Chimeric Protein Expression System for Secretion Yield
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Solution Overview
Problem
Mammalian cell-based systems for protein production face challenges such as low yield and contamination risks due to differences in post-translational modifications and protein folding between prokaryotic and eukaryotic cells, particularly for non-secreted proteins which require cell sacrifice for harvesting and have limited intracellular capacity.
Innovation Solution
Exploiting genetic signals for post-translational fate and secretion machinery of host cells by using signal peptides from non-mammalian bulk-secreted proteins to enhance or induce secretion of both secreted and non-secreted proteins, improving yield and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If mammalian cell-based systems are used for protein production, then protein folding and post-translational modifications are improved, but production yield decreases
Solution Approach 1:
The invention extracts and utilizes the natural secretion capability of mammalian cells by fusing the target protein (whether naturally secreted or non-secreted) with a signal peptide that directs it to the secretory pathway. This allows the protein to be secreted into the culture medium, separating the production function from the cell itself and enabling harvest without cell sacrifice.
Solution Approach 2:
The signal peptide acts as an intermediary element that mediates between the target protein and the cell's secretory machinery. By introducing this intermediary sequence, non-secreted proteins are redirected through the secretory pathway, enabling their export to the medium while maintaining the benefits of mammalian cell expression.
2Manufacturing precision
If non-secreted proteins are produced in mammalian cells, then protein integrity is maintained, but harvesting requires cell sacrifice
Solution Approach 1:
The invention extracts the protein from the cell interior by redirecting it to the secretory pathway through signal peptide fusion. The protein is effectively taken out of the cell during its natural secretion process, allowing harvesting from the culture medium without requiring cell lysis or sacrifice.
Solution Approach 2:
Instead of harvesting intracellular proteins by sacrificing cells (the conventional approach), the invention inverts the strategy by directing proteins to be secreted extracellularly. This reversal of the retention approach transforms a destructive harvesting process into a non-destructive one.
3Productivity
If intracellular capacity is increased for recombinant protein, then production yield improves, but protein synthesis is attenuated to protect cell integrity
Solution Approach 1:
The invention continuously extracts secreted protein from the cell interior through the secretory pathway into the culture medium. This prevents accumulation of intracellular protein that would trigger protective attenuation mechanisms, allowing sustained high-level expression without compromising cell integrity.
Solution Approach 2:
The secretory pathway provides a continuous route for protein export, maintaining a steady flow of recombinant protein from the endoplasmic reticulum through the Golgi to the cell surface. This continuous action prevents intracellular saturation and allows uninterrupted protein synthesis at high levels.
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
Significantly enhances the secretion of naturally secreted proteins and induces secretion of non-secreted proteins, leading to higher yields and improved protein production efficiency without the need for cell sacrifice, while maintaining protein integrity and reducing contamination risks.
Implementation Method 1
proteins that are naturally secreted from the cell are more straightforward to produce in cell factories because the recombinant protein is also secreted by the host
Implementation Method 2
The translation of mRNA occurs on ribosomes and ribosomes are only located in the cytoplasm
Implementation Method 3
All proteins destined for secretion by eukaryotic cells must pass through, in turn, the endoplasmic reticulum (ER) and the Golgi apparatus before being packaged into membrane bound vesicles that allow secretion
Data Source
AI summary
The present invention relates to a method of producing a target protein, which method comprises expressing said protein in a host cell which contains a nucleic acid molecule which encodes a chimeric protein, said chimeric protein comprising a signal peptide from a non-mammalian bulk-secreted protein and said target protein; nucleic acids, vectors, host cells and kits for carrying out the method are also described.