Pseudomonas fluorescens Sec Secretion for Recombinant Protein Folding
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Solution Overview
Problem
Current methods for producing recombinant proteins in bacteria often result in improperly folded or aggregated proteins due to the formation of inclusion bodies and difficulties in forming disulfide bonds, leading to inactive proteins, especially for eukaryotic proteins, which limits the yield of properly processed and active recombinant proteins for therapeutic use.
Innovation Solution
Utilizing the Pseudomonas fluorescens Sec secretion system and newly identified secretion signals, such as those from phosphate binding protein, Outer Membrane Porin E, azurin, Lys-Arg-Orn binding protein, and lipoprotein B, to target and process recombinant proteins in the periplasm, enhancing their folding and secretion into the extracellular environment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If recombinant proteins are produced in bacterial cytoplasm, then production volume is high, but proteins form inclusion bodies and aggregate, reducing activity
Solution Approach 1:
The patent extracts the protein from the problematic cytoplasmic environment and relocates it to the periplasmic space through secretion systems. By using signal peptides to direct proteins out of the cytoplasm and into the periplasm, the invention removes the harmful aggregation environment while maintaining high production capability.
Solution Approach 2:
The patent introduces signal peptides as intermediary elements that mediate protein translocation from the cytoplasm to the periplasm. These signal sequences act as guides that facilitate the movement of recombinant proteins through secretion systems, enabling them to reach the periplasmic environment where they can fold properly without aggregating.
2Manufacturing precision
If proteins are secreted to the periplasm, then disulfide bond formation is facilitated and folding is improved, but production yield may decrease
Solution Approach 1:
The patent changes the environmental parameters by relocating protein synthesis and folding to the periplasmic space, which has different chemical conditions (oxidizing environment, different pH, presence of chaperones) that favor disulfide bond formation and proper folding. This parameter change resolves the folding problem while the secretion systems maintain adequate production levels.
3Reliability
If secretion systems are used, then protein solubility and activity increase, but system complexity increases
Solution Approach 1:
The patent utilizes universal secretion systems and signal peptides that can be applied to multiple different recombinant proteins. The same periplasmic targeting mechanism works for various protein types, making the system broadly applicable rather than requiring protein-specific complex mechanisms for each case.
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
This approach significantly increases the production of properly processed and active recombinant proteins, improving their yield and solubility, with up to 50% of total protein produced being correctly processed and active, facilitating high-level expression suitable for commercial scale.
Implementation Method 1
Gram-negative bacteria have evolved numerous systems for the active export of proteins across their dual membranes
Implementation Method 2
The most common form of secretion of peptides with a signal sequence involves the Sec system
Implementation Method 3
Secretion into the periplasmic space also has the well known effect of facilitating proper disulfide bond formation
Data Source
AI summary
Improved methods and prokaryotic expression systems for producing recombinant proteins using Sec-system secretion signal peptides are described using Pseudomonas fluorescens Sec secretion systems. Specific novel Sec-system secretion signal peptides are described, as are fusion proteins and coding sequences for improved secretion of recombinant proteins and peptides.