Journal
PROTEIN JOURNAL
Volume 32, Issue 3, Pages 172-182Publisher
SPRINGER
DOI: 10.1007/s10930-013-9474-5
Keywords
Crocodilians; cDNA cloning; Hemoglobin; Heme insertion; Protein expression
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Funding
- Royal Golden Jubilee (RGJ) Ph.D Program of Thailand Research Fund
- National Research University Project of Thailand, Office of the Higher Education Commission
- Protein and Proteomics Research Group at the Department of Biochemistry, Faculty of Science, Khon Kaen University
- Mahidol University, Thailand
- Department of Bioscience, School of Agriculture, Tokai University, Japan
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The first report of complete nucleotide sequences for alpha- and beta-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding alpha- and beta-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length alpha-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the beta-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both alpha- and beta-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 (HbO(2)). 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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