coli (RBD-1) lacks the disulfide bond formation and glycosylation. However, RBD of SARS-CoV-2 expressed in E. coli and has been used as a cost-effective antigen for worldwide serological testing. Moreover, RBD (N318-V510) of SARS-CoV-2 S protein was expressed by E. Some studies suggest that RBD of SARS-CoV S protein expressed by E. coli is one of the most popular bacterial hosts for heterologous protein expression. Bacterial expression systems for heterologous protein expression have the advantages of easy use, low cost, short generation times, and scalability. However, the low expression yield and high cost of RBD could not meet the need of the therapeutics and vaccine development. RBD has been expressed in the eukaryotic cell expression systems, including baculovirus-insect cells, yeast cells, and mammalian cells (e.g. RBD has a central twisted antiparallel β-sheet formed by five strands decorated with secondary structure elements and loops.
The glycan moieties have a relevant role in the in vivo protein folding process, stability, and immunogenicity of RBD. N-glycosylation and O-glycosylation were both observed by analysis of RBD. The apparent molecular mass of RBD was determined to be ∼34 kDa, whereas that of the RBD amino acid sequence alone was ∼27 kDa. RBD contains 220 amino acid residues with nine cysteine residues and two N -glycosylation sites. Moreover, an advantage of the RBD-based vaccine is its ability to minimize the host immunopotentiation. Thus, RBD of SARS-CoV-2 S protein is an appealing antigen for vaccine development, which elicits most neutralizing antibodies during SARS-CoV-2 infection. In addition, it can bind to a host receptor, angiotensin-converting enzyme 2 (ACE2) through the receptor-binding domain (RBD). It serves as a target for development of antibodies, entry inhibitors and vaccines. S protein plays the most important roles in viral attachment, fusion, and entry. SARS-CoV-2 contains four structural proteins, including spike (S), envelope, membrane and nucleocapsid proteins. There is an urgent quest to develop effective therapeutics and preventive vaccines against SARS-CoV-2. COVID-19 prominently affects the respiratory tract, with the initial symptoms of common cold, fever, dry cough, fatigue, nasal congestion, sore throat, and diarrhea to severe pneumonia. COVID-19 is now widespread around the globe and spreads readily with an exponential increase in recent days. SARS-CoV-2 is responsible for a disruptive worldwide viral pandemic and a severe respiratory disease known as COVID-19. SARS-CoV-2 belongs to the β-coronavirus genus, which is a member of the SARS-related coronavirus. Fourier-transform infrared spectroscopy.Thus, RBD-1 was expected to apply in the vaccine development, screening drugs and virus test kit. RBD-1 could strongly bind ACE2 with a dissociation constant (K D) of 2.98 × 10 –8 M. In particular, the major β-sheet content of RBD-1 was almost unaltered. The secondary structure and tertiary structure of RBD-1 were largely maintained without glycosylation.
RBD-1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD-2). coli and purified by a Ni Sepharose Fast Flow column. In the present study, RBD-1 was expressed by E. coli (RBD-1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. RBD expressed in mammalian cells suffers from low expression yield and high cost. RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines.
Spike protein of SARS-CoV-2 mediates viral entry into host cells by binding ACE2 through the receptor-binding domain (RBD). SARS-CoV-2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID-19.
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