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Expression and Purification of pAG-MNase for CUT&RUN
1Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan. riverwin@naro.affrc.go.jp.
Methods in Molecular Biology (Clifton, N.J.)
|November 5, 2024
Summary
Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a chromatin profiling method. This study details the purification and hyperactive enzymatic assessment of the recombinant pAG-MNase protein for large-scale analyses.
Area of Science:
- Molecular Biology
- Epigenetics
- Biochemistry
Background:
- Chromatin profiling is essential for understanding gene regulation.
- Existing methods face challenges in efficiency and scalability.
- Cleavage Under Targets and Release Using Nuclease (CUT&RUN) offers a novel approach.
Purpose of the Study:
- To describe the expression and purification of recombinant pAG-MNase.
- To assess the enzymatic activity of the purified pAG-MNase.
- To evaluate its utility for large-scale chromatin analyses.
Main Methods:
- Expression and purification of recombinant pAG-MNase protein.
- Assessment of micrococcal nuclease (MNase) enzymatic activity.
- Application of CUT&RUN methodology using purified pAG-MNase.
Main Results:
- Successfully expressed and purified recombinant pAG-MNase.
- Demonstrated hyperactive enzymatic function of pAG-MNase.
- Validated the efficacy of pAG-MNase for CUT&RUN assays.
Conclusions:
- Purified recombinant pAG-MNase is a highly active enzyme.
- This hyperactive pAG-MNase enables efficient large-scale CUT&RUN analyses.
- The optimized protein enhances chromatin profiling capabilities.
