Related Experiment Video
Updated: Jul 8, 2026
Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Expression, purification, and refolding of an optimized SARS-CoV-2 receptor binding domain in E. coli
Anamika Biswas1, Arighna Sarkar1, Sreejith Raran-Kurussi1
1Tata Institute of Fundamental Research Hyderabad, 36/P Gopanpally, Serilingampally, Hyderabad, Telangana 500046, India.
Abstract:
The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a significant risk to global health, warranting the formulation of efficient preventive and therapeutic measures to tackle its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein of coronaviruses plays a pivotal role in viral attachment and entry into host cells. The receptor-binding domain (RBD) of the SARS-CoV-2 S protein has demonstrated a robust binding affinity to ACE2 receptors in humans. Consequently, it has become a prime target for therapeutic interventions using antibodies, vaccines, or other designed inhibitors. We engineered an RBD sequence with refined ORF boundaries guided by structural insights, which enabled efficient in vitro refolding. This highlights the critical role of precise expression cassette design in a plasmid, extending beyond conventional parameters such as promoter or fusion tag selection. Using customized refolding procedures, we obtained 10-12 mg of active protein from a one-liter LB culture. The biological activity of the refolded RBD was confirmed by monitoring its interaction with the designed LCB1 miniprotein ligand by surface plasmon resonance, wherein they exhibited significant affinity levels as reflected by their dissociation constants (KD values <10 nM). The resulting RBD could be an ideal target for designing potent COVID-19 antivirals.
Insights
Researchers engineered a SARS-CoV-2 spike protein RBD sequence for efficient refolding and high yield. This active protein demonstrates strong binding affinity, offering a promising target for developing new COVID-19 antivirals.
Area of Science:
- Biochemistry
- Virology
- Structural Biology
Background:
- COVID-19, caused by SARS-CoV-2, requires effective therapeutics.
- The SARS-CoV-2 spike protein's RBD binds to human ACE2 receptors, making it a key target for interventions.
- Efficient production of functional RBD is crucial for developing targeted therapies.
Purpose of the Study:
- To engineer a SARS-CoV-2 RBD sequence for improved in vitro refolding and high-yield production.
- To confirm the biological activity and binding affinity of the refolded RBD.
- To assess the potential of the engineered RBD as a therapeutic target.
Main Methods:
- Engineered RBD sequence with refined ORF boundaries based on structural insights.
- Developed customized refolding procedures for protein expression.
- Utilized surface plasmon resonance (SPR) to confirm biological activity and binding affinity with LCB1 miniprotein ligand.
Main Results:
- Achieved high yield (10-12 mg/L) of active, refolded RBD protein.
- Confirmed significant binding affinity of the RBD to the LCB1 ligand (KD <10 nM) via SPR.
- Demonstrated the importance of precise expression cassette design for protein refolding.
Conclusions:
- The engineered RBD sequence and refolding protocol enable efficient production of biologically active protein.
- The high binding affinity confirms its potential as a target for therapeutic development.
- This work provides a foundation for designing novel COVID-19 antiviral strategies targeting the RBD.
