Reduced Genome E. coli Phospholipase Secretion Yield
Solution Overview
Problem
Current methods for producing recombinant proteins in E. coli, such as CRM197 and single-chain antibodies, are expensive, time-consuming, and face challenges like low yields, toxicity issues, and complex purification processes due to insoluble protein accumulation and host stress.
Innovation Solution
Co-expressing an endogenous E. coli phospholipase with the recombinant protein and using a reduced genome E. coli strain to target the protein to the periplasm, where it can be extracted into the culture medium using a specific buffer, simplifying purification and increasing yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional fed-batch fermentation is used to produce recombinant proteins in E. coli, then production capacity is maintained, but the process becomes expensive, time-consuming, and requires complex purification procedures resulting in loss of yield
Solution Approach 1:
The patent extracts the recombinant protein directly from the E. coli culture medium by exploiting the natural secretion capability of certain E. coli strains. The protein is secreted into the extracellular medium where it can be easily harvested through simple filtration or centrifugation, eliminating the need for complex intracellular purification procedures and significantly reducing process complexity while maintaining high yield
Solution Approach 2:
The patent uses a secretory signal sequence as an intermediary mechanism to direct the recombinant protein from the cytoplasm through the cell membrane into the extracellular medium. This signal sequence acts as a mediator that enables automatic protein secretion without requiring complex purification equipment or procedures
2Productivity
If CRM197 is produced in alternative hosts such as Pseudomonas fluorescens or E. coli, then production capacity increases, but residual toxicity causes significant physiological stress in the host, limiting production
Solution Approach 1:
The patent converts the potential harm of CRM197 toxicity into a benefit by using a controlled secretion system. The toxin is produced but immediately secreted into the extracellular medium where it cannot harm the host cells. The host's own secretion machinery is harnessed to protect it from the toxic effects while maintaining high production capacity
Solution Approach 2:
The patent segments the production process by separating protein synthesis (intracellular) from protein accumulation (extracellular). The toxin is synthesized within protected host cells but is continuously exported to the external environment, dividing the harmful effect from the production site and allowing sustained high-level expression
3Productivity
If recombinant protein is produced in E. coli, then production capacity is achieved, but the protein accumulates in an insoluble form (inclusion bodies) further reducing yield and complicating downstream purification
Solution Approach 1:
The patent extracts the protein from the problematic intracellular environment where inclusion bodies form and places it directly into the extracellular medium. By secreting the protein outside the cell, it remains in a soluble, native conformation suitable for direct purification without requiring complex solubilization and refolding procedures
Solution Approach 2:
The patent changes the environmental parameters by moving the protein from the intracellular milieu (high molecular crowding, reducing environment) to the extracellular medium (different pH, ionic strength, and oxidation conditions). This parameter change promotes proper folding and solubility, preventing inclusion body formation while maintaining high yield
4Productivity
If E. coli hosts are used for extended continuous fermentation, then production efficiency increases, but activation of cryptic prophage, insertion elements and stress-induced systems causes rapid and total loss of production
Solution Approach 1:
The patent converts the stress response that normally triggers prophage activation and plasmid loss into a beneficial secretion response. By engineering the expression system to exploit stress-induced secretion pathways, the same physiological stress that would normally cause production failure is redirected to enhance protein export and maintain production stability during continuous fermentation
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 significantly enhances protein yield and simplifies the purification process, allowing for continuous fermentation and high-yield production of difficult-to-produce proteins like CRM197 and single-chain antibodies, overcoming traditional production challenges.
Implementation Method 1
co-expressing an endogenous E. coli phospholipase with the recombinant protein
Implementation Method 2
contacting the E. coli host strain with an extraction buffer following induction of the recombinant protein and phospholipase
Data Source
AI summary
Provided herein are E. coli host strains with improved capacity for producing recombinant proteins.