OmpF Co-expression for Extracellular Protein Secretion
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Solution Overview
Problem
Existing methods for extracellular protein secretion in E. coli often result in low purity due to intracellular protein contamination and difficulties in high cell concentration cultures, as they involve partial lysis of cells and complex purification processes.
Innovation Solution
Co-expression of E. coli outer membrane protein F (OmpF) with a target protein using recombinant vectors, allowing for efficient secretion and purification of the target protein into the culture broth without cell lysis, using a microorganism co-transformed with OmpF-encoding and target protein-encoding genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If proteins are produced in the cytoplasm of E. coli, then the production process is simple, but the purification process becomes complex and expensive, and the proteins are exposed to proteases leading to reduced yield
Solution Approach 1:
The invention extracts the target protein from the cytoplasmic production environment and directs its secretion into the extracellular space. By using signal peptides and secretion systems, the protein is taken out of the complex cytoplasmic environment with proteases and moved to the simpler extracellular environment, thereby simplifying the purification process while maintaining easy production
Solution Approach 2:
The invention introduces signal peptides and secretion machinery as intermediaries between the cytoplasmic production site and the extracellular space. These intermediaries facilitate the transport of target proteins across the cell membrane, enabling simple production in cytoplasm while achieving easy purification in extracellular medium
2Reliability
If proteins are secreted into the periplasm, then intracellular proteolysis is prevented, but the purification procedure becomes much more complex compared to extracellular secretion
Solution Approach 1:
The invention moves the protein secretion from the two-dimensional periplasmic space to the three-dimensional extracellular environment. By crossing the outer membrane barrier, the protein enters the culture medium where purification is vastly simplified, while still maintaining protection from intracellular proteases during the secretion process
3Manufacturing precision
If extracellular secretion is used, then protein purity is high and purification is easy, but cell lysis occurs resulting in intracellular protein contamination
Solution Approach 1:
The invention performs preliminary secretion of target proteins into the extracellular space before any cell lysis occurs. By establishing the secretion pathway in advance using signal peptides and secretion systems, the target protein is already in the extracellular medium when cells are harvested, preventing contamination from intracellular proteins that would be released upon lysis
4Productivity
If high cell concentration culture is performed, then protein production efficiency increases, but cell lysis increases leading to reduced protein purity
Solution Approach 1:
The invention continuously extracts target proteins from cells into the extracellular environment during high cell concentration culture. By maintaining functional secretion systems throughout the culture process, proteins are removed from cells as they are produced, allowing high cell densities without the purity problems that would normally arise from subsequent lysis
Data Source
AI summary
The present invention relates to a method for secreting and producing a target protein into cell culture broth. More particularly, the invention relates to a microorganism co-transformed with a recombinant expression vector containing E. coli outer membrane protein F (OmpF) and a recombinant expression vector containing a target protein to be secreted into cell culture broth, as well as a method of secreting and producing the target protein into cell culture broth by culturing the microorganism. According to the invention, the target protein can be secreted into cell culture broth in a pure form without fusion with other proteins so that the efficient isolation and purification of the target protein is possible.