Recovery of histidine-tagged nucleocapsid protein of Newcastle disease virus using immobilised metal affinity chromatography

• Main Authors: Tan, Yan Peng, Tau, Chuan Ling, Tan, Wen Siang, Yusoff, Khatijah, Tey, Beng Ti
• Format: Article
• Language: English; English
• Published: Elsevier 2006
• Online Access: http://psasir.upm.edu.my/id/eprint/5608/1/Recovery%20of%20histidine.pdf; http://psasir.upm.edu.my/id/eprint/5608/; 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.