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Updated: Aug 12, 2026
09:01
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Rapid creation of a novel protein function by in vitro coevolution
1Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA.
Journal of Molecular Biology
|April 28, 2005
Summary
We developed an in vitro coevolution method to create novel protein functions. This technique successfully engineered human estrogen receptor alpha ligand-binding domain variants with new corticosterone activity.
Area of Science:
- Protein engineering
- Molecular biology
- Biochemistry
Background:
- Engineering novel protein functions is challenging with existing methods.
- Directed evolution offers a pathway for protein modification.
- Nuclear hormone receptors are crucial drug targets.
Purpose of the Study:
- To develop a novel in vitro coevolution method for creating new protein functions.
- To engineer human estrogen receptor alpha ligand-binding domain (hERalphaLBD) for corticosterone activity.
Main Methods:
- Designed a hypothetical pathway for target function.
- Employed stepwise directed evolution using random mutagenesis.
- Screened approximately 10^6 variants over four rounds.
- Utilized testosterone and progesterone as intermediate ligands.
Main Results:
- Successfully engineered two hERalphaLBD variants with corticosterone activity.
- Identified four critical mutations for the novel ligand activity.
- Observed mutations outside the ligand-binding pocket influencing activity.
- Demonstrated the creation of new ligand activity not achievable by other methods.
Conclusions:
- In vitro coevolution is a powerful tool for engineering novel protein functions.
- This method can overcome limitations of traditional protein engineering approaches.
- The findings provide insights into the evolution of nuclear hormone receptors.
- The approach is broadly applicable for engineering biological molecules and systems.
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