CRM197 Periplasmic Expression with OmpC Signal Targeting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing CRM197 protein are not industrially profitable and lack a stable, high-yield production process, particularly in E. coli strains, leading to insoluble forms that are difficult to convert into soluble forms effectively.
Innovation Solution
An expression system in E. coli BL21 T7XB strain using a low copy number origin of replication, medium strength ribosome binding site, and an inducible promoter sequence, combined with a signal peptide for periplasmic targeting, along with controlled culture conditions, to achieve stable and high expression levels of CRM197.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRM197 is produced in E. coli BL21 wild-type strain, then production capacity is achieved, but expression levels remain unstable and low
Solution Approach 1:
The patent modifies multiple parameters of the expression system including using a low copy number origin of replication (p15A) instead of high copy number, employing a medium strength ribosome binding site with optimized sequence (AGGAGGA), and using a moderate strength promoter (T7lac). These parameter changes collectively achieve stable and reproducible high-level expression of CRM197 in E. coli BL21 T7XB strain, resolving the contradiction between productivity and reliability.
2Productivity
If high expression levels of CRM197 are achieved in E. coli, then production yield increases, but the protein forms insoluble aggregates
Solution Approach 1:
The patent employs a combination of expression parameters including low copy number plasmid (p15A origin), medium strength ribosome binding site (AGGAGGA), and moderate promoter strength (T7lac) to achieve balanced expression levels that prevent protein aggregation. This parameter optimization enables production of soluble CRM197 at high yields, resolving the contradiction between productivity and manufacturing precision.
3Manufacturing precision
If CRM197 is secreted into periplasmic space, then soluble form is obtained, but expression levels are difficult to stabilize
Solution Approach 1:
The patent uses a specific combination of expression parameters including the T7lac promoter, p15A low copy number origin, and AGGAGGA ribosome binding site, together with the OmpC signal peptide for periplasmic targeting. This optimized parameter set achieves both high solubility in periplasmic form and stable, reproducible expression levels across multiple fermentations, resolving the contradiction between manufacturing precision and reliability.
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 method provides a high and stable expression yield of CRM197 in the periplasmic space, enabling efficient purification and commercial use, suitable for vaccine and therapeutic development.
Implementation Method 1
The protein CRM197 and signal sequence, OmpC, enabling the targeting of protein CRM197 towards the periplasmic space
Data Source
AI summary
The present invention relates to a method for producing a periplasmic form of SEQ ID NO: 12, to the expression vector encoding SEQ ID NO: 12 and signal sequence SEQ ID NO: 2, enabling the targeting of SEQ ID NO: 12 towards the periplasmic space, as well as to the strain transformed by the expression vector.