Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI), Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, 40002, Thailand.
Protein J. 2013 Mar;32(3):172-82. doi: 10.1007/s10930-013-9474-5.
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 Co(2+)-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.
本研究首次报道了来自暹罗鳄血红蛋白（Crocodylus siamensis）的α-和β-珠蛋白链的完整核苷酸序列。使用简并引物和快速扩增 cDNA 末端法，通过 RT-PCR 克隆编码α-和β-珠蛋白的 cDNA。全长α-珠蛋白 cDNA 包含一个开放阅读框，编码 141 个氨基酸残基，而β-珠蛋白 cDNA 包含一个开放阅读框，编码 146 个氨基酸残基。通过在大肠杆菌（E. coli）中的异源表达也证实了两种α-和β-珠蛋白 cDNA 克隆的真实性。这是首次在原核系统中产生重组暹罗鳄珠蛋白。此外，通过使用 Co(2+)-charged Talon 树脂进行亲和层析，将血红素基团插入重组蛋白中，并对结合血红素的蛋白进行纯化。结合血红素的蛋白在 415nm 处出现最大吸收峰，表明重组蛋白结合了氧并形成了活性氧合血红蛋白（HbO2）。结果表明，重组暹罗鳄珠蛋白在原核系统中成功表达，并具有作为配体结合蛋白的活性。
