Mutated PAL Protein Expression for Recombinant Protein Release
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Solution Overview
Problem
Existing bacterial strains used for recombinant protein production, such as E. coli mutants with defects in the Tol-Pal complex or overexpression of proteins like TolAIII, cause significant cell lysis and membrane destabilization, leading to reduced viability and contamination, making them unsuitable for high-density fermentation and complex protein production.
Innovation Solution
A bacterial strain expressing a mutated peptidoglycan-associated lipoprotein (Pal protein) lacking a membrane anchor, which permeabilizes the outer membrane to release recombinant proteins without causing severe cell lysis, using a functional promoter to control the expression of the mutated Pal protein and recombinant protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If tol- and pal-deletion mutants are used to release periplasmic proteins into the fermentation medium, then protein release is achieved, but cell viability is severely compromised and growth defects occur
Solution Approach 1:
The invention extracts only the essential function of the Pal protein (facilitating protein release from periplasm to medium) while removing the harmful membrane-destabilizing aspect. This is achieved by deleting the membrane anchor domain of Pal, leaving a soluble version that can promote protein release without compromising cell envelope integrity.
Solution Approach 2:
The soluble Pal protein acts as an intermediary that mediates protein release without directly compromising membrane integrity. It facilitates the transfer of periplasmic proteins to the extracellular medium through a mechanism that does not involve direct membrane disruption, thus maintaining cell viability while achieving protein secretion.
2Productivity
If TolAIII is overexpressed to destabilize the cell envelope and increase protein release, then more protein is released into the medium, but cell viability decreases by a factor of 1000
Solution Approach 1:
The invention extracts only the essential function of the Pal protein (facilitating protein release from periplasm to medium) while removing the harmful membrane-destabilizing aspect. This is achieved by deleting the membrane anchor domain of Pal, leaving a soluble version that can promote protein release without compromising cell envelope integrity.
Solution Approach 2:
The invention converts the potentially harmful membrane-destabilizing activity of full-length Pal into a beneficial soluble form. By removing the membrane anchor, the protein's ability to facilitate release is preserved while its capacity to cause membrane damage is eliminated, turning a harmful function into a beneficial one.
3Productivity
If bacteriocin release proteins are overexpressed to degrade the outer membrane and release proteins, then protein release is achieved, but severe membrane defects and cell lysis occur
Solution Approach 1:
The invention extracts only the essential function of the Pal protein (facilitating protein release from periplasm to medium) while removing the harmful membrane-destabilizing aspect. This is achieved by deleting the membrane anchor domain of Pal, leaving a soluble version that can promote protein release without compromising cell envelope integrity.
Solution Approach 2:
The invention changes the physical state and localization parameter of the Pal protein from membrane-anchored to soluble. This parameter change fundamentally alters the protein's interaction with the cell envelope, enabling it to facilitate release without causing the severe membrane defects associated with membrane-destabilizing proteins.
Data Source
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AI summary
The invention relates to a bacterial strain containing an open reading frame which codes for a recombinant protein, thereby controlling a functional promoter. The invention is characterized in that the bacterial strain contains an open reading frame which codes for a muted peptidoglycan-associated lipoprotein (PAL protein), thereby controlling a functional promoter, wherein the PAL protein is muted such that the PAL protein does not contain a membrane anchor for the outer cell membrane of the bacterium. The invention additionally relates to a plasmid which codes for a recombinant protein and the muted PAL protein and to a method for the fermentative production of recombinant protein using the bacterial strain according to the invention. In this manner, product yields in the culture residue which are increased compared to the prior art can be achieved without leading to a substantial die-off of the bacterial cells.