Recombinant Bacterial Strain with Reduced Tsp Activity
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Solution Overview
Problem
Bacterial cells, such as E. coli, face challenges in producing protease-sensitive proteins due to the degradation by bacterial proteases like Tsp, leading to reduced yields of active protein.
Innovation Solution
A recombinant gram-negative bacterial cell strain with reduced Tsp activity, co-expressing DsbC, and a mutated spr gene to improve protein production by minimizing proteolysis and maintaining cell health.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Tsp protease activity is reduced to minimize protein degradation, then protein yield and integrity are improved, but cell growth and viability are worsened due to thermosensitive growth defects
Solution Approach 1:
The patent introduces DsbC as an intermediary chaperone protein that mediates between the reduced Tsp activity and protein folding requirements. DsbC compensates for the loss of Tsp's chaperone function by facilitating proper protein folding in the periplasm, thereby maintaining cell growth while allowing reduced Tsp protease activity to preserve protein yield
Solution Approach 2:
The patent employs a temperature-dependent regulatory system that changes the activity parameters of Tsp protease based on growth phase. During exponential growth, Tsp is more active to support cell division, while during stationary phase, Tsp activity is reduced to minimize degradation of accumulated recombinant proteins, thus resolving the contradiction between cell growth and protein preservation
2Manufacturing precision
If Tsp protease activity is reduced to preserve protease-sensitive proteins, then protein integrity is improved, but proteolysis of essential substrates increases affecting cell physiology
Solution Approach 1:
The patent introduces DsbC as an intermediary that takes over the essential proteolytic functions of Tsp for substrates like Penicillin-binding protein 3 and phage tail proteins. DsbC acts as a mediator that performs the necessary proteolysis for cell physiology while the reduced Tsp activity preserves the integrity of recombinant proteins of interest
3Productivity
If protease-deficient strains are used to increase protein yield, then productivity is improved, but cell viability and growth rate are reduced
Solution Approach 1:
The patent applies local quality by differentiating the functional requirements in different cellular compartments. Tsp protease activity is selectively reduced in the periplasm to preserve recombinant proteins, while essential proteolytic functions are maintained through DsbC and other proteases to ensure cell viability and growth rate
Solution Approach 2:
The patent creates a composite proteolytic system combining multiple proteases (Tsp, DsbC, DegP, Lon) with different activity profiles and substrate specificities. This composite system allows selective proteolysis of recombinant proteins to be minimized while maintaining essential proteolytic functions for cell physiology, thus resolving the contradiction between productivity 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 strain achieves higher yields of intact proteins with reduced proteolytic fragments, improved cell growth, and increased protein production rates, particularly in the periplasm, while maintaining correct protein folding and secretion.
Implementation Method 1
Protein disulphide isomerase is an enzyme that catalyzes the formation and breakage of disulphide bonds between cysteine residues within proteins as they fold
Implementation Method 2
Bacterial proteases act to degrade the recombinant protein of interest, thereby often significantly reducing the yield of active protein
Implementation Method 3
The spr gene is designated as UniProtKB/Swiss-Prot POAFV4 (SPR_ECOLI). Hara et al. isolated thermoresistant revertants containing extragenic suppressor (spr) mutations
Data Source
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AI summary
The present invention provides a recombinant gram-negative bacterial cell, characterized in that the cell: a. comprises a recombinant polynucleotide encoding DsbC; and b. has reduced Tsp protein activity compared to a wild-type cell.