Soluble Recombinant Plasmodium falciparum CSP Expression
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Solution Overview
Problem
Current methods for expressing recombinant Plasmodium falciparum circumsporozoite proteins in E. coli face challenges due to high AT content, rare codons, and complex protein structure, leading to insoluble protein expression and the need for extensive refolding, which complicates the production of a functional and pure vaccine antigen.
Innovation Solution
Development of novel recombinant Plasmodium falciparum circumsporozoite proteins with optimized nucleotide sequences and expression vectors for E. coli, allowing for the production of soluble, high-purity proteins without denaturing or refolding, using bacterial cells like E. coli SHUFFLE™ strain, and incorporating expression tags for efficient purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant Plasmodium falciparum circumsporozoite proteins are expressed in E. coli using current methods, then protein production is achieved, but the proteins become insoluble and require extensive refolding
Solution Approach 1:
The patent applies parameter changes by optimizing the nucleotide sequence of the circumsporozoite protein gene for E. coli expression. This includes modifying codon usage to match E. coli preferences, adjusting GC content, and optimizing other sequence parameters to enable soluble protein expression without refolding requirements
Solution Approach 2:
The patent creates an optimized copy of the circumsporozoite protein gene with modified nucleotide sequences that maintain the amino acid sequence while improving expression characteristics in E. coli, resulting in soluble protein production
2Stability of the object's composition
If high AT content and rare codons are present in the nucleotide sequence, then the native gene structure is maintained, but protein expression in E. coli is compromised
Solution Approach 1:
The patent changes the nucleotide sequence parameters including codon usage frequency, GC content, and other sequence characteristics to optimize for E. coli expression while maintaining the encoded amino acid sequence, thereby resolving the conflict between native structure and expression productivity
3Productivity
If extensive refolding is required for the expressed protein, then protein production is achieved, but manufacturing complexity and time increase
Solution Approach 1:
The patent performs preliminary optimization of the gene sequence before expression to ensure the protein is produced in soluble form directly, eliminating the need for subsequent refolding steps and reducing manufacturing time
4Productivity
If the protein is expressed as insoluble, then production is achieved, but purity and functionality for vaccine use are reduced
Solution Approach 1:
The patent optimizes expression parameters through nucleotide sequence modification to produce soluble, properly folded protein directly in E. coli, achieving high purity suitable for vaccine applications without extensive purification and refolding procedures
Data Source
AI summary
The present invention provides novel nucleotide sequence and other constructs used for expression of novel recombinant P. falciparum circumsporozoite proteins in bacterial cells such as E. coli. Processes are provided for producing a soluble recombinant P. falciparum CSP from E. coli. Methods to produce a human-grade, highly immunogenic anti-malaria vaccine based on CSP are shown. The novel recombinant P. falciparum circumsporozoite protein by itself or in combination with other malaria antigens or adjuvants can form the basis of an effective malaria vaccine.