Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography.
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.
Chong
Fui Chin
FC
Department of Chemical and Natural Resources Engineering, Faculty of Engineering, Universiti Malaysia Pahang, Kuantan, Pahang, Malaysia.
Tan
Wen Siang
WS
Biak
Dayang Radiah Awang
DR
Ling
Tau Chuan
TC
Tey
Beng Ti
BT
D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry*; D045405:Nipah Virus / Q000235:genetics*; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D008670:Metals; D019590:Nucleocapsid Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine