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| Home > Institute Collections > W500 > Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli. |
| Home > Institute Collections > W500 > Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli. |
| Journal Article | DKFZ-2025-02472 |
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2022
bio-protocol.org
Sunnyvale, CA
Abstract: Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract.
Keyword(s): Bacterial two hybrid ; Chloramphenicol Acetyl Transferase ; Protein-protein interaction ; Self-ubiquitylation ; Ubiquitin E3-ligase ; Ubiquitylation readout
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