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Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and
1The Florey Institute of Neuroscience and Mental Health and The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Cellular High-throughput Encapsulation Solubilization and Screening (CHESS) enables protein engineering under harsh conditions. This method successfully generated thermostable and detergent-resistant green fluorescent protein (GFP) mutants, expanding directed evolution capabilities.
Area of Science:
- Protein engineering
- Biotechnology
- Molecular biology
Background:
- Directed evolution techniques like phage display link genes to proteins for screening.
- Existing methods are limited in harsh industrial conditions (high temp, detergents).
- Cellular High-throughput Encapsulation Solubilization and Screening (CHESS) was developed for detergent-stable G protein-coupled receptors.
Purpose of the Study:
- To demonstrate the general applicability of CHESS for engineering soluble proteins.
- To characterize CHESS microcapsule stability and permeability.
- To engineer thermostable and detergent-resistant green fluorescent protein (GFP) mutants.
Main Methods:
- Encapsulating library-transformed bacterial cells in detergent-resistant polymers.
- Using CHESS to screen for detergent-stable protein mutants.
- Characterizing microcapsule properties and engineering GFP variants.
Main Results:
- CHESS microcapsules demonstrated stability and permeability suitable for screening.
- Thermostable and sodium dodecyl sulfate (SDS)-resistant GFP mutants were successfully generated.
- This marks the first instance of engineering soluble proteins using CHESS.
Conclusions:
- CHESS is a versatile directed evolution method applicable to soluble proteins.
- The technique overcomes limitations of traditional methods in harsh environments.
- CHESS expands the scope of protein engineering for industrial and research applications.
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