SARS MERS, Sf9
Description
Introduction to SARS-MERS S2 Recombinant Protein Produced in Sf9 Cells
The term "SARS MERS, Sf9" refers to recombinant proteins derived from the spike (S) glycoproteins of SARS-CoV and MERS-CoV, produced using the Spodoptera frugiperda (Sf9) insect cell-baculovirus expression system. These proteins are critical for studying coronavirus-host interactions, vaccine development, and therapeutic antibody design. The Sf9 system is favored for its ability to perform post-translational modifications (e.g., glycosylation) while maintaining high protein yield and purity .
Expression System
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Host cells: Sf9 insect cells infected with recombinant baculovirus .
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Construct design:
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Purification: Proprietary chromatographic techniques yield >85% purity .
Biophysical Properties
| Property | MERS-CoV S2 (Sf9) | SARS-CoV S2 (Sf9) |
|---|---|---|
| Molecular mass | 60.7 kDa | ~140 kDa (trimer) |
| Glycosylation | N-linked | N-linked |
| Stability | 4°C (short-term), -20°C (long-term) | Similar, with trimerization domain |
MERS-CoV S2 Subunit
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Domain organization:
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Role in fusion: Mediates viral and host membrane fusion via HR1-HR2 six-helix bundle formation .
SARS-CoV S2 Subunit
Vaccine Development
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Virus-like particles (VLPs): Co-expression of MERS-CoV S, E, and M proteins in Sf9 cells generates VLPs that induce neutralizing antibodies in macaques .
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Immunogenicity: MERS-CoV VLPs elicit robust humoral and cellular immune responses .
Therapeutic Antibody Design
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Nanobodies: Sf9-produced MERS-CoV RBD (residues 367–606) enables isolation of ultrapotent neutralizing nanobodies (e.g., Nb14, IC₅₀ = 0.0014 μg/mL) .
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Epitope mapping: Structural studies reveal conserved regions for cross-neutralizing antibodies .
Comparative Analysis of SARS-CoV and MERS-CoV S Proteins
| Feature | SARS-CoV (Sf9) | MERS-CoV (Sf9) |
|---|---|---|
| Receptor-binding domain | ACE2 | DPP4 |
| S1/S2 cleavage site | Arg667/Ser668 | Arg747/Ser748 |
| RBD location | Residues 318–510 | Residues 367–588 |
| HR1 length | 122 residues | 121 residues |
Data derived from structural and functional studies .
Challenges and Innovations
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Stability issues: Sf9-produced S proteins require glycerol (10%) and PBS (pH 7.4) to prevent aggregation .
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Glycan shielding: N-linked glycans on S2 modulate immune evasion and receptor binding .
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Synergistic therapies: Combining nanobodies (e.g., Nb14 + Nb9) reduces MERS-CoV pseudovirus infection by 2-fold .
Product Specs
The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) has been a concern since April 2012, with cases reported globally. This virus belongs to the coronavirus family, known for causing illnesses ranging from the common cold to severe respiratory syndromes like SARS (severe acute respiratory syndrome). These illnesses can be life-threatening with high mortality rates. MERS-CoV, a novel strain in this family, leads to serious pneumonia and acute respiratory distress, often with a high fatality rate. As of January 27th, 2015, the World Health Organization (WHO) has documented 956 human infections and 351 deaths linked to MERS-CoV. The number of infections caused by this new coronavirus strain is predicted to rise. Similar to other coronaviruses, a large surface spike glycoprotein is crucial to the virus's structure. Situated on the virion's surface, this protein facilitates binding and entry into the host cell. The spike protein is divided into two domains: S1 and S2. The S1 domain is responsible for cell targeting and interaction, while the S2 domain mediates membrane fusion. A receptor binding domain located within the C-terminal of the S1 domain is a key focus for vaccine research and serves as a target for diagnostic development.
Recombinant SARS MERS protein, expressed in Sf9 insect cells using a baculovirus system, is a single, glycosylated polypeptide chain. This protein consists of 1285 amino acids (spanning from positions 18 to 1296), resulting in a molecular weight of 141.6 kDa. A 6-amino acid His-tag is fused to the C-terminus of the SARS MERS protein to facilitate purification, which is achieved through proprietary chromatographic methods.
The provided SARS MERS solution has a concentration of 0.25 mg/ml. It is formulated in a solution containing 10% glycerol and Phosphate-Buffered Saline (PBS) with a pH of 7.4.
For optimal storage, keep the SARS MERS protein vial at 4°C if it will be fully used within 2 to 4 weeks. For extended storage, it is recommended to freeze the protein at -20°C. To further enhance stability during long-term storage, the addition of a carrier protein such as 0.1% HSA (human serum albumin) or BSA (bovine serum albumin) is advisable. It is important to avoid repeated cycles of freezing and thawing to maintain protein integrity.
