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Development of an improved mammalian overexpression method for human CD62L
Haley A Brown1, Gwynne Roth1, Genevieve Holzapfel1
1Structural Immunology Section, Lab of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, United States.
Protein Expression and Purification
|October 7, 2014
Summary
We developed a faster method for producing recombinant proteins using glutamine synthetase (GS) expression systems. This new approach significantly reduces cell line construction time from months to two, enabling quicker protein production.
Area of Science:
- Biotechnology
- Molecular Biology
- Mammalian Cell Culture
Background:
- Mammalian recombinant protein expression systems, such as the glutamine synthetase (GS)-based system, are valuable for producing therapeutic proteins.
- A major limitation of existing stable mammalian expression systems is the extended timeframe required for cell line development, typically 4-6 months.
- Previous GS-based systems relied on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX) for high-level protein production.
Purpose of the Study:
- To significantly reduce the construction time of stable mammalian cell lines for recombinant protein production.
- To investigate the efficacy of single-round in situ amplification as an alternative to multi-round amplification.
- To develop a more efficient method for evaluating recombinant protein expression levels in stable cell lines.
Main Methods:
- Implemented single-round in situ gene amplification to replace multi-round amplification in the GS-based expression system.
- Utilized Chinese hamster ovary (CHO) cells for the development of recombinant cell lines.
- Developed and validated a methionine sulfoximine (MSX) resistance assay as an alternative to ELISA for assessing expression levels.
Main Results:
- Shortened the cell line construction time from 4-6 months to 2 months.
- Achieved comparable recombinant protein yields (approximately 5 mg/L of soluble CD62L) using single-round amplification as with multi-round methods.
- Demonstrated the utility of the MSX resistance assay for evaluating stable recombinant CHO cell line expression.
Conclusions:
- Single-round in situ amplification is an effective strategy to accelerate the development of stable mammalian cell lines for recombinant protein production.
- The developed MSX resistance assay provides a viable and potentially faster alternative to ELISA for screening and evaluating expression levels.
- This optimized system offers a significant improvement in turn-around time without compromising protein yield.
