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Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
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Development of an experimental method of systematically estimating protein expression limits in HEK293 cells
Yoshihiro Mori1, Yuki Yoshida2, Ayano Satoh3
1Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
Scientific Reports
|March 18, 2020
Summary
Scientists developed a new method to measure protein expression limits in human cells. Exceeding these limits, especially for proteins in organelles, can cause cell death, aiding in the identification of toxic proteins.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Protein overexpression can lead to cellular defects, but the mechanisms remain unclear.
- Identifying a protein's expression limit is crucial for understanding these mechanisms.
Purpose of the Study:
- To develop an experimental method for estimating the expression limits of target proteins in HEK293 cells.
- To investigate the consequences of exceeding these expression limits.
Main Methods:
- Utilized human embryonic kidney (HEK293) cells and high-copy plasmid DNA with a cytomegalovirus promoter.
- Quantified expression limits by measuring green fluorescent protein (GFP) levels fused to target proteins via the P2A self-cleaving sequence.
- Assessed expression limits for GFP, GFP with organelle-targeting signals, and proteins involved in vesicular trafficking.
Main Results:
- The expression limit for a model GFP was approximately 5.0% of total protein, with sustained overexpression leading to cell death.
- Expression limits were significantly lower for mitochondrially targeted (1.6%) and ER-localized (0.38%) GFPs.
- Proteins involved in vesicular trafficking exhibited much lower expression limits compared to a red fluorescent protein.
Conclusions:
- The developed method provides a valuable tool for estimating protein expression limits.
- This method can help define toxic proteins and elucidate the consequences of protein overexpression.
- Organelle localization and protein function (e.g., vesicular trafficking) influence protein expression tolerance.
