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Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
Published on: March 25, 2015
Engineering single Cys2His2 zinc finger domains using a bacterial cell-based two-hybrid selection system
Stacey Thibodeau-Beganny1, Morgan L Maeder, J Keith Joung
1Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|August 4, 2010
Summary
This study details updated protocols for selecting novel DNA-binding zinc finger proteins using a bacterial two-hybrid system. This method is effective for identifying proteins with specific DNA-binding properties.
Area of Science:
- Molecular Biology
- Protein Engineering
- Biotechnology
Background:
- Custom DNA-binding proteins are crucial for gene regulation and genome engineering.
- Phage display and bacterial two-hybrid systems are common methods for selecting proteins with specific DNA-binding affinities.
- Previous work established the bacterial two-hybrid system as a viable alternative to phage display for zinc finger selection.
Purpose of the Study:
- To provide updated, detailed protocols for utilizing the bacterial two-hybrid system for zinc finger selection.
- To facilitate the identification of synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities.
Main Methods:
- Utilizing a bacterial cell-based two-hybrid system for protein selection.
- Employing large randomized libraries of Cys2His2 zinc finger domains.
- Implementing selection methodologies to identify desired DNA-binding specificities.
Main Results:
- The bacterial two-hybrid system demonstrates effectiveness in selecting zinc fingers with desired specificities.
- Updated protocols are provided for the practical application of this selection method.
Conclusions:
- The bacterial two-hybrid system offers an effective and detailed approach for identifying novel DNA-binding zinc finger proteins.
- These protocols can aid researchers in protein engineering and synthetic biology applications.
