CRM197 Protein Periplasmic Secretion Folding
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Solution Overview
Problem
Current methods for producing recombinant CRM197 protein in bacterial hosts often result in misfolded or insoluble forms, which can compromise the protein's immunogenicity and stability.
Innovation Solution
A method involving the use of an expression plasmid with a CRM197 nucleotide sequence and a secretion signal sequence, such as pectate lyase B (PelB), to direct the CRM197 protein to the periplasmic space of E. coli, where it can fold correctly and form appropriate disulfide bonds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRM197 protein is produced in bacterial hosts using conventional methods, then protein production is achieved, but the protein becomes misfolded or insoluble
Solution Approach 1:
The patent introduces a secretion signal sequence as an intermediary element that directs the CRM197 protein to the periplasmic space of E. coli. This mediator enables the protein to access an environment conducive to proper folding and disulfide bond formation, resolving the contradiction between production and proper folding/solubility
Solution Approach 2:
The patent changes the cellular location parameter where the protein is produced, directing it to the periplasmic space rather than the cytoplasm. This parameter change creates an oxidizing environment that facilitates correct disulfide bond formation and protein folding, thereby improving both manufacturing precision and reliability
2Productivity
If CRM197 protein is produced at high levels, then productivity increases, but protein misfolding and insolubility worsen
Solution Approach 1:
The secretion signal sequence acts as a mediator that facilitates proper protein folding even at high expression levels by directing the protein to the periplasmic space where folding conditions are more favorable, thus maintaining manufacturing precision while achieving high productivity
3Ease of manufacture
If CRM197 protein is produced in cytoplasm, then production is simple, but disulfide bond formation is impaired
Solution Approach 1:
The secretion signal sequence serves as an intermediary that automatically directs the protein to the periplasmic space without requiring complex manual intervention. This maintains ease of manufacture while ensuring correct disulfide bond formation through the oxidizing environment of the periplasm
Solution Approach 2:
The protein sequence itself contains the secretion signal that enables it to self-direct to the appropriate cellular compartment for proper folding and disulfide bond formation, eliminating the need for complex external manipulation while maintaining production simplicity
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
This approach enables the production of properly folded, soluble CRM197 protein with correct disulfide linkages, enhancing its immunogenicity and stability, and facilitating its use in vaccine development.
Implementation Method 1
A method involving the use of an expression plasmid with a CRM197 nucleotide sequence and a secretion signal sequence, such as pectate lyase B (PelB), to direct the CRM197 protein to the periplasmic space of E. coli, where it can fold correctly and form appropriate disulfide bonds.
Data Source
AI summary
The present invention relates to a method for producing CRM197 recombinant protein in cells. The method comprises culturing a cell comprising an expression plasmid with a polynucleotide and inducing expression of the CRM197 protein.