Engineered Bacteria Secreting Therapeutic Proteins via Periplasmic Chaperones
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Solution Overview
Problem
The expression of recombinant polypeptides, particularly those with disulfide bonds, in engineered bacteria often results in low production yields and misfolded proteins, making it challenging to achieve clinically effective levels for therapeutic and diagnostic applications.
Innovation Solution
The use of engineered bacteria comprising a heterologous nucleic acid sequence encoding a signal recognition particle (SRP) pathway signal sequence, a CsgGE export signal sequence, and a therapeutic polypeptide, which facilitates proper folding and increased expression and export of recombinant polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant polypeptides are expressed in engineered bacteria, then production yield is improved, but protein folding quality deteriorates
Solution Approach 1:
The patent introduces periplasmic chaperone proteins (Skp, SurA, DegP) as intermediary molecules that facilitate proper protein folding in the periplasmic space. These chaperones act as mediators between the expressed recombinant polypeptides and the folding environment, preventing misfolding and aggregation while maintaining high production yields.
Solution Approach 2:
The patent changes the subcellular localization parameter by targeting recombinant polypeptides to the periplasmic space using signal sequences. This parameter change (from cytoplasmic to periplasmic expression) creates an oxidizing environment favorable for disulfide bond formation and proper protein folding, while the introduced chaperones further optimize folding conditions.
2Stability of the object's composition
If disulfide bonds are formed in recombinant polypeptides, then protein stability is improved, but expression efficiency deteriorates
Solution Approach 1:
The patent changes the redox environment parameter by expressing proteins in the periplasmic space, which has a more oxidizing environment compared to the cytoplasm. This parameter change enables spontaneous disulfide bond formation and proper protein stability while maintaining high expression efficiency through the use of periplasmic signal sequences and chaperone assistance.
3Reliability
If recombinant polypeptides are secreted by engineered bacteria, then therapeutic effectiveness is improved, but production complexity deteriorates
Solution Approach 1:
The patent extracts the signal sequence portion of native secreted proteins and attaches it to recombinant polypeptides. This extraction and reapplication of the signaling function enables the recombinant proteins to be automatically secreted by the bacterial secretion machinery without requiring complex external secretion systems or purification steps.
Solution Approach 2:
The patent uses periplasmic chaperone proteins as intermediaries that assist in the proper folding and preparation of recombinant polypeptides for secretion. These chaperones mediate the transition from cytoplasmic synthesis to periplasmic folding and eventual secretion, simplifying the overall production process while ensuring therapeutic quality.
Data Source
AI summary
Engineered bacteria that secrete therapeutic polypeptides, pharmaceutical compositions comprising the bacteria, methods for producing recombinant polypeptides, and methods for using the bacteria for diagnostic and therapeutic purposes are provided.