Journal of Microbiology

Research Support, Non-U.S. Gov't

Comparative Evaluation of Three Purification Methods for the Nucleocapsid Protein of Newcastle Disease Virus from Escherichia coli Homogenates: Yan Peng Tan , Tau Chuan Ling , Khatijah Yusoff , Wen Siang Tan , Beng Ti Tey; J. Microbiol. 2005;43(3):295-300.; DOI: https://doi.org/2210 [pii]

Abstract: In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni^2^+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.

Production of the Nucleocapsid Protein of Newcastle Disease Virus in Escherichia coli and its Assembly into Ring- and Nucleocapsid-like Particles: Chiew Ling Kho , Wen Siang Tan , Khatijah Yusoff; J. Microbiol. 2001;39(4):293-299.

Abstract: The nucleocapsid (NP) protein of Newcastle disease virus (NDV) and its derivative (NP_cfus ) containing the myc region and six histidine residues fused to its C-terminus were expressed abundantly in Escherichia coli. The proteins were purified by sucrose gradient centrifugation. Both the NP and NP_cfus proteins self-assembled into ring-like particles with a diameter of 24 +- 2 nm around a central hole of 7 +- 1 nm. Some of these ring-like particles stacked together to form nucleocapsid-like structures which are heterogeneous in length with a diameter of 20 +- 2 nm and a central hollow of 5 +- 1 nm. Only a very small amount of the monomers in the particles was linked by inter-molecular disulfide bonds. Fusion of the C-terminal end to 29 amino acids inclusive of the myc epitope and His-tag did not impair ring assembly but inhibited the formation of the long herringbone structures. Immunogold labeling of the particles with the anti-myc antibody showed that the C-terminus of the NP_cfus protein is exposed on the surface of these ring-like particles.

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