GS Knockout HEK293 Cell Line for High-Productivity Protein Selection
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Solution Overview
Problem
The HEK293 cell line, widely used for protein production, lacks a stable system for continuous high-concentration production of target proteins, due to high resistance against MSX in the GS/MSX system caused by endogenous expression of glutamine synthetase, limiting the selection efficiency of high-production clones.
Innovation Solution
A glutamine synthetase (gs) gene knockout transgenic HEK293 cell line is developed using CRISPR/Cas9 technology to eliminate MSX resistance, allowing for efficient cell line selection and increased productivity by increasing glutamine dependency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If HEK293 cell line is used for protein production, then human originated protein quality is improved, but MSX resistance due to endogenous GS expression reduces selection efficiency
Solution Approach 1:
The patent extracts and removes the endogenous glutamine synthetase (GS) gene from the HEK293 cell line using CRISPR/Cas9 gene editing technology. This eliminates the source of MSX resistance while preserving the human-originated protein production capability of the cell line, thereby resolving the contradiction between protein quality and selection efficiency
Solution Approach 2:
The patent changes the GS enzyme activity parameter from high (endogenous expression) to zero (knockout state). This parameter change eliminates MSX resistance, allowing efficient selection of high-production clones while maintaining the cell line's ability to produce high-quality human-originated proteins
2Productivity
If GS/MSX system is used for cell line selection, then productivity per cell is improved, but endogenous GS expression causes MSX resistance reducing selection efficiency
Solution Approach 1:
The patent converts the harmful effect of endogenous GS expression (MSX resistance) into a beneficial feature by creating a GS-knockout cell line that is highly sensitive to MSX. This allows the GS/MSX system to work more effectively, enabling efficient selection of high-productivity clones through amplified MSX sensitivity rather than resistance
3Productivity
If transient transfection system is used in HEK293, then screening of target protein is improved, but stable continuous high concentration production is not achieved
Solution Approach 1:
The patent performs preliminary gene knockout of the endogenous GS gene before establishing stable cell lines for continuous production. This preliminary action eliminates MSX resistance upfront, enabling subsequent stable transfection and continuous high-concentration production without the interference of endogenous GS expression, thereby achieving both screening efficiency and production stability
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
The gs knockout HEK293 cell line enhances the selection efficiency and productivity of target proteins, such as antibodies and cytokines, by overcoming MSX resistance, enabling stable high-concentration production and expanding market opportunities.
Implementation Method 1
A glutamine synthetase (gs) gene knockout transgenic HEK293 cell line is developed using CRISPR/Cas9 technology
Data Source
AI summary
The present invention relates to a novel GS (glutamine synthetase) gene knock out transgenic HEK293 (human embryonic kidney 293) cell line and a production method of a target protein using the said transgenic HEK293 cell line. Particularly, the present inventors eliminated the expression of GS in the HEK293 cells in order to overcome a barrier of the cell line selection caused by the over-expression of GS, for producing a target protein by GS/MSX system, by which the efficiency of the cell line selection for the high production of a target protein would be increased and accordingly the protein production by the selected cell line would be increased, suggesting that the human originated transgenic HEK293 cell line could be efficiently used for the production of a target protein.