Chong Fui Chin, Tan Wen Siang, Biak Dayang Radiah Awang, Ling Tau Chuan, Tey Beng Ti
Department of Chemical and Natural Resources Engineering, Faculty of Engineering, Universiti Malaysia Pahang, Kuantan, Pahang, Malaysia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 15;877(14-15):1561-7. doi: 10.1016/j.jchromb.2009.03.048. Epub 2009 Apr 7.
Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.
尼帕病毒（NiV）的核衣壳（N）蛋白是用于诊断NiV感染的一种潜在血清学标志物。在本研究中，应用HisTrap 6 Fast Flow填充床柱这一快速高效的纯化系统，从澄清的原料中纯化NiV的重组组氨酸标签N蛋白。研究了NiV的N蛋白与镍琼脂糖6 Fast Flow结合及洗脱条件的优化。在pH 7.5、表面流速1.25 cm/min时实现了最佳结合。通过用不同浓度（50、150、300和500 mM）的咪唑进行分步洗脱，成功回收了结合的N蛋白。在扩大规模的镍琼脂糖6 Fast Flow固定化金属亲和色谱（IMAC）填充床柱中，按照方法探索获得的优化条件，从0.4 mg/ml的入口N蛋白浓度中捕获并洗脱NiV的N蛋白。使用IMAC填充床柱纯化组氨酸标签N蛋白的产率为68.3%，纯化倍数为7.94。
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