The purity of this protein is greater than 85%, as determined by SDS-PAGE analysis.
Middle East respiratory syndrome coronavirus, Human betacoronavirus 2c EMC/2012, MERS-CoV, MERS, MERSCoV SP, Spike glycoprotein, S glycoprotein, S, Spike protein, E2, Peplomer protein
Sf9, Baculovirus cells.
YVDVGPDSVK SACIEVDIQQ TFFDKTWPRP IDVSKADGII YPQGRTYSNI TITYQGLFPY QGDHGDMYVY SAGHATGTTP QKLFVANYSQ DVKQFANGFV VRIGAAANST GTVIISPSTS ATIRKIYPAF MLGSSVGNFS DGKMGRFFNH TLVLLPDGCG TLLRAFYCIL EPRSGNHCPA GNSYTSFATY HTPATDCSDG NYNRNASLNS FKEYFNLRNC TFMYTYNITE DEILEWFGIT QTAQGVHLFS SRYVDLYGGN MFQFATLPVY DTIKYYSIIP HSIRSIQSDR KAWAAFYVYK LQPLTFLLDF SVDGYIRRAI DCGFNDLSQL HCSYESFDVE SGVYSVSSFE AKPSGSVVEQ AEGVECDFSP LLSGTPPQVY NFKRLVFTNC NYNLTKLLSL FSVNDFTCSQ ISPAAIASNC YSSLILDYFS YPLSMKSDLS VSSAGPISQF NYKQSFSNPT CLILATVPHN LTTITKPLKY SYINKCSRLL SDDRTEVPQL VNANQYSPCV SIVPSTVWED GDYYRKQLSP LEGGGWLVAS GSTVAMTEQL QMGFGITVQY GTDTNSVCPK LEFANDTKIA SQLGNCVEYS LYGVSGRGVF QNCTAVGVRQ QRFVYDAYQN LVGYYSDDGN YYCLRACVSV PVSVIYDKET KTHATLFGSV ACEHISSTMS QYSRSTRSML KRRDSTYGPL QTPVGCVLGL VNSSLFVEDC KLPLGQSLCA LPDTPSTLTP RSVRSVPGEM RLASIAFNHP IQVDQLNSSY FKLSIPTNFS FGVTQEYIQT TIQKVTVDCK QYVCNGFQKC EQLLREYGQF CSKINQALHG ANLRQDDSVR NLFASVKSSQ SSPIIPGFGG DFNLTLLEPV SISTGSRSAR SAIEDLLFDK VTIADPGYMQ GYDDCMQQGP ASARDLICAQ YVAGYKVLPP LMDVNMEAAY TSSLLGSIAG VGWTAGLSSF AAIPFAQSIF YRLNGVGITQ QVLSENQKLI ANKFNQALGA MQTGFTTTNE AFQKVQDAVN NNAQALSKLA SELSNTFGAI SASIGDIIQR LDVLEQDAQI DRLINGRLTT LNAFVAQQLV RSESAALSAQ LAKDKVNECV KAQSKRSGFC GQGTHIVSFV VNAPNGLYFM HVGYYPSNHI EVVSAYGLCD AANPTNCIAP VNGYFIKTNN TRIVDEWSYT GSSFYAPEPI TSLNTKYVAP QVTYQNISTN LPPPLLGNST GIDFQDELDE FFKNVSTSIP NFGSLTQINT TLLDLTYEML SLQQVVKALN ESYIDLKELG NYTYYNKWPH HHHHH
Q&A
What are the fundamental structural differences between SARS-CoV, MERS-CoV, and SARS-CoV-2?
This structural variation is particularly significant in the receptor-binding domains (RBDs). Despite SARS-CoV and SARS-CoV-2 S1 units sharing approximately 78% sequence similarity, they display robust secondary structure differences, particularly in their protein/receptor domains. These structural distinctions contribute to the different receptor-binding affinities and transmissibility profiles of these viruses .
How do receptor binding mechanisms differ between SARS-CoV, MERS-CoV, and SARS-CoV-2?
The three coronaviruses utilize different cellular receptors and binding mechanisms:
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MERS-CoV: The spike glycoprotein targets dipeptidyl peptidase 4 (DPP4). Specifically, the receptor-binding subdomain of MERS-CoV RBD interacts with DPP4 β-propeller but not its intrinsic hydrolase domain .
