Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography

Tan, Lik Chern Melvin; Chua, Anthony Jin Shun; Goh, Li Shan Liza; Pua, Shu Min; Cheong, Yuen Kuen; Ng, Mah Lee

doi:10.1016/j.pep.2010.06.015

Cited by 24 publications

(16 citation statements)

References 40 publications

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“…In the current study 10 μg total proteins, with a majority of VHSV structural proteins, have been injected versus 2–100 μg of recombinant domain III. This could explain in part these contrasted results [6], [9], [11], [43]. However it has to be mentioned that the SP G E WNV TM G antigen, although almost undetectable in Western blot was anyway in sufficient amount to induce detectable antibody responses.…”

Section: Discussionmentioning

confidence: 84%

A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

Nzonza

Lecollinet

Chat³

et al. 2014

PLoS ONE

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West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

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Section: Discussionmentioning

confidence: 84%

A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

Nzonza

Lecollinet

Chat³

et al. 2014

PLoS ONE

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“…Various recombinant proteins produced using microbes and higher organisms are used as a therapeutic agents and vaccine candidates ( 12 – 14 ). The expression host system mostly used for recombinant dengue virus proteins include bacteria ( 15 ), yeast ( 16 ), mammalian cells ( 17 ), insect cells ( 18 ), transgenic animals ( 19 ), and transgenic plants ( 20 ). The recombinant proteins are mainly produced with the help of genetic engineering techniques.…”

Section: Introductionmentioning

confidence: 99%

Recent Developments in Recombinant Protein–Based Dengue Vaccines

Tripathi

Shrivastava

2018

Front. Immunol.

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Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant proteins of therapeutic and prophylactic importance have been successfully produced in microbial and higher expression host systems. Since there is no specific antiviral therapy available against dengue, the prevention by vaccination is the mainstay in reducing the disease burden. Therefore, efficacious vaccines are needed to control the spread of dengue worldwide. Dengue is an emerging viral disease caused by any of dengue virus 1–4 serotypes that affects the human population around the globe. Dengue virus is a single stranded RNA virus encoding three structural proteins (capsid protein, pre-membrane protein, and envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). As the only licensed dengue vaccine (Dengvaxia) is unable to confer balanced protection against all the serotypes, therefore various approaches for development of dengue vaccines including tetravalent live attenuated, inactivated, plasmid DNA, virus-vectored, virus-like particles, and recombinant subunit vaccines are being explored. These candidates are at different stages of vaccine development and have their own merits and demerits. The promising subunit vaccines are mainly based on envelope or its domain and non-structural proteins of dengue virus. These proteins have been produced in different hosts and are being investigated for development of a successful dengue vaccine. Novel immunogens have been designed employing various strategies like protein engineering and fusion of antigen with various immunostimulatory motif to work as self-adjuvant. Moreover, recombinant proteins can be formulated with novel adjuvants to enhance the immunogenicity and thus conferring better protection to the vaccinees. With the advent of newer and safer host systems, these recombinant proteins can be produced in a cost effective manner at large scale for vaccine studies. In this review, we summarize recent developments in recombinant protein based dengue vaccines that could lead to a good number of efficacious vaccine candidates for future human use and ultimately alternative dengue vaccine candidates.

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“…Some of the important parameters for enhancing protein production are medium composition, cultivation conditions, inducer concentration, induction time and duration, and mode of cultivation that can be optimized to get recombinant protein with high yield 19 , 21 . The purification of recombinant enzyme is necessary with minimum possible steps to avoid loss of activity or degradation and achieve the required level of purity for therapeutic studies 22 , 23 . In general, scale up cultivation process at the optimal condition increases the expression and production level of enzyme to many times in comparison to shake flask.…”

Section: Introductionmentioning

confidence: 99%

Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus

Saha

Acharya

Priya

et al. 2018

Sci Rep

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Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and <10% respectively. We used this cell free HTS system to screen panel of metal ions and its conjugate which revealed zinc acetate as a potential candidate, which was further found to inhibit CHIKV in Vero cells. Scale-up process has not been previously reported for any of the arboviral nonstructural enzymes. The successful scale-up method for viral protease together with a HTS assay could lead to the development of industrial level large-scale screening platform for identification of protease inhibitors against emerging and re-emerging viruses.

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Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography

Cited by 24 publications

References 40 publications

A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

Recent Developments in Recombinant Protein–Based Dengue Vaccines

Development of nsP2 protease based cell free high throughput screening assay for evaluation of inhibitors against emerging Chikungunya virus

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