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Purification and Characterization of Ovotransferrin from Crocodylus siamensis

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Abstract

Ovotransferrin (OTf) is the major glycoprotein in reptile egg whites. However, knowledge concerning its functional and biological properties remains limited. In this study, OTf from Crocodylus siamensis was purified and characterized. The proteins were precipitated with 80 % ammonium sulfate and then purified by anion exchange chromatography followed by hydrophobic interaction chromatography. The purified crocodile ovotransferrin (cOTf) had a molecular weight of 79 kDa. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) indicated multiple isoforms of cOTf, which had isoelectric points ranging from 6.0 to 6.8. cOTf was N-linked glycosylated protein identified by using PNGase F deglycosylation technique. Optimal autoproteolysis of cOTf occurred under acidic conditions and pH values more than 5, which differs from that of OTf.

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Abbreviations

OTf:

Ovotransferrin

cOTf:

Crocodile ovotransferrin

DTT:

Dithiothreitol

DEAE:

Diethylaminoethanol

TBST:

Tris-buffered saline and Tween 20

TBS:

Tris-buffered saline

NP-40:

Nonidet P 40

PNGase F:

Peptide-N glycosidase F

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Acknowledgments

This work was financially supported by the Synchrotron Light Research Institute (Public Organization), Thailand. We would like to thank the Protein and Proteomic Research Group of the Faculty of Science of Khon Kaen University and Thailand National Research University for their funding support and research facilities. Sriracha Moda Co., Ltd. provided the crocodile (Crocodylus siamensis) eggs, which were necessary for this research. Finally, we gratefully acknowledge the support of the National Research University Project of Thailand for their assistance.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

    Sukanya Chaipayang, Napus Heamatorn, Likhid Keha, Sakda Daduang, Prasan Swatsitang & Sompong Thammasirirak

  2. Protein and Proteomics Research Group, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand

    Sukanya Chaipayang, Napus Heamatorn, Likhid Keha, Sakda Daduang & Sompong Thammasirirak

  3. Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima, 30000, Thailand

    Chomphunuch Songsiriritthigul

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

    Apisak Dhiravisit

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  1. Sukanya Chaipayang
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  2. Napus Heamatorn
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Correspondence to Sompong Thammasirirak.

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Chaipayang, S., Heamatorn, N., Keha, L. et al. Purification and Characterization of Ovotransferrin from Crocodylus siamensis . Protein J 32, 89–96 (2013). https://doi.org/10.1007/s10930-012-9461-2

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  • DOI: https://doi.org/10.1007/s10930-012-9461-2

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