Single-Chain RBD-Dimer Stabilization via Linker Peptides
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Solution Overview
Problem
Current vaccines and treatments for β-coronaviruses, such as MERS-CoV and SARS-CoV-2, are inadequate, and existing methods for producing RBD-dimer proteins are unstable due to disulfide bond formation, leading to low yields and reduced immunogenicity.
Innovation Solution
The development of a single-chain RBD-dimer protein structure by linking identical or substantially identical RBD-monomer proteins through flexible regions at the N-terminal and C-terminal, using linker sequences like GGS, to stabilize the dimer formation and enhance expression and immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RBD-dimer proteins are produced using traditional disulfide bond formation methods, then dimer structure is achieved, but production stability is poor and yield is low
Solution Approach 1:
The patent divides the RBD-dimer protein into two separate expression boxes, each containing the gene sequence for one RBD monomer. This segmentation allows independent expression and assembly of monomers into dimers, improving production stability and yield by avoiding the limitations of traditional disulfide bond formation methods.
Solution Approach 2:
The patent introduces a linker peptide sequence as an intermediary between the two RBD monomers. This linker facilitates controlled dimer formation through flexible connections rather than relying on unstable disulfide bonds, thereby enhancing both production stability and immunogenicity while maintaining the functional dimer structure.
2Reliability
If traditional RBD-dimer proteins are used, then dimer structure is formed, but immunogenicity is reduced
Solution Approach 1:
The linker peptide acts as an intermediary that enhances immunogenicity by providing flexible connections between monomers, allowing better antigen presentation to the immune system. The linker's flexible nature creates a more immunogenic structure compared to rigid disulfide bonds, while the overall complexity remains manageable through modular design.
Solution Approach 2:
The patent changes the structural parameters of the dimer by introducing flexible linker regions instead of fixed disulfide bonds. This parameter change increases the conformational flexibility and immunogenicity of the protein while maintaining a relatively simple modular structure that is easier to produce and characterize.
3Reliability
If single-chain RBD-dimer is constructed with linker sequences, then expression stability is improved, but structural complexity increases
Solution Approach 1:
The single-chain RBD-dimer is segmented into modular components: two identical RBD monomer sequences separated by a standardized linker peptide. This segmentation approach improves expression stability by allowing independent folding of monomers while the linker provides controlled connectivity, reducing the complexity of expressing and characterizing the full dimer structure.
Data Source
AI summary
The embodiments of the present disclosure relate to antigens of β-coronaviruses, preparation methods and uses thereof. The amino acid sequence of the antigen of the β-coronavirus includes an amino acid sequence arranged in a (A-B)-(A-B) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B) pattern or an amino acid sequence arranged in a (A-B)-(A-B′) pattern or an amino acid sequence arranged in a (A-B)-C-(A-B′) pattern. The antigen of the β-coronavirus has a single-chain dimer structure. A single-chain dirtier expressed according to examples of the present disclosure is stable in content and has excellent immunogenicity as an antigen of a β-coronavirus, and a vaccine prepared by using the single-chain dimer as an antigen of a β-coronavirus can elicit high-titer neutralizing antibodies in mice.