CRM197 Periplasmic Targeting in E. coli Expression
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Solution Overview
Problem
Current methods for producing CRM197, a nontoxic form of diphtheria toxin, face challenges such as low protein abundance and inefficient expression systems, particularly in E. coli, leading to suboptimal yields and improper folding, which hinder its use in vaccine production.
Innovation Solution
A method involving the use of a heterologous signal peptide to target CRM197 to the periplasm of E. coli cells, with a designed cleavage site and specific expression conditions, enables the production of soluble, folded CRM197 at high yields by optimizing secretion and folding processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRM197 is expressed in E. coli using conventional expression systems, then the expression level is low, but the complexity of the expression system is simple
Solution Approach 1:
The invention divides the CRM197 expression system into functional modules: a heterologous signal peptide sequence for periplasmic targeting, a cleavage site sequence for signal peptide removal, and the CRM197 coding sequence. This modular design enables optimized expression while maintaining system simplicity.
Solution Approach 2:
The invention introduces a heterologous signal peptide as an intermediary element that mediates the targeting of CRM197 to the periplasm. This intermediary enables proper localization and folding without requiring complex expression system modifications.
2Quantity of substance
If CRM197 is produced in high quantities, then the protein abundance increases, but the proper folding decreases leading to aggregates
Solution Approach 1:
The invention performs preliminary targeting of CRM197 to the periplasm via a heterologous signal peptide before full-scale production. This preliminary localization ensures proper folding environment is established, allowing high-yield production of correctly folded protein without aggregate formation.
Solution Approach 2:
The invention changes the localization parameter of CRM197 from cytoplasmic to periplasmic by introducing a heterologous signal peptide. This parameter change creates an oxidizing environment favorable for disulfide bond formation and proper folding, enabling high-concentration production of folded protein.
3Productivity
If the signal peptide is retained in the final protein, then the secretion efficiency is high, but the protein purity decreases
Solution Approach 1:
The invention extracts or removes the heterologous signal peptide from the final CRM197 product by incorporating a cleavage site sequence. This allows the signal peptide to perform its secretion function and then be removed, ensuring high protein purity in the final product.
Solution Approach 2:
The heterologous signal peptide is discarded after performing its secretion function. The cleavage site enables selective removal of the signal peptide while recovering the pure CRM197 protein, separating the transient secretion function from the final product.
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 results in high yields of properly folded CRM197, with at least 50% of the protein being soluble and not present in aggregates, effectively addressing the limitations of previous expression systems and enhancing vaccine production capabilities.
Implementation Method 1
CRM197 is fused to a heterologous signal peptide that targets CRM197 to the periplasm of the E. coli cell
Implementation Method 2
the nucleic acid encodes a cleavage site between the signal peptide that targets CRM197 to the periplasm and the CRM197 protein
Implementation Method 3
enables the production of soluble, folded CRM197 at high yields by optimizing secretion and folding processes
Data Source
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AI summary
The present invention provides novel methods of producing diphtheria toxin. In particular, the present invention provides novel methods of producing nontoxic forms of diphtheria toxin, e.g., CRM197. The present invention also provides novel compositions comprising diphtheria toxin or nontoxic forms of diphtheria toxin, e.g., CRM197.