Mammalian Expression Vector Signal Sequence Optimization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing recombinant proteins in mammalian cells, such as CHO cells, face challenges in efficiently secreting non-secretion competent polypeptides, like antibody chains, due to the unpredictability of signal sequences across species, leading to moderate expression levels and complex purification processes.
Innovation Solution
An expression cassette is developed with a promoter linked to a DNA sequence encoding a signal peptide, specifically selected from sequences like SEQ ID No. 10 or 9, to facilitate efficient secretion of heterologous proteins in mammalian host cells, including CHO cells, by targeting the secretory pathway and ensuring proper processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native signal sequences are used for recombinant protein secretion in mammalian cells, then the protein can be targeted to the secretory pathway, but secretion efficiency is low and unpredictable due to species-specific incompatibility
Solution Approach 1:
The patent changes the amino acid sequence parameters of the signal peptide to create a human-optimized sequence (V19) that is compatible with mammalian cell secretory pathways. By modifying the signal peptide sequence while maintaining its functional properties, the invention achieves reliable and efficient secretion of recombinant proteins in mammalian cells, resolving the species-specific incompatibility issue.
Solution Approach 2:
The patent introduces a human-optimized signal peptide V19 as an intermediary element that mediates between the heterologous gene and the mammalian cell secretory system. This optimized signal peptide acts as a bridge that enables efficient recognition and processing by the human secretory pathway, improving both secretion efficiency and reliability.
2Productivity
If intracellular proteins are expressed at high levels, then protein production increases, but purification becomes complex requiring cell lysis and multiple isolation steps
Solution Approach 1:
The patent extracts the protein from the intracellular compartment and directs it to the secretory pathway for extracellular release. By using the optimized signal peptide V19, the recombinant protein is secreted into the culture medium, separating it from the complex intracellular environment and enabling simpler purification processes that avoid cell lysis and multiple isolation steps.
Solution Approach 2:
The patent employs the cell's own secretory machinery to automatically export the recombinant protein to the extracellular medium. The optimized signal peptide V19 directs the protein through the endoplasmic reticulum and Golgi apparatus, utilizing the cell's inherent secretion capabilities to deliver the protein externally, thereby simplifying downstream purification.
3Ease of operation
If secretion is achieved using conventional signal sequences, then protein can be released into culture medium, but secretion function is insufficient due to poor operability of native signal sequences in host cells
Solution Approach 1:
The patent optimizes the signal peptide sequence parameters to create V19, which has improved amino acid composition and structural properties suitable for human cell recognition and processing. This parameter optimization enhances both the functionality and efficiency of protein secretion in mammalian cells.
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 results in improved secretion and increased mean antibody concentrations, with signal sequence V19 showing a particularly strong increase, demonstrating enhanced protein production and simplifying purification by secreting proteins into the extracellular medium.
Implementation Method 1
This 'preprotein' form includes an additional peptide sequence at the amino-terminus which is required to target the nascent peptide chain to the endoplasmatic reticulum to enable its entry into the secretory pathway
Implementation Method 2
Successful protein secretion requires effective translocation of the protein across the endoplasmatic reticulum or plasma membrane
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention relates to a mammalian cell based expression and secretion system and the expression and secretion of recombinant proteins by using secretory signal peptides. The present invention also relates to an expression cassette useful for the secretion of a heterologous gene from a mammalian cell, in particular a CHO cell. The present invention is also directed to a method of secreting a heterologous protein from mammalian cells such as CHO cells.