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Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea

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Abstract

Background

Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this study aimed to produce the recombinant tropomyosin protein from S. olivacea and subsequently investigate its IgE reactivity.

Methods and Results

The tropomyosin gene was cloned and expressed in the Escherichia coli system, followed by SDS-PAGE and immunoblotting test to identify the allergenic potential of the recombinant protein. The 855-base pair of tropomyosin gene produced was found to be 99.18% homologous to Scylla serrata. Its 284 amino acids matched the tropomyosin of crustaceans, arachnids, insects, and Klebsiella pneumoniae, ranging from 79.03 to 95.77%. The tropomyosin contained 89.44% alpha-helix folding with a tertiary structure of two-chain alpha-helical coiled-coil structures comprising a homodimer heptad chain. IPTG-induced histidine tagged-recombinant tropomyosin was purified at the size of 42 kDa and confirmed as tropomyosin using anti-tropomyosin monoclonal antibodies. The IgE binding of recombinant tropomyosin protein was reactive in 90.9% (20/22) of the sera from crab-allergic patients.

Conclusions

This study has successfully produced an allergenic recombinant tropomyosin from S. olivacea. This recombinant tropomyosin may be used as a specific allergen for the diagnosis of allergy.

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Data availability

Data deposition (mud crab tropomyosin identification number) is made in the GenBank repository found at https://www.ncbi.nlm.nih.gov/. Other data is available from the authors for non-commercial purposes on reasonable request.

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Acknowledgements

The authors wish to thank the Ministry of Higher Education Malaysia (MOE) for the financial funding of this research through the Fundamental Research Grant Scheme (FRGS 2016-0085-102-02).

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Authors and Affiliations

  1. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900, Tanjong Malim, Perak, Malaysia

    Nur Farah Hani Azemi, Rosmilah Misnan, Bun Poh Keong, Marina Mokhtar, Nurhaida Kamaruddin, Wong Chee Fah & Haidr Msahir Ateshan

  2. Disease Control Division, Ministry of Health Malaysia, 62590, Putrajaya, Malaysia

    Zailatul Hani Mohamad Yadzir & Hani Mohamad Yadzir

  3. Allergy and Immunology Research Centre, Institute for Medical Research, Jalan Pahang, 50588, Kuala Lumpur, Malaysia

    Zailatul Hani Mohamad Yadzir, Hani Mohamad Yadzir, Faizal Bakhtiar, Noormalin Abdullah & Masita Arip

  4. Department of Biology, Mohsen Al-Hakim School, Thi-Qar Education Directorate, Nasiriyah, Iraq

    Haidr Msahir Ateshan

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  1. Nur Farah Hani Azemi
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Correspondence to Rosmilah Misnan.

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11033_2021_6661_MOESM1_ESM.tif

IgE immunoblotting of crude extract of Scylla olivacea. Lane M, Vivantis Chromatein Prestained Protein Ladder (Vivantis Technologies, USA); lane N, immunoblot using serum from a non-allergic individual; and lane B, blank; lanes 1-22, immunoblots showing binding of IgE from different serum samples.Supplementary file1 (TIF 1669 kb)

11033_2021_6661_MOESM2_ESM.tif

Agarose gel electrophoresis of PCR re-amplification of tropomyosin gene of S. olivacea (o). Marker using 100 bp DNA ladder (Thermo Scientific, USA). Supplementary file2 (TIF 748 kb)

11033_2021_6661_MOESM3_ESM.tif

Overexpression of recombinant tropomyosin protein from S. olivacea. S; soluble, IS; insoluble. Supplementary file3 (TIF 5958 kb)

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Azemi, N.F.H., Misnan, R., Keong, B.P. et al. Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea. Mol Biol Rep 48, 6709–6718 (2021). https://doi.org/10.1007/s11033-021-06661-x

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