Stabilizing SARS-CoV-2 Spike Protein Mutations for Yeast Display
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Solution Overview
Problem
Current technologies face challenges in stabilizing the prefusion 'up' conformation of SARS-CoV-2 Spike protein, which is crucial for effective vaccine formulations and serological diagnostic testing, while also struggling with low expression yields in recombinant systems and inefficient display on yeast surfaces.
Innovation Solution
Identification and implementation of novel stabilizing mutations in the SARS-CoV-2 Spike protein to enhance its stability in the prefusion 'up' conformation, combined with the development of optimized yeast surface display systems and growth media to facilitate the expression and display of type I viral fusion proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If stabilizing mutations are introduced to the SARS-CoV-2 Spike protein to lock it in the prefusion 'up' conformation, then the conformational stability and vaccine efficacy are improved, but the protein expression yields in recombinant systems deteriorate
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid sequences at specific positions (e.g., P987-988 proline mutations, N331 mutations) to stabilize the prefusion conformation. These sequence parameter changes were screened in yeast display systems to identify mutations that maintain both conformational stability and adequate expression levels, resolving the contradiction between stabilization and productivity.
2Extent of automation
If conventional yeast surface display systems are used to screen Spike protein variants, then the screening process can be performed, but the display efficiency and throughput deteriorate due to inadequate growth and induction conditions
Solution Approach 1:
The patent optimizes yeast display parameters including galactose concentration (0.2-2% w/v), induction temperature (16-37°C), and pH (4.0-8.0) to improve Spike protein display efficiency. These parameter optimizations enable reliable high-throughput screening while maintaining the automated FACS-based workflow.
3Ease of manufacture
If the Spike protein is expressed in mammalian suspension culture systems, then the protein can be produced, but the expression yields deteriorate compared to well-expressing proteins
Solution Approach 1:
The patent segments the full-length Spike protein into ectodomain variants (S-ecto) for expression in yeast systems, and uses targeted mutagenesis to create stabilized versions. This segmentation and stabilization approach enables production in more efficient recombinant systems while maintaining immunogenicity for vaccine applications.
Data Source
AI summary
The invention relates to one or more mutations configured to stabilize the prefusion “up” protomer trimeric Spike protein from SARS-CoV-2. The inventive technology further relates to systems, methods, and compositions to display one or more proteins on the surface of a yeast cell. Specifically, in one embodiment the invention relates to systems, methods, and compositions to display one or more Spike protein from SARS-CoV-2 on the surface of a yeast cell, and more preferably a Spike protein from SARS-CoV-2 stabilized in its prefusion conformation on the surface of a yeast cell.