Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli

Abstract

The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.

Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells

“…Thus, this observation is in good agreement with other paramyxovirus N proteins, in which the C termini are susceptible to proteolytic removal (14,15,26,29,30).…”

“…The NiV N protein produced in E. coli is mainly found in inclusion bodies (30). The solubility of the recombinant protein can be improved by expression under the control of different promoters and hosts, truncating the protein, and changing the temperature of the growing culture.…”

Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain

“…(42), of the four paramyxovirus P multimerization domain structures. The residues in a basic patch of Sendai virus P implicated in L binding are indicated (39), as are the residues in a corresponding basic patch of Nipah virus P. It is unknown if L binds this site or a different site in P, as noted for the rhabdovirus vesicular stomatitis virus (43) (B) Model of the organization of the Nipah virus replication machinery, roughly to scale, based on dimensions visualized by electron microscopy (Nipah virus nucleocapsid [44] and vesicular stomatitis virus L [44,45]) and X-ray crystallography (this structure and the X domain of measles virus P [46]). For clarity, Nipah virus P is illustrated in either its polymerase cofactor function, in which it is bound to L and the nucleocapsid (left), or in its role as a chaperone for nascent N 0 (right).…”

Serodiagnosis Using Recombinant Nipah Virus Nucleocapsid Protein Expressed in Escherichia coli

“…Eshaghi, Tan, and colleagues reported that Nipah virus N (NiV-N) protein expressed in insect cells and Escherichia coli assembled into different types of structures of different lengths, including spherical, ring-like, and herringbone-like particles, under electron microscopy and that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition (7,16,20). But since only a limited number of swine samples were tested, more studies are required to assess the use of the recombinant N protein in routine diagnosis, especially for human samples.…”