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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
Published on: November 16, 2016
Directed evolution of proteins in vitro using compartmentalization in emulsions
Eric A Davidson1, Paulina J Dlugosz, Matthew Levy
1University of Texas at Austin, Austin, Texas, USA.
Current Protocols in Molecular Biology
|July 4, 2009
Summary
Directed evolution of proteins is achieved using in vitro compartmentalization, enabling selection of proteins that bind target molecules. This method allows screening of large libraries for desired protein functions, even those toxic in vivo.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Directed evolution is a powerful technique for protein engineering.
- In vitro compartmentalization (IVC) offers a platform for high-throughput screening.
- Existing methods may have limitations in library size and selectable functions.
Purpose of the Study:
- To describe a protocol for directed evolution of proteins using IVC.
- To enable selection of proteins with specific binding capabilities.
- To provide a foundation for selecting other protein functionalities.
Main Methods:
- Utilizing in vitro transcription and translation (IVTT) reactions within water droplets in an oil emulsion.
- Encapsulating individual reactions to create compartments for protein expression and selection.
- Recovering genes encoding proteins that successfully bind to a target molecule.
Main Results:
- Demonstrated a method for selecting proteins that bind a target molecule.
- Facilitated the recovery of genes encoding the selected protein variants.
- Established a protocol adaptable for selecting other protein functionalities, such as catalysis.
Conclusions:
- In vitro compartmentalization provides an effective platform for directed protein evolution.
- This method allows for screening of very large libraries.
- It enables selection for functions not accessible through in vivo methods.
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