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

Abstract

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.

RECOVERY OF CYCLODEXTRIN GLUCANOTRANSFERASE (CGTase) USING IMMOBILIZED METAL CHELATING AFFINITY CHROMATOGRAPHY

“…Results from this study were superior to those obtained by Clemmitt et al (2000), who purified β-galactosidase from E. coli homogenate (via an expanded bed adsorption) with DBC values of 0.78 mg/mL. In another study of purification of histidine-tagged nucleocapsid protein of Nipah virus via IMAC (Chong et al, 2009), a DBC value of 2.5 mg/ml was obtained using Nickel Sepharose FF, which was also lower compared to this study. Deviations such as these were explained by Sharma et al (2001) as being due to the suitability or deviation of the interaction of the protein with the resin, which depends heavily on the nature of the protein.…”

Modulation of protease activity to enhance the recovery of recombinant nucleocapsid protein of Nipah virus

“…The amount of intact N protein (63 kDa) from various samples were estimated by comparing the intensity of the bands from Western blots (probed with antihistidine antibody) with the help of an internal standard of the N protein purified using sucrose gradient ultracentrifugation [24]. The bands were quantified by using the Volume Tools from the Bio-Rad imaging devices supported by the Quantity One 1 software (Bio-Rad).…”

A Novel Silica-Based Metal Chelate Stationary Phase—L-Glutamic Acid–Copper(II)

“…Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions INTRODUCTION Immobilized metal affinity chromatography (IMAC), also known as metal chelate affinity chromatography (MCAC), was first proposed by Porath et al (1) in 1975. The method has been widely used in the isolation and purification of proteins due to the selective affinity of metal chelate column for proteins and peptides (2)(3)(4)(5)(6). A series of immobilized metal chelate ligands was developed in order to improve the selectivity and stability of metal chelate column (4,(7)(8).…”