Mutated Malaria CSP Proteins for High-Yield Expression
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Solution Overview
Problem
Current mammalian expression systems yield low levels of Plasmodium falciparum Circumsporozoite Protein (CSP) proteins, posing challenges for malaria vaccine development, with existing vaccines like RTS,S only targeting the pre-erythrocytic stage and lacking essential epitopes for broader immunity.
Innovation Solution
Development of truncated and mutated CSP proteins with increased stability and expression in mammalian cells by mutating specific domains, such as KKNSR to SSNSS or SSNSA, to enhance yield up to 300-fold, allowing for fusion with various epitopes and potential cleavage, and delivery with lipid nanoparticles or adjuvants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type CSP protein is expressed in mammalian cells, then the protein can be produced, but the expression yield is extremely low (50 μg/L)
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid sequences in the CSP protein, particularly in the N-terminal domain. Mutations such as KKNSR to SSNSS or SSNSA directly upstream of the SLGENDD cleavage site, and C5S mutations to prevent dimerization, fundamentally alter the protein's biochemical parameters to achieve 100-300 fold increases in expression yield in mammalian cells.
2Reliability
If full-length CSP protein is used, then all epitopes are present, but expression levels remain low and protein stability is poor
Solution Approach 1:
The patent applies segmentation by dividing the full-length CSP protein into functional domains. Truncated versions retain the immunogenic repeat regions (NANP repeats) and C-terminal domain while removing or modifying the N-terminal domain that causes low expression. This segmentation preserves essential epitopes for immune recognition while eliminating problematic regions, achieving both high expression and vaccine efficacy.
Solution Approach 2:
The patent extracts and removes specific problematic segments from the CSP protein sequence. The N-terminal domain containing the KKNSR motif and free cysteine (C5) that cause low expression and dimerization is taken out or mutated. This extraction eliminates the harmful effects while preserving the immunogenic core regions needed for vaccine function.
3Adaptability or versatility
If CSP protein is expressed in mammalian cells, then eukaryotic expression system advantages are achieved, but expression yields are extremely low compared to other systems
Solution Approach 1:
The patent optimizes parameters for mammalian cell expression by introducing specific mutations that are compatible with eukaryotic translation and folding mechanisms. The SSNSS/SSNSA mutations and C5S modifications improve protein stability and prevent aggregation in mammalian cells, enabling yields of 1-10 mg/L which is 100-300 fold higher than wild-type, while maintaining the advantages of eukaryotic expression systems.
Data Source
AI summary
Mutated and/or truncated malarial circumsporozoite proteins (CSP) and associated nucleic acids that are more stable and highly expressed in mammalian cells are described. The mutated and/or truncated CSP and associated nucleic acids can be expressed to produce malaria vaccine antigens.