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09:02
Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment
Published on: April 20, 2018
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A Well-Controlled BioID Design for Endogenous Bait Proteins
Giel Vandemoortele1,2, Delphine De Sutter1,2, Aline Moliere1,2
1VIB Center for Medical Biotechnology, VIB , B-9000 Ghent , Belgium.
Journal of Proteome Research
|December 12, 2018
Summary
This study introduces a novel genome engineering method for endogenous protein proximity labeling using CRISPR/Cas9 technology. It enables precise BioID analysis of native proteins, overcoming limitations of traditional cell line models.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Engineered clonal cell lines risk artifacts due to clonal variation.
- BioID (proximity labeling) traditionally uses ectopic protein expression.
- Existing methods struggle with artifacts from clonal variation and overexpression.
Purpose of the Study:
- To develop a genome engineering strategy for endogenous protein BioID.
- To create an isogenic cell population for studying native protein interactions.
- To overcome limitations of classical BioID approaches.
Main Methods:
- Genome engineering to introduce a T2A-BirA* module at the C-terminus of endogenous p53.
- Utilizing a Cas9-cytidine deaminase base editor for precise modification.
- Quantitative proteomics to analyze protein proximity.
Main Results:
- Successfully generated an isogenic population expressing a functional p53-BirA* fusion protein.
- Demonstrated significant benefits over classical ectopic expression of p53-BirA*.
- Provided a well-controlled view of endogenous p53 proximal proteins in colon carcinoma cells.
Conclusions:
- This novel application of base editors expands the CRISPR/Cas9 toolbox.
- The method enables reliable BioID analysis of endogenous proteins.
- It offers a valuable addition for synthetic biology and proteomic studies.
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