Yeast-Based SARS-CoV VLP Expression for Soluble Multi-Subunit Production
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Solution Overview
Problem
Current methods for expressing SARS-CoV virus-like proteins face challenges such as insoluble expression and aggregation due to high cysteine and hydrophobicity, making it difficult to produce and purify, especially in prokaryotic systems, and existing eukaryotic expression systems struggle with efficient co-expression of multi-subunit VLPs like S, M, and E proteins for effective vaccine development.
Innovation Solution
The development of a yeast-based expression system using codon optimization and co-expression strategies with episomal and integrative vectors to produce recombinant multi-subunit VLPs of SARS-CoV, specifically targeting S, M, and E proteins, ensuring efficient expression, high yields, and maintaining stoichiometric ratios for effective vaccine production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If SARS-CoV proteins are expressed in prokaryotic systems, then production cost is reduced, but the proteins become insoluble and aggregate due to high cysteine and hydrophobicity
Solution Approach 1:
The patent uses a yeast-based expression system as an intermediary between prokaryotic simplicity and eukaryotic protein folding capability. Yeast provides eukaryotic post-translational modification machinery that resolves the solubility issue while maintaining relative ease of manufacture compared to mammalian cell systems.
Solution Approach 2:
The patent applies codon optimization to adapt the SARS-CoV protein sequences for efficient expression in yeast. By changing the codon usage parameters to match yeast preferences, the system achieves high-level expression of soluble, functional proteins without aggregation.
2Reliability
If eukaryotic expression systems are used to produce soluble SARS-CoV proteins, then protein solubility is improved, but co-expression efficiency of multi-subunit VLPs decreases
Solution Approach 1:
The patent segments the co-expression system into two distinct vector types: integrative vectors for stable, long-term expression of structural proteins (S, M, E) and episomal vectors for flexible, high-level expression of additional components. This segmentation allows each vector type to optimize for its specific function, resolving the co-expression efficiency problem.
Solution Approach 2:
Instead of using a single complex expression system, the patent inverts the approach by using multiple simpler vector systems working in parallel. The integrative and episomal vectors complement each other, with the integrative vector providing stability and the episomal vector providing high-level expression capability.
3Productivity
If multiple episomal vectors are used for co-expression, then gene delivery is improved, but foreign DNA load in cells increases
Solution Approach 1:
The patent segments the genetic material into two categories: integrated DNA (in the yeast genome via integrative vectors) and extrachromosomal DNA (episomal vectors). This segmentation allows the system to deliver multiple genes efficiently while managing the foreign DNA load by distinguishing between stable integrated sequences and transient episomal sequences.
Solution Approach 2:
The patent extracts the high-copy-number expression function into episomal vectors, separating it from the genomic integration requirement. This allows high-level expression of specific genes without requiring multiple genomic integrations, thereby reducing the overall foreign DNA load in the cell genome.
4Productivity
If transmembrane region is deleted to improve expression level, then protein expression increases, but VLP assembly and budding capability is lost
Solution Approach 1:
The patent applies local quality modification by selectively removing only the transmembrane anchor regions from the SARS-CoV proteins while preserving all other functional domains. This localized modification allows the proteins to be expressed at high levels in the secretory pathway while retaining their ability to assemble into VLPs that can bud from the cell surface.
Solution Approach 2:
The patent creates modified copies of the SARS-CoV proteins with deleted transmembrane regions. These copied proteins maintain the essential structural and assembly functions but lack the membrane-anchoring capability, allowing them to be expressed and assembled into VLPs that can be secreted efficiently.
Data Source
AI summary
The present invention relates to expression of SARS-CoV like virus proteins [S, M and E] proteins; recombinant polynucleotides, polypeptides; constructs, virus-like particles (VLPs); immunogenic compositions or vaccines comprising Virus Like Particles (VLPs). Method of producing the VLPs/expressing the multi-subunit virus like proteins and method for co-expression of multi-subunit and virus like proteins (VLPs) are also provided. The present invention also provides strategies, methods, systems, kits and combinations for scalable expression, purification and enhanced production of the virus like proteins of SARS-CoV while maintaining their size range and composition. Such multi-subunit VLPs can be utilized to make immunogenic compositions or vaccines.