Development and Application of the Split-CAT System for Protein-Protein Interaction
and Ubiquitylation Studies
Our research leverages the split-chloramphenicol acetyl transferase (Split-CAT) assay
to advance our understanding of protein-protein interactions (PPIs) and ubiquitylation
within E. coli. This innovative assay system, built on synthetic biology principles,
enables efficient detection of PPIs and ubiquitylation through the assembly of split-CAT
fragments, which confer antibiotic resistance upon reconstitution. By merging these
studies, we explore critical areas:
UBE3B Self-Ubiquitylation and Regulation The initial application of the Split-
CAT system demonstrated that UBE3B, a ubiquitin E3-ligase, undergoes self-
ubiquitylation that regulates its activity through oligomerization. This process,
which occurs without degradation in E. coli, highlights UBE3B's functional
modulation, providing insights into mechanisms underlying Kaufman
oculocerebrofacial syndrome (KOS) (Levin-Kravets et al., Journal of Molecular
Biology, 2021) see publication​
Protocol for Protein-Protein Interaction Analysis with Split-CAT Expanding
the assay, we published a protocol detailing the steps for analyzing both
ubiquitylation and PPIs in E. coli. This resource outlines optimal conditions for
growth selection, including using the SAMPLE data acquisition tool for monitoring
microbial growth. This protocol enables researchers to systematically assess
PPIs without purification, enhancing the utility of Split-CAT in detecting weak
interactions (Florentin et al., Bio-protocol, 2022) see publication​
Proximal Co-Translation for Weak PPI Detection We recently introduced a
proximal co-translation approach, enhancing the detection of ultra-weak
interactions by expressing interacting partners from a single mRNA. This method
increases PPI detection sensitivity in split-CAT systems, confirmed through
interactions involving Ub-binding domains (UBDs) like the ENTH domain. This
co-translation system showed promise across diverse weak PPI cases, including
Rpn10 and PLK4 complexes, providing a novel strategy for PPI studies
(Kordonsky et al., International Journal of Molecular Sciences, 2024)
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Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-
Journal of Molecular Biology, 2021
Ubiquitylation-Dependent Regulation of UBE3B
Journal of Molecular Biology, 2021
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Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-ProteinInteractions and Ubiquitylation in Escherichia coli
Link to paper
Bio-protocol, 2022
​
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Proximal Co-Translation Facilitates Detection of Weak Protein-Protein
Interactions
Link to paper
International Journal of Molecular Sciences, 2024