E. coli-Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins
Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.
Key wordsBacterial genetics Selection Identification Expression and purification of ubiquitylated proteins
We are very thankful to A Weissman, A Chitnis, R Vierstra, I Dikic, D Katzmann and S Misra, R Klevit, and Scott Emr for the fruitful discussions and for providing reagents.
We also thank S Ben-Aroya and D Rotin for validation experiments in cells and D Wolf and O Kleifeld for mass spectrometry analyses. This research was supported by grants from the Israeli Science Foundation (grant no. 1695/08, 464/11, and 651/16), from the EC FP7 Marie Curie International Reintegration Grant (PIRG03-GA-2008-231079), and from the Israeli Ministry of Health (5108) to GP.
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