Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli

Nipah virus (NiV) possesses a nonsegmented, single-stranded negative sense RNA genome that contains six structural genes arranged in the order of 3' N-P-M-F-G-L 5'. The nucleocapsid (N) gene of Nipah virus isolated from swine was amplified from the viral RNA by reverse transcription polymerase chain reaction (RT-PCR). The nucleocapsid (N) gene of Nipah virus was cloned into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a 63 kDa fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. The N gene sequence shared 99% homology with that of Nipah virus isolated from human. The coding region of N protein of NiV was cloned into different vectors and subsequently introduced into different E. coli strains. The yield of the N protein produced in different vectors and different hosts was compared. It was found that the amount of N protein expressed by the pTrcHis2 vector containing the trc promoter in E. coli SG 935 was four-fold higher than that of vector pRSETB and pGEX-4T-l in E. coli strain BL 21 series. Deletion of the N- or/and C-terminal region of the N protein revealed that the N-terminal region plays a role in N protein solubility, but a mutant (MN50fus) containing the second half of the N protein showed the highest expression level in all the three E. coli strains. Lowering the growth temperature of E. coli cell cultures to 25°C improved the solubility of the full-length and truncated Nfus protein from 50% to 80%. This study addresses the fundamental problems encountered in production of Nipah viral N protein in E. coli which may be useful as an alternative antigen for the detection of anti-NiV in swine

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

The nucleocapsid (N)protein of Nipah virus (NiV) is a major constituent of the viral proteins which play a role in encapsidation, regulating the transcription and replication of the viral genome. To investigate the use of a fusion system to aid the purification of the recombinant N protein for structural studies and potential use a diagnostic reagent, the NiV n gene was cloned into the pFastBacHT vector and and his tagged fusion protian was expressed in Sf9 insect cells by recombinant baculovirus. Western blot analysis of the recombinant fusion protein with anti-Niv antibodies produced a band of approximately 62 kDa. At time course study showed that the highest level of expression was achived after 3 days of incubation. Electron microscopic analysis of the NiV recombinant N fusion protein purified on a nickel- nitrilotriacetic asid resin column revealed different types of structures, including spherical, ring-like, particles. The light-scattering measurements of the recombinant N protein also confirmed the polydispersity of the sample with hydrodynamic radii of small and large types. The optical density spectra of the purified recombinant fusion protein reavealed a high A 260 / A 280 ratio, indicating the presence of nucleic acids. Western blotting and enzyme-linked immunosorbent assay result showed that the recombinant N protein exhibited the antigenic sites and conformation necessary for specific antigen-antibody recognition

An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM

Solubility, Immunogenicity and Physical Properties Of the Nucleocapsid Protein of Nipah Virus Produced in Escherichia Coli.

The nucieocapsid (N) protein of Nipah virus (Niv) can be produced in three Escherichia coli starins (TOP10,BL (DEB) and S G935) under the control of trc promoter, However, most of the product existed in the form of insolubie inclusion bodies. There was no improvement in the solubility of the products when this protein was placed under thecontrol of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21 (DE3) cell culture. 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 herring bone-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

N-terminally His-tagged hepatitis B core antigens: construction, expression, purification and antigenicity

The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg

An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles

Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production of Escherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h−1. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h−1) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C

Mutagenesis of the nucleocapsid protein of nipah virus involved in capsid assembly.

The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30–32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation