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Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis)

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Abstract

The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co2+-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.

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Abbreviations

GSP:

Gene specific primers

Hb:

Hemoglobin

IPTG:

Isopropyl-β-D-thiogalactopyranoside

RACE-PCR:

Rapid amplification of cDNA ends

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Acknowledgments

This research was supported by the Royal Golden Jubilee (RGJ) Ph.D Program of Thailand Research Fund and National Research University Project of Thailand, Office of the Higher Education Commission through financial support. We would also like to thank the Protein and Proteomics Research Group at the Department of Biochemistry, Faculty of Science, Khon Kaen University, Mahidol University, Thailand and Department of Bioscience, School of Agriculture, Tokai University, Japan for their supports. We wish to acknowledge the support of the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University, for their assistance.

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Authors and Affiliations

  1. Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

    Preeyanan Anwised, Thai Kabbua & Sompong Thammasirirak

  2. Sriracha Moda Farm, Chon Buri, 20110, Thailand

    Theeranan Temsiripong

  3. Faculty of Humanities and Social Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand

    Apisak Dhiravisit

  4. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Sarawut Jitrapakdee

  5. Department of Bioscience, School of Agriculture, Tokai University, Aso, Kumamoto, 869-1404, Japan

    Tomohiro Araki & Kazunari Yoneda

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  1. Preeyanan Anwised
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  2. Thai Kabbua
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  8. Sompong Thammasirirak
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Correspondence to Sompong Thammasirirak.

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Anwised, P., Kabbua, T., Temsiripong, T. et al. Molecular Cloning and Expression of α-Globin and β-Globin Genes from Crocodile (Crocodylus siamensis). Protein J 32, 172–182 (2013). https://doi.org/10.1007/s10930-013-9474-5

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  • DOI: https://doi.org/10.1007/s10930-013-9474-5

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