Salmonid Alphavirus E2 Protein Refolding for Vaccine Immunogenicity
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Solution Overview
Problem
Current bacterial expression systems for producing Salmonid Alphavirus E2-protein result in low immunogenicity and the formation of undesirable inclusion bodies, making it difficult to induce effective protection against Salmonid Alphavirus infection in fish.
Innovation Solution
The E2-protein is expressed in a bacterial system, denatured in urea, and refolded in the presence of L-arginine to produce a soluble and highly immunogenic form capable of inducing protection in fish, overcoming the limitations of inclusion body formation and low immunogenicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If E2-protein is expressed in a bacterial expression system, then large amounts of protein can be produced inexpensively, but the protein forms inclusion bodies and has low immunogenicity
Solution Approach 1:
The patent applies preliminary action by performing denaturation of the inclusion bodies before refolding. The E2-protein is first denatured in 6M urea to unfold the aggregated inclusion bodies, then refolded in the presence of L-arginine to restore proper conformation. This preliminary denaturation step enables subsequent successful refolding and restores immunogenicity while maintaining the productivity benefits of bacterial expression
Solution Approach 2:
The patent uses L-arginine as an intermediary substance during the refolding process. L-arginine acts as a mediator that facilitates proper protein folding by preventing aggregation and promoting correct disulfide bond formation. This intermediary agent enables the transformation from insoluble inclusion bodies to soluble, immunogenic protein without changing the bacterial expression system
2Reliability
If E2-protein is isolated from the virus, then the protein can be used for vaccine preparation, but the process is time-consuming and expensive
Solution Approach 1:
The patent uses copying by expressing the E2-protein gene recombinantly in bacterial systems. Instead of isolating the protein from the actual virus (which is time-consuming and expensive), the gene encoding E2-protein is inserted into bacterial expression vectors and produced in E. coli. This copying approach through recombinant expression dramatically reduces production time and cost while maintaining protein quality for vaccine applications
3Stability of the object's composition
If inclusion bodies are denatured and refolded using conventional methods, then solubility is improved, but immunogenicity remains insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the chemical environment during refolding. Specifically, it changes the presence of L-arginine (at concentrations of 0.1-1.0M) and controls urea concentration during the refolding process. These parameter changes in the refolding buffer composition enable proper protein folding and disulfide bond formation, resulting in both improved solubility and restored immunogenicity that conventional refolding methods fail to achieve
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The refolded SAV E2-protein provides at least 75% relative percentage protection against Salmonid Alphavirus infection when administered in low amounts with standard adjuvants, significantly improving vaccine efficacy compared to inclusion body-based vaccines.
Implementation Method 1
denatured in urea
Implementation Method 2
refolded in the presence of L-arginine
Data Source
AI summary
The present invention relates to Salmonid alphavirus E2 -protein expressed in a bacterial expression system, its use in medicine, vaccines comprising such protein, methods for the preparation of such proteins and methods for the preparation of vaccines comprising such proteins.