Transposon-Mediated Protein Expression in Suspension CHO Cells
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Solution Overview
Problem
Current methods for producing recombinant proteins in mammalian cells are inefficient and require significant time and effort, with low productivity and stability of introduced genes, and existing systems face challenges in achieving accurate post-translational modifications and high expression levels.
Innovation Solution
A method involving the introduction of an expression vector with a DNA encoding the protein of interest and attenuated selectable marker genes flanked by Tol1 or Tol2 transposon sequences into suspension CHO cells, allowing for transient transposase-mediated integration and suspension culture to enhance protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional expression vectors are introduced into mammalian cells, then protein production can be achieved, but productivity is low and gene stability is poor
Solution Approach 1:
The patent uses transposon sequences as intermediary elements that mediate the integration of the expression vector into the mammalian cell genome. The transposon-based integration system acts as a bridge between the exogenous expression vector and the host chromosome, enabling stable incorporation while maintaining high expression levels. This intermediary mechanism resolves the contradiction by providing both stable integration (reliability) and efficient expression (productivity).
Solution Approach 2:
The patent modifies the integration mechanism by changing from conventional random integration to transposon-mediated targeted integration. This parameter change in the integration method improves both gene stability through controlled insertion and productivity through optimized expression vector design, resolving the trade-off between these two parameters.
2Reliability
If transposon sequences are used for gene integration, then gene stability improves, but integration efficiency may be reduced
Solution Approach 1:
The patent applies preliminary action by pre-designing the expression vector with transposon sequences already incorporated into its structure before introduction into the cell. This preliminary preparation ensures that once the vector enters the cell, the transposon-mediated integration can proceed efficiently without requiring additional steps, thus maintaining both high gene stability and integration efficiency.
3Quantity of substance
If time-consuming cell line development is performed to achieve high expression, then protein yield increases, but development time and cost increase
Solution Approach 1:
The patent employs preliminary action by pre-configuring the expression vector with transposon sequences that enable direct, efficient integration into the genome upon introduction. This preliminary design eliminates the need for time-consuming cell line development and screening processes, allowing high protein yield to be achieved much faster through straightforward transfection and selection.
Solution Approach 2:
The patent applies the skipping principle by bypassing the conventional lengthy cell line development process. The transposon-mediated integration system allows the procedure to rush through to high-expression cell lines much faster than traditional methods, significantly reducing the time loss while achieving the same or better protein yields.
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 reduces the time to establish high-expression cell lines and increases recombinant protein yield, providing a more efficient and stable production method for mammalian-derived proteins.
Implementation Method 1
integrating the gene fragment inserted between the pair of transposon sequences into a chromosome of the mammalian cell
Data Source
Figure 1
AI summary
This invention relates to a method for producing a protein of interest, comprising introducing an expression vector which comprises a gene fragment comprising a DNA encoding the protein of interest and a selectable marker gene and also comprises a pair of transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell; integrating the gene fragment inserted between the pair of transposon sequences into a chromosome of the mammalian cell; obtaining a suspension mammalian cell producing the protein of interest; and suspension-culturing the suspension mammalian cell, and a suspension mammalian cell which expresses the protein of interest by the method.