Getting protein factories to run—How deubiquitinating enzymes moonlight as Fubi proteases nchembio
. For mammalian cell expression, full-length cDNA sequences for Flag–USP7, Flag–USP10 , Flag–USP16, Flag–USP36, Flag–USP42 and Flag–USP48 were cloned into pOPINE. USP45 was expressed from pCMV-Flag-USP45 . Full-length Fubi-S30 was expressed with an N-terminal HA tag and a C-terminal Flag tag from pOPINE. Point mutations were generated using site-directed mutagenesis.
Ubiquitin, Fubi and other Ubl thioesters were obtained using intein-mediated C-terminal functionalization, chitin-binding affinity purification and size exclusion. Lysis was performed on ice by sonication in buffer A supplemented with DNase. Protease inhibitors were included in the lysis buffer for Fubi and all other Ubl modifiers, and 5% glycerol was present in all buffers for Fubi and NEDD8 purification and functionalization. Lysate was cleared by centrifugation and filtering .
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