Nur Farah Hani Azemi1, Rosmilah Misnan2, Bun Poh Keong1, Marina Mokhtar1, Nurhaida Kamaruddin1, Wong Chee Fah1, Zailatul Hani Mohamad Yadzir3,4, Hani Mohamad Yadzir3,4, Faizal Bakhtiar4, Noormalin Abdullah4, Masita Arip4, Haidr Msahir Ateshan1,5. 1. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900, Tanjong Malim, Perak, Malaysia. 2. Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris, 35900, Tanjong Malim, Perak, Malaysia. rosmilah@fsmt.upsi.edu.my. 3. Disease Control Division, Ministry of Health Malaysia, 62590, Putrajaya, Malaysia. 4. Allergy and Immunology Research Centre, Institute for Medical Research, Jalan Pahang, 50588, Kuala Lumpur, Malaysia. 5. Department of Biology, Mohsen Al-Hakim School, Thi-Qar Education Directorate, Nasiriyah, Iraq.
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.
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.
Authors: Anas M Abdel Rahman; Andreas L Lopata; Robyn E O'Hehir; John J Robinson; Joseph H Banoub; Robert J Helleur Journal: J Mass Spectrom Date: 2010-04 Impact factor: 1.982