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SARS-CoV and SARS-CoV-2: Both bind to angiotensin-converting enzyme 2 (ACE2), but SARS-CoV-2 demonstrates higher binding affinity. Crystal structure analysis shows that while SARS-CoV and SARS-CoV-2 RBDs share high structural similarity in their core subdomains, they diverge notably in their receptor-binding subdomains .
These differences in receptor recognition mechanisms directly impact host tropism, transmissibility, and pathogenesis of the respective viruses.
What is the Sf9 cell system and how is it applied in coronavirus research?
The Sf9 cell system is derived from Spodoptera frugiperda (fall armyworm) and serves as a fundamental component of the baculovirus expression vector system (BEVS). In coronavirus research, this system offers several methodological advantages:
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Sf9 cells are used to express recombinant proteins, including coronavirus spike proteins and receptor binding domains, with proper folding and post-translational modifications .
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The BEVS/Sf9 system enables high-level expression of viral proteins that maintain their biological activity, making it valuable for structural studies and vaccine development .
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For coronavirus vaccine development specifically, Sf9 cells have been utilized to produce recombinant COVID-19 vaccines expressing the SARS-CoV-2 spike protein receptor binding domain (S-RBD) .
The system's safety profile is enhanced because baculoviruses only parasitize invertebrates and their products are highly safe to mammals, making this expression system particularly valuable for vaccine production .
How do SARS, MERS, and COVID-19 compare epidemiologically?
These three coronavirus diseases exhibit distinct epidemiological profiles:
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Severity spectrum: COVID-19 cases range from mild to severe, while SARS and MERS cases typically present with more severe clinical manifestations .
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Transmissibility: COVID-19 demonstrates higher transmissibility than both SARS and MERS, contributing to its pandemic spread .
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Mortality rates: MERS has the highest mortality rate among the three, followed by SARS, with COVID-19 having a comparatively lower but still significant mortality rate .
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Geographic distribution: While COVID-19 has achieved global pandemic status, MERS has remained largely confined to the Middle East and Arabian Peninsula, and SARS primarily affected Asia and North America during its outbreak .
All three diseases are caused by zoonotic coronaviruses, with evidence suggesting transmission between animals and humans as confirmed by WHO .
What methodological approaches are used to determine secondary structures of coronavirus spike proteins?
Researchers employ several complementary techniques to elucidate the secondary structures of coronavirus spike proteins:
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Infrared (IR) spectroscopy analysis: This technique allows researchers to identify and quantify secondary structure elements by analyzing the amide I band absorption. Each secondary structure element (α-helix, β-sheet, β-turn, random coil) exhibits characteristic absorption bands. The area of each absorption band is proportional to the relative amount of the corresponding secondary structure, enabling percentage estimation through the ratio of integrated intensity .
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X-ray crystallography: This method is used to determine high-resolution three-dimensional structures of RBDs alone or in complex with their receptors. For example, the MERS-CoV RBD bound to human DPP4 was resolved at 3.0 Å resolution, revealing the core and receptor-binding subdomains of the viral protein .
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Chimeric protein design: To facilitate crystallization of challenging viral proteins, researchers design chimeric constructs. For example, a chimeric RBD using the core from SARS-CoV RBD as a crystallization scaffold and the receptor-binding motif (RBM) from the target virus enables structural studies of otherwise difficult-to-crystallize proteins .
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Sequence alignment and structural bioinformatics: Comparative analyses of protein sequences combined with structural predictions help identify critical residues and motifs involved in receptor binding .
How effective are heterologous boosting strategies with Sf9 cell-derived vaccines compared to homologous approaches?
Recent clinical trials provide evidence for the superior effectiveness of heterologous boosting with Sf9 cell-derived vaccines:
In a controlled trial with 85 adult participants who had previously received inactivated vaccines, a heterologous boost with the Sf9 cells recombinant vaccine demonstrated significant advantages over homologous boosting with CoronaVac:
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Immunogenicity: The geometric mean titer (GMT) of binding antibodies to the receptor-binding domain of prototype SARS-CoV-2 on day 28 post-booster was dramatically higher with Sf9 cells vaccine (100,683.37) compared to CoronaVac (9,451.69), p < 0.001 .
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Neutralizing antibody response: GMTs of neutralizing antibodies against pseudo SARS-CoV-2 viruses increased by 22.23–75.93 folds from baseline after Sf9 cells boosting, compared to only 3.29–10.70 folds with CoronaVac. Similar superior performance was observed against live viruses (68.18–192.67 folds vs. 19.67–37.67 folds) .
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Cellular immunity: A more robust Th1 cellular response was observed with the Sf9 cells booster (mean IFN-γ+ spot-forming cells per 2 × 105 PBMCs: 26.66 vs. 13.59) .
