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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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Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells
Irene Nigi1, Louise Fairall1, John W R Schwabe1
1Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom.
Current Protocols in Protein Science
|November 2, 2017
Summary
Mammalian expression systems, like HEK 293F cells, offer a viable alternative for producing soluble eukaryotic proteins and protein complexes for structural studies when prokaryotic systems fail. This method yields milligram quantities of human protein complexes.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Prokaryotic expression systems are common for protein structural studies due to cost and speed.
- However, they often fail to produce soluble eukaryotic proteins and protein complexes, leading to low yields or inclusion bodies.
- Mammalian expression systems are perceived as difficult and costly, limiting their use.
Purpose of the Study:
- To present a straightforward method for expressing and purifying milligram quantities of human protein complexes.
- To highlight the utility of HEK 293F cells as an efficient mammalian expression system.
- To address the underutilization of mammalian systems for recombinant protein production.
Main Methods:
- Utilizing HEK 293F cells grown in suspension culture.
- Transient transfection of HEK 293F cells with appropriate expression plasmids.
- Development of a protocol for expressing and purifying milligram quantities of a human protein complex.
Main Results:
- HEK 293F cells can be easily cultured and efficiently transfected.
- Significant yields of soluble, natively folded human protein complexes were achieved.
- The described method enables the production of milligram quantities suitable for structural studies.
Conclusions:
- HEK 293F cells provide a practical and efficient system for producing complex eukaryotic proteins.
- This method overcomes limitations of prokaryotic expression systems for certain proteins.
- Mammalian expression systems, particularly HEK 293F cells, are a valuable and underutilized resource for protein science.
