An easy pipeline for one-step purification of SARS-CoV-2 nucleocapsid protein from insect cell suspension culture

Camargo, Brenda Rabello de; Silva, Leonardo A. da; Oliveira, A. S. de; Ribeiro, Bergmann Morais

doi:10.1016/j.jviromet.2021.114341

Cited by 7 publications

(6 citation statements)

References 25 publications

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“…Thus, it would be very interesting to test whether SARS-CoV-2 ORF3a is included in virion. One of the well-accepted methods to test this possibility is to subject purified SARS-CoV-2 viral particles to chromatography, by which it can precisely detect whether ORF3a protein is associated with virion ( Shen et al, 2005 ; de Camargo et al, 2022 ). Other confirmed or possible roles of SARS-CoV-2 ORF3a in the SARS-CoV-2 viral life cycle are illustrated in Figure 2 .…”

Section: Roles Of Sars-cov-2 Orf3a In Viral Life Cyclementioning

confidence: 99%

Understanding the Role of SARS-CoV-2 ORF3a in Viral Pathogenesis and COVID-19

et al. 2022

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The ongoing SARS-CoV-2 pandemic has shocked the world due to its persistence, COVID-19-related morbidity and mortality, and the high mutability of the virus. One of the major concerns is the emergence of new viral variants that may increase viral transmission and disease severity. In addition to mutations of spike protein, mutations of viral proteins that affect virulence, such as ORF3a, also must be considered. The purpose of this article is to review the current literature on ORF3a, to summarize the molecular actions of SARS-CoV-2 ORF3a, and its role in viral pathogenesis and COVID-19. ORF3a is a polymorphic, multifunctional viral protein that is specific to SARS-CoV/SARS-CoV-2. It was acquired from β-CoV lineage and likely originated from bats through viral evolution. SARS-CoV-2 ORF3a is a viroporin that interferes with ion channel activities in host plasma and endomembranes. It is likely a virion-associated protein that exerts its effect on the viral life cycle during viral entry through endocytosis, endomembrane-associated viral transcription and replication, and viral release through exocytosis. ORF3a induces cellular innate and pro-inflammatory immune responses that can trigger a cytokine storm, especially under hypoxic conditions, by activating NLRP3 inflammasomes, HMGB1, and HIF-1α to promote the production of pro-inflammatory cytokines and chemokines. ORF3a induces cell death through apoptosis, necrosis, and pyroptosis, which leads to tissue damage that affects the severity of COVID-19. ORF3a continues to evolve along with spike and other viral proteins to adapt in the human cellular environment. How the emerging ORF3a mutations alter the function of SARS-CoV-2 ORF3a and its role in viral pathogenesis and COVID-19 is largely unknown. This review provides an in-depth analysis of ORF3a protein’s structure, origin, evolution, and mutant variants, and how these characteristics affect its functional role in viral pathogenesis and COVID-19.

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Section: Roles Of Sars-cov-2 Orf3a In Viral Life Cyclementioning

confidence: 99%

Understanding the Role of SARS-CoV-2 ORF3a in Viral Pathogenesis and COVID-19

et al. 2022

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“…The efficient lactosebased autoinduction method reported in this study is an easy-to-adapt method that does not require continuous monitoring of the culture density for the addition of inducers such as IPTG. Other groups have reported the production of recombinant SARS-CoV-2 nucleocapsid protein in different hosts such as E. coli, insects, and mammalian cells [17,[22][23][24][25][26][27]. Li et al reported the purification of nucleocapsid protein from E. coli after refolding of inclusion bodies with a yield of approximately 50 mg/liter fermentation broth [22].…”

Section: Discussionmentioning

confidence: 99%

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Verma¹,

Abhishek²

2022

J App Biol Biotech

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SARS-CoV-2 encodes a nucleocapsid protein that binds to the single-stranded RNA genome present inside the viral particles. It is one of the predominant proteins expressed during infection and tends to be highly immunogenic in infected individuals. Consequently, it has been recognized as an important diagnostic marker for the development of antigen or antibody detection-based diagnostic platforms for COVID-19. In this work, we have described cloning, autoinduction-based expression, and purification of full-length SARS-CoV-2 nucleocapsid protein using Escherichia coli as the heterologous host. We have characterized the purified protein using commercial rapid antigen test kits and indirect ELISA to demonstrate its suitability as bait for diagnostic assays. The purified recombinant protein reported in this study can be useful for numerous applications including the development of monoclonal or polyclonal antibodies, studying protein structure and function, and mapping B-and T-cell epitopes for designing suitable vaccines and therapeutics.

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“…In other studies, a SARS-CoV-2 nucleocapsid protein was produced in BEV using a suspension culture of insect cells [ 36 ]. A total of 1 mg of protein was purified from 50 mg of crude protein.…”

Section: Application Of Baculovirus Expression Vector System (Bev) Fo...mentioning

confidence: 99%

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

Azali

Mohamed

Harun

et al. 2022

Journal of Genetic Engineering and Biotechnology

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Background The baculovirus expression vector system has been developed for expressing a wide range of proteins, including enzymes, glycoproteins, recombinant viruses, and vaccines. The availability of the SARS-CoV-2 genome sequence has enabled the synthesis of SARS-CoV2 proteins in a baculovirus-insect cell platform for various applications. Main body of the abstract The most cloned SARS-CoV-2 protein is the spike protein, which plays a critical role in SARS-CoV-2 infection. It is available in its whole length or as subunits like S1 or the receptor-binding domain (RBD). Non-structural proteins (Nsps), another recombinant SARS-CoV-2 protein generated by the baculovirus expression vector system (BEV), are used in the identification of new medications or the repurposing of existing therapies for the treatment of COVID-19. Non-SARS-CoV-2 proteins generated by BEV for SARS-CoV-2 diagnosis or treatment include moloney murine leukemia virus reverse transcriptase (MMLVRT), angiotensin converting enzyme 2 (ACE2), therapeutic proteins, and recombinant antibodies. The recombinant proteins were modified to boost the yield or to stabilize the protein. Conclusion This review covers the wide application of the recombinant protein produced using the baculovirus expression technology for COVID-19 research. A lot of improvements have been made to produce functional proteins with high yields. However, there is still room for improvement and there are parts of this field of research that have not been investigated yet.

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An easy pipeline for one-step purification of SARS-CoV-2 nucleocapsid protein from insect cell suspension culture

Cited by 7 publications

References 25 publications

Understanding the Role of SARS-CoV-2 ORF3a in Viral Pathogenesis and COVID-19

Understanding the Role of SARS-CoV-2 ORF3a in Viral Pathogenesis and COVID-19

Production of SARS-CoV-2 nucleocapsid protein in Escherichia coli and its characterization

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

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