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Purification of ubiquitylated proteins in E. coli


Ubiquitylation regulates nearly all-cellular pathways, but degradation and deubiquitylation make it difficult to isolate the ubiquitylated isoforms for biochemical and biophysical studies. We coded the entire eukaryotic ubiquitylation apparatus in E. coli, which lacks deubiquitylases and therefore ubiquitylated proteins are stable. Two different affinity-tags, one on ubiquitin and a second on the protein of interest, facilitate the purification of the modified substrate. We demonstrated that this synthetic biology approach produces proteins with native, stable ubiquitin modifications, and generated numerous plasmids with different E2s, E3s and substrates. The system produces milligram quantities of highly pure ubiquitylated protein for downstream biochemistry or crystallography studies.

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