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ORF-selector ESPRIT: a second generation library screen for soluble protein expression employing precise open reading
Yingfeng An1, Hayretin Yumerefendi, Philippe J Mas
1Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
Journal of Structural Biology
|April 26, 2011
Summary
We enhanced ORF-selector ESPRIT to screen protein truncation libraries, identifying soluble, high-yield protein constructs. This method efficiently produces milligram quantities of purifiable proteins for structural biology and screening applications.
Area of Science:
- Protein biochemistry
- Molecular biology
- Structural biology
Background:
- Identifying soluble protein constructs is crucial for structural and functional studies.
- Challenging proteins often yield insoluble or low-quantity products using standard expression methods.
Purpose of the Study:
- To present ORF-selector ESPRIT, a significantly enhanced technology for screening protein truncation libraries.
- To facilitate the identification and production of soluble, high-yielding protein constructs from difficult targets.
Main Methods:
- Employing incremental gene truncation libraries to access internal protein domains.
- Utilizing an unbiased, intein-based open reading frame selection to ensure in-frame DNA inserts.
- Automated, two-step screening of tens of thousands of constructs for high expression using robotic systems.
Main Results:
- Benchmarking with p85α protein successfully isolated all known domains.
- Screening of human kinase IKK1 yielded a purified predicted internal domain.
- Demonstrated production of multi-milligram quantities of purifiable proteins.
Conclusions:
- ORF-selector ESPRIT provides an integrated and efficient route for producing soluble proteins from challenging genes.
- The technology supports quantities suitable for structural biology, screening, and immunization studies.
- Enables the study of diverse systems, including multi-subunit complexes, due to high genetic diversity sampling.
