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A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets
Jiří Zahradník1, Debabrata Dey1, Shir Marciano1
1Weizmann Institute of Science, Herzl St. 234, Rehovot 7610001, Israel.
ACS Synthetic Biology
|November 23, 2021
Summary
Researchers enhanced yeast display technology using protein engineering, developing brighter reporters and improved expression for faster, more effective protein selection and engineering. This advanced platform enables better protein surface display and optimization.
Area of Science:
- Biotechnology
- Molecular Biology
- Protein Engineering
Background:
- Yeast display is a powerful technique for protein engineering and selection.
- Existing methods have limitations in sensitivity and efficiency.
- Need for improved reporters and expression systems.
Purpose of the Study:
- To enhance the yeast display method through DNA and protein engineering.
- To develop novel surface exposure-tailored reporters (eUnaG2 and DnbALFA).
- To create a versatile platform for diverse protein expression and labeling strategies.
Main Methods:
- Engineered yeast display vectors with new reporters (eUnaG2, DnbALFA).
- Optimized reporter fluorescence, thermostability, and protein expression levels.
- Developed a complex plasmid system for varied protein expression.
- Compared new platform with traditional labeling methods (pCTcon2, c-myc).
Main Results:
- Optimized eUnaG2 showed 5x brighter fluorescence and 10°C higher thermostability.
- Optimized DnbALFA exhibited 10x higher expression.
- Achieved up to 5x better cell separation and higher binding affinities.
- Demonstrated consistent superior expression signals over c-myc labeling across 16 proteins.
- Successfully engineered a previously non-expressing protein (ATG16L1 WD40 domain).
- Rapidly selected high-affinity binders for two protein targets.
Conclusions:
- The enhanced yeast display platform offers significant improvements in simplicity, speed, and cost-effectiveness.
- New applications enable monitoring protein surface exposure and secretion pathway retention.
- The method facilitates engineering of difficult-to-express proteins and generation of novel protein-protein interactions.
- Qualitative enhancement in yeast display applicability for various research and development needs.
