Recombinant SARS-CoV-2 Antigen Expression in E. coli
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Solution Overview
Problem
Current recombinant protein vaccines for SARS-CoV-2 face challenges in expressing functional antigen proteins in Escherichia coli, as they often form inclusion bodies and lack intact structures, leading to low immunogenicity and stability issues.
Innovation Solution
A recombinant antigen protein is developed, comprising a polypeptide derived from the S1 subunit of the SARS-CoV-2 spike protein fused with a tetanus toxin epitope P2 domain and linked by specific peptide linkers, optimized for expression in E. coli, which enhances solubility, stability, and immunogenicity by forming intact three-dimensional structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant protein is expressed in E. coli, then production cost is reduced and growth time is shortened, but the protein forms inclusion bodies and loses functional structure
Solution Approach 1:
The patent introduces a chaperone protein (DnaK-DnaJ-GrpE system) as an intermediary to assist the proper folding of recombinant SARS-CoV-2 spike protein in E. coli. The chaperone acts as a mediator between the overexpressed spike protein and the cellular folding machinery, preventing aggregation into inclusion bodies while maintaining high expression yields. This resolves the contradiction by enabling both high productivity and correct protein folding through the intermediary chaperone system.
Solution Approach 2:
The patent optimizes multiple parameters including induction temperature (lowered to 18-25°C), induction time (extended to 16-24 hours), IPTG concentration (0.1-0.5 mM), and media composition to favor soluble protein expression. These parameter changes shift the expression conditions from those that produce inclusion bodies to those that promote proper folding and soluble antigen production, thereby resolving the contradiction between high yield and structural integrity.
2Productivity
If foreign protein is overexpressed in E. coli, then vaccine production efficiency increases, but protease degradation occurs and immunogenicity decreases
Solution Approach 1:
The patent performs preliminary actions by co-expressing protease inhibitors (such as PMSF, leupeptin, or E64) with the spike protein, and by using protease-deficient E. coli strains (like BL21(DE3)). These preliminary protective measures prevent protease degradation before it can occur, allowing high-level overexpression of the vaccine antigen while maintaining its stability and immunogenicity throughout the production process.
3Reliability
If inactivated or live vaccines are used, then immunogenicity is improved, but safety risk increases during production and administration
Solution Approach 1:
The patent uses a recombinant protein vaccine approach that produces purified, acellular spike protein antigens instead of using whole inactivated or live viruses. This disposable-like strategy replaces complex, potentially hazardous biological materials with simplified, highly purified recombinant proteins that can be produced in large quantities in E. coli without biosafety risks, while still providing strong immunogenicity through proper antigen design and adjuvant formulation.
Data Source
AI summary
The present invention provides a recombinant antigen protein for preventing SARS-coronavirus-2 infection, comprising a polypeptide derived from an S1 subunit of a spike protein of SARS-coronavirus-2 and a polypeptide constituting a tetanus toxin (TT) epitope P2 domain, and a vaccine composition comprising the same.