Yeast expression and characterization of SARS-CoV N protein

Cao, Shishu; Wang, Haihe; Luhur, Arthur; Wong, Sek‐Man

doi:10.1016/j.jviromet.2005.06.010

Cited by 6 publications

(4 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We observed that during extraction of inclusion bodies, IMAC purification and upon storage at 4 • C, NP exhibited some degradation which was also reported previously (Tang et al, 2005). This is due to the low stability of NP of SARS-CoV since it is the only SARS-CoV protein which contains no Cys residues (Cao et al, 2005). It is also reported that SARS virus looses its infectious capacity at 56 • C and the low stability of the NP may be at least one of the important determinants for the thermosensitivity of SARS virus (Duan et al, 2003).…”

supporting

confidence: 83%

Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene

Das¹,

Suresh²

2006

Journal of Virological Methods

View full text Add to dashboard Cite

The severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) is one of the predominant antigenic protein and the most abundant shed antigen throughout the SARS-CoV infection. This feature makes it a suitable molecular target for diagnostic applications. In this study the full length codon optimized NP gene and its subfragment gene segment was cloned in a bacterial expression vector. The full length NP could be expressed in E. coli at very high level within inclusion bodies. The inclusion bodies were successfully solubilized, purified under denaturing conditions employing IMAC column and refolded. The non-glycosylated NP was used to immunize mice for hybridoma development. The polyclonal antiserum from animals immunized with this recombinant NP protein was found to specifically recognize the NP and its subfragments, thus demonstrating the immunogenic nature of the recombinant protein. The NP antigen or a subfragment could be useful for developing a sensitive serum diagnostic assay to monitor SARS-CoV outbreaks by detecting the early human anti-SARS antibodies. In addition, the availability of the NP fragments could facilitate epitope mapping of anti-NP monoclonals for identifying suitable sandwich pairs.

show abstract

supporting

confidence: 83%

Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene

Das¹,

Suresh²

2006

Journal of Virological Methods

View full text Add to dashboard Cite

show abstract

“…In this study, we explore the possibility of using yeast expression system as an efficient and low-cost option to supplement the bacterial system. Previous reports have demonstrated the feasibility of using the P. pastoris as host to express the SARS-CoV proteins, including the membrane [9] and the nucleocapsid [10,11] protein. Although Lu et al [12] expressed a small fragment of the S1 domain (amino acid residues 251-561) in the P. pastoris, they had not demonstrated the ability of this fragment to specifically pulldown ACE2 from cell lysate.…”

Section: Discussionmentioning

confidence: 99%

Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris

et al. 2008

View full text Add to dashboard Cite

To establish a rapid and economical method for the expression of viral proteins in high yield and purity by Pichia pastoris, the S protein of the SARS-CoV was selected in this study. Six S glycoprotein fragments were expressed in Escherichia coli BL21 and yeast KM71H strains. After purification by affinity chromatography, the protein identities were confirmed by western blot analysis, N-terminal sequencing and mass spectrometry. The proteins expressed in E. coli were low in solubility and bound by GroEL. They still formed soluble aggregates even when the GroEL was removed by urea. The proteins expressed in P. pastoris were relatively soluble. The maximal yield of the RBD reached 46 mg/l with purity greater than 95%. Pull-down assay revealed that ACE2 was specifically captured from cell lysate, indicating that the RBD was biologically active. The glycosylated and deglycosylated RBD was then subjected to SEC and results showed that deglycosylated RBD formed soluble aggregates again. Taken together, pure and biological active RBD of the S protein could be expressed in P. pastoris, and the P. pastoris expression platform will be a good alternative for the expression of viral proteins, in particular, the highly glycosylated surface proteins that mediate the tissue tropism and viral entry.

show abstract

“…Structural studies of SARS-CoV, MERS-CoV, and SARS-CoV-2 SARS-CoV is a coronavirus with a diameter of approximately 100 nm. Coronaviruses are the largest +ssRNA viruses and contain at least 14 ORFs, 16,55 among which five main ORFs encode the replicase (Rep), and the S, E, M, and N proteins. 56 protein combines with viral RNA to form a nucleocapsid, which is involved in the replication of SARS-CoV and is the most abundant protein in virus-infected cells.…”

Section: Epidemiological Analysis and Symptoms Of Sars Mers And Cormentioning

confidence: 99%

Overview of lethal human coronaviruses

Chen

Tian

et al. 2020

Sig Transduct Target Ther

255

236

View full text Add to dashboard Cite

Coronavirus infections of multiple origins have spread to date worldwide, causing severe respiratory diseases. Seven coronaviruses that infect humans have been identified: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. Among them, SARS-CoV and MERS-CoV caused outbreaks in 2002 and 2012, respectively. SARS-CoV-2 (COVID-19) is the most recently discovered. It has created a severe worldwide outbreak beginning in late 2019, leading to date to over 4 million cases globally. Viruses are genetically simple, yet highly diverse. However, the recent outbreaks of SARS-CoV and MERS-CoV, and the ongoing outbreak of SARS-CoV-2, indicate that there remains a long way to go to identify and develop specific therapeutic treatments. Only after gaining a better understanding of their pathogenic mechanisms can we minimize viral pandemics. This paper mainly focuses on SARS-CoV, MERS-CoV, and SARS-CoV-2. Here, recent studies are summarized and reviewed, with a focus on virus-host interactions, vaccine-based and drug-targeted therapies, and the development of new approaches for clinical diagnosis and treatment.

show abstract

Yeast expression and characterization of SARS-CoV N protein

Cited by 6 publications

References 13 publications

Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene

Copious production of SARS-CoV nucleocapsid protein employing codon optimized synthetic gene

Expression of SARS-coronavirus spike glycoprotein in Pichia pastoris

Overview of lethal human coronaviruses

Contact Info

Product

Resources

About