Recovery of histidine-tagged nucleocapsid protein of Newcastle disease virus using immobilised metal affinity chromatography
Tan, Yan Peng and Tau, Chuan Ling and Tan, Wen Siang and Yusoff, Khatijah and Tey, Beng Ti (2006) Recovery of histidine-tagged nucleocapsid protein of Newcastle disease virus using immobilised metal affinity chromatography. Process Biochemistry, 41 (4). pp. 874-881. ISSN 0032-9592
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Official URL: http://dx.doi.org/10.1016/j.procbio.2005.11.003
An immobilised metal affinity packed bed adsorption chromatography (IMA-PBAC) for the purification of recombinant nucleocapsid protein (NP) of Newcastle disease virus (NDV) directly from clarified feedstock was developed. The XK 16/20 (i.d. = 16 mm) was used as a packed bed column and Streamline chelating adsorbent immobilised with Ni2+ ion was used as IMA adsorbent. This purification method has resulted in a 59% adsorption and 5.6% recovery of NP protein. Adsorbed NP proteins were successfully recovered using a two-step elution protocol which employed elution buffer 1 containing 50 mM imidazole to eliminate contaminating proteins and elution buffer 2 containing 350 mM imidazole to recover the NP protein at pH 8 with flow velocity of 10 cm h−1. About 70% of the adsorbed NP protein was eluted. The purity of the recovered NP protein was about 70% and the volume of processing fluid was reduced by a factor of 4. The antigenic features of purified NP proteins were confirmed by enzyme-linked immunosorbent assay (ELISA) analysis.
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