Mutated Tsp and DegP Proteases for Recombinant Protein Yield
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Solution Overview
Problem
Current bacterial strains used for recombinant protein production, such as E. coli, face challenges in producing protease-sensitive proteins due to the degradation by bacterial proteases like Tsp and DegP, leading to reduced yields and potential metabolic disruptions from knockout mutations and antibiotic resistance markers.
Innovation Solution
Development of recombinant E. coli strains with mutated Tsp and DegP genes that have reduced protease activity, using targeted mutations at the gene start and stop codons to minimize genome disruption, thereby reducing proteolysis and maintaining chaperone activity for proper protein folding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If bacterial proteases (Tsp, DegP) are knocked out to reduce proteolysis of recombinant proteins, then protein yield is improved, but cell growth rate deteriorates and metabolic disruptions occur
Solution Approach 1:
The invention applies local quality by creating site-specific point mutations in the Tsp and DegP protease genes rather than complete knockouts. Specific amino acid residues involved in proteolytic activity are mutated to reduce protease function locally while preserving the overall protein structure and chaperone functions. This localized modification allows the bacteria to maintain adequate protease activity for normal cellular functions while reducing degradation of recombinant proteins.
Solution Approach 2:
The invention changes the functional parameters of Tsp and DegP proteases by mutating specific amino acid residues (e.g., Tsp S430A, D441A, K455A; DegP S210A, H105A, D135A) to alter their proteolytic activity. These parameter changes reduce the enzymes' ability to degrade recombinant proteins while maintaining their chaperone activity, thus improving protein yield without severely impacting cell growth.
2Stability of the object's composition
If complete protease knockout is performed to eliminate proteolytic degradation, then recombinant protein stability is improved, but chaperone activity is lost and protein folding is compromised
Solution Approach 1:
The invention applies local quality by making targeted point mutations at specific amino acid residues that are critical for proteolytic activity but not for chaperone function. For example, mutating the catalytic triad residues (Ser, His, Asp) in DegP reduces protease activity while preserving the chaperone domain's ability to facilitate protein folding. This selective local modification allows differential preservation of enzyme functions.
Solution Approach 2:
The invention segments the functional domains of Tsp and DegP proteins by differentially affecting their protease and chaperone activities through specific point mutations. The mutations are designed to disrupt the proteolytic catalytic site while leaving the chaperone binding and folding domains intact, effectively separating these two functions within the same protein molecule.
3Productivity
If traditional protease-deficient strains are used to produce recombinant proteins, then proteolytic degradation is reduced, but genome disruption from knockout markers causes metabolic burdens
Solution Approach 1:
The invention extracts the harmful proteolytic degradation function from Tsp and DegP enzymes through point mutations, while deliberately retaining the beneficial chaperone functions. This selective extraction of function avoids the need for complete gene knockout and eliminates the requirement for antibiotic resistance markers or other genomic disruption elements, simplifying the genetic modification approach.
Solution Approach 2:
The invention uses simple point mutations rather than complex knockout constructs with antibiotic resistance markers. This approach is metaphorically 'cheap' in terms of genetic engineering complexity and avoids the metabolic burden of maintaining and expressing marker genes, making the system more elegant and suitable for therapeutic protein production where genome integrity is important.
Data Source
Figure 1a~1b
Figure 1c
Figure 2
AI summary
A recombinant gram-negative bacterial cell comprising one or more of the following mutated protease genes: a. a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene; b. a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene; and c. a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity ; wherein the cell is isogenic to a wild-type bacterial cell except for the mutated Tsp gene and/or mutated ptr gene and/or mutated Deg P gene and optionally a polynucleotide sequence encoding a protein of interest.