Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2009-05, Vol.877 (14-15), p.1561-1567
• Main Authors: Chong, Fui Chin, Tan, Wen Siang, Biak, Dayang Radiah Awang, Ling, Tau Chuan, Tey, Beng Ti
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity - methods; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; General pharmacology; Histidine - genetics; Histidine - metabolism; Immobilized metal affinity chromatography; Medical sciences; Metals - chemistry; Nipah virus; Nipah Virus - genetics; Nucleocapsid protein; Nucleocapsid Proteins - genetics; Nucleocapsid Proteins - isolation & purification; Nucleocapsid Proteins - metabolism; Pharmacology. Drug treatments; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2009.03.048

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 1.25cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4mg/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.