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A specific protease cleavage site, such as the one recognized by HRV 3C protease (Leu-Glu-Val-Leu-Phe-GlnGly-Pro), can be engineered between the GST tag and the target protein

There are three enzymes contributing to UPP: E1 (Ub-activating enzyme) and E2s (Ub-carrier) prepare Ub for conjugation, but the main enzyme in the process is the E3 (Ub-protein ligase) which transfers the activated ubiquitin to the protein substrate to be degraded [66]

doi: 10.1016/j.freeradbiomed.2020.02.025