CRM197 Periplasmic Signal Sequence for Low-Toxicity E. Coli Expression
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Solution Overview
Problem
Existing methods for producing CRM197 protein in E. coli face challenges in achieving efficient expression and secretion into the periplasm while minimizing toxicity to the host cells.
Innovation Solution
Optimized signal sequences, represented by specific amino acid sequences (SEQ ID NO: 13 to SEQ ID NO: 21), are designed to enhance translation and secretion of CRM197 protein into the periplasm of E. coli, using nucleic acids and expression vectors like pHex1.3, which include optimized CRM197 DNA and signal sequences, and recombinant microorganisms to facilitate high-yield production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRM197 protein is expressed in E. coli using conventional methods, then production is achieved, but toxicity to host cells occurs and secretion efficiency into periplasm is low
Solution Approach 1:
The patent uses a periplasmic targeting signal sequence as an intermediary element that directs CRM197 protein to the periplasmic space. This mediator component enables controlled localization of the toxic protein away from the cytoplasm, reducing harm to host cells while maintaining production efficiency. The signal sequence acts as a bridge between the toxic protein and the safe periplasmic compartment.
2Manufacturing precision
If CRM197 protein is secreted into periplasm, then purity and productivity are improved, but appropriate signal sequence selection is critical and complex
Solution Approach 1:
The patent optimizes specific parameters of the signal sequence, including amino acid composition, hydrophobicity distribution, and length. By systematically adjusting these parameters, the invention achieves efficient periplasmic targeting without requiring overly complex signal sequences. The optimized signal sequence contains specific structural features that enhance targeting efficiency while maintaining simplicity.
3Productivity
If conventional signal sequences are used, then expression is achieved, but secretion efficiency into periplasm is insufficient
Solution Approach 1:
The patent incorporates pre-optimized signal sequences that have been designed beforehand to ensure efficient periplasmic targeting. These preliminary optimized sequences include specific structural features and amino acid compositions that guarantee reliable secretion. By preparing and testing signal sequences in advance, the invention ensures consistent and efficient secretion without requiring complex optimization during production.
Data Source
AI summary
The disclosure relates to a signal sequence for expressing a CRM197 protein in Escherichia coli and secreting same into the periplasm, and use thereof, more specifically, to: a signal sequence for expressing a CRM197 protein; a nucleic acid for coding the signal sequence; a nucleic acid construct or expression vector comprising the nucleic acid and a CRM197 protein gene; a recombinant microorganism having the nucleic acid construct or expression vector introduced therein; and a CRM197 protein production method comprising a step for culturing the recombinant microorganism. A CRM197 protein having the same physicochemical/immunologic properties as the protein isolated from the parent bacteria may be expressed even in Escherichia coli of which a redox potential is not adjusted, and a CRM197 protein having high periplasmic secretion efficiency may be produced even without shifting the pH of a culture medium in order to increase secretion into the periplasm in CRM197 protein production.