHis2-TOPO vector and the recombinant protein containing a His-tag was produced in Escherichia coli. SDS-PAGE and Western blot analysis using the anti-His antibody confirmed the protein expression. An optimization study of E. coli fermentation showed that the optimal cultivation temperature was 37°C, while the optimal induction time for P protein expression was at 9 h with 1 mM IPTG. Solubility analysis showed that E. coli cultivated at 37°C produced the highest fraction (70%) of soluble P protein. The recombinant P protein was purified from clarified E. coli lysate using an immobilized metal affinity chromatography (IMAC) technique to a purity of 92.67%, with a purification factor of 11.58. The purified P protein strongly reacted with the anti-NiV swine sera collected during a NiV outbreak, suggesting its potential as a diagnostic reagent.

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Authors and Affiliations

  1. Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia

    Shamala Salvamani, Wen Cheng Ng & Wen Siang Tan

  2. Department of Chemical and Environmental Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia

    Beng Ti Tey

  3. Institute of Bioscience, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia

    Beng Ti Tey & Wen Siang Tan

  4. School of Engineering, Monash University, Jalan Lagoon Selatan, 46150, Bandar Sunway, Selangor, Malaysia

    Beng Ti Tey

Authors
  1. Shamala Salvamani
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  2. Beng Ti Tey
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  3. Wen Cheng Ng
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  4. Wen Siang Tan
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Correspondence to Wen Siang Tan.

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Salvamani, S., Tey, B.T., Ng, W.C. et al. Production and purification of the phosphoprotein of Nipah virus in Escherichia coli for use in diagnostic assays. Biotechnol Bioproc E 16, 1166–1172 (2011). https://doi.org/10.1007/s12257-011-0095-6

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  • DOI: https://doi.org/10.1007/s12257-011-0095-6

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