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Protective effectiveness: Heterologous boosting with the Sf9 cells vaccine demonstrated approximately twice the protective effectiveness against symptomatic COVID-19 compared to homologous boosting (68.18% vs. 36.59%, p = 0.004) .
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Safety profile: The Sf9 cells vaccine showed a comparable or potentially better safety profile with a post-booster adverse events rate of 20.45% compared to 31.71% for CoronaVac (p = 0.279) .
What are the critical residues in receptor binding domains that determine host tropism and binding affinity in coronaviruses?
Critical residues in coronavirus RBDs play decisive roles in determining receptor specificity, binding affinity, and consequently, host tropism:
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MERS-CoV: Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are critical for viral binding to DPP4 and entry into target cells. These residues form specific interactions with the β-propeller domain of DPP4 .
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SARS-CoV: A critical arginine residue on the side loop of the SARS-CoV RBM forms a strong salt bridge with ACE2. Additionally, another arginine in the core structure interacts with glycan. The variable loop between two disulfide-bond-forming cysteines in the ACE2-binding ridge contains residues crucial for receptor interaction .
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SARS-CoV-2: The RBD of SARS-CoV-2 exhibits structural differences in the receptor-binding subdomain compared to SARS-CoV, despite high similarity in the core subdomain. These differences, particularly in the extended β-sheets of SARS-CoV-2, contribute to its higher binding affinity for ACE2 .
What protein expression and purification protocols are most effective for coronavirus structural studies?
For high-quality coronavirus protein expression and purification, researchers should consider the following methodological approach:
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Expression system selection: The Sf9 insect cell/baculovirus system has proven effective for expressing coronavirus spike proteins and RBDs with proper folding and post-translational modifications .
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Protein design considerations:
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Purification protocol:
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Validation methods:
This systematic approach ensures the production of high-quality viral proteins suitable for structural studies, binding assays, and vaccine development.
How do the advantages of insect cell-based protein expression systems compare to other platforms for coronavirus vaccine development?
The baculovirus expression vector system (BEVS) using Sf9 insect cells offers several distinct advantages for coronavirus vaccine development:
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Safety profile: Baculoviruses only parasitize invertebrates, making their products highly safe for mammals – a critical consideration for vaccine development .
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Expression efficiency: BEVS facilitates high-level expression of recombinant proteins, enabling efficient production of viral antigens .
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Protein quality: The system correctly folds and translates recombinant proteins with appropriate post-translational modifications, preserving biological activity crucial for eliciting protective immune responses .
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Established regulatory acceptance: BEVS has already been applied in approved vaccines for human use, including influenza vaccines, demonstrating regulatory precedent .
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Immunogenicity benefits: Vaccines expressed in insect cells may confer unique immunogenicity advantages. As demonstrated in clinical trials, the Sf9 cells recombinant COVID-19 vaccine with aluminum hydroxide adjuvant showed strong immunogenicity even with a traditional adjuvant, possibly attributable to the insect cell expression system .
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Broader population targeting: Recombinant subunit vaccines produced in insect cells potentially target a wider population range, with enhanced safety profiles for individuals with low immunity, the elderly, and children .
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Logistical advantages: These vaccines typically offer easier storage and logistics compared to some other vaccine platforms .
Product Science Overview
The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) has highlighted the importance of understanding the mechanisms behind viral entry into host cells. Both viruses belong to the betacoronavirus genus and share similarities in their spike (S) proteins, which play a crucial role in mediating viral entry by binding to host cell receptors.
The spike protein is a large, trimeric glycoprotein that protrudes from the viral surface. It is composed of two subunits: S1 and S2. The S1 subunit contains the receptor-binding domain (RBD), which is responsible for binding to the host cell receptor, while the S2 subunit facilitates membrane fusion, allowing the viral genome to enter the host cell.
Recombination is a common phenomenon among coronaviruses, leading to the exchange of genetic material between different viral strains. This process can result in the emergence of new viral variants with altered properties, including changes in receptor binding and host range. In the case of SARS-CoV and MERS-CoV, recombination events have been observed in the RBD of the spike protein, which can significantly impact the virus’s ability to infect host cells .
SARS-CoV primarily uses angiotensin-converting enzyme 2 (ACE2) as its entry receptor, while MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4). However, some studies suggest that recombination events between these viruses could potentially lead to the emergence of new variants capable of using alternative receptors, thereby expanding their host range .
Understanding the recombination events and structural changes in the spike protein is crucial for the development of effective vaccines. Recombinant spike proteins, which are engineered to mimic the native structure of the viral spike, are being investigated as potential vaccine candidates. These recombinant proteins can elicit strong immune responses, providing protection against viral infection .
