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Comprehensive Screening of a Light-Inducible Split Cre Recombinase with Domain Insertion Profiling
Nathan Tague1,2, Virgile Andreani1,2, Yunfan Fan3
1Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, United States.
ACS Synthetic Biology
|October 3, 2023
Summary
We developed a new pooled library method for rapidly creating and screening split proteins, enabling precise post-translational control. This approach simplifies engineering stimulus-responsive proteins for various applications.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Protein Engineering
Background:
- Inducible protein function via split proteins offers post-translational control.
- Current methods for engineering stimulus-responsive split proteins are labor-intensive and require specialized expertise.
Purpose of the Study:
- To develop a streamlined method for generating and screening split protein constructs.
- To enable rapid engineering of stimulus-responsive proteins with inducible post-translational control.
Main Methods:
- Utilized a pooled library approach for parallel generation and screening of split protein constructs.
- Employed sequencing for readout of split protein library results.
- Developed a Bayesian computational approach to enhance prediction accuracy of split protein behavior.
Main Results:
- Successfully applied the method to Cre recombinase with optogenetic dimers, generating comprehensive split site data.
- Demonstrated a streamlined process for creating inducible post-translational control of proteins.
Conclusions:
- The pooled library and computational approach significantly accelerates the engineering of stimulus-responsive split proteins.
- This method provides a broadly applicable strategy for achieving precise post-translational control of protein function.
