Mlv votu

Ingelvac PRRS® nsp2 nucleotides 1342-1986, coding for nsp2 aa 1-215, were inserted downstream of a 6x histidine tag, codon optimized for expression in E. coli, and used to synthesize the viral OTU polypeptide (MLV vOTU) (GenScript, Piscataway, NJ). A JXwn06 vOTU polypeptide representing nsp2 aa 1-215 was produced in a like manner. The coding sequence for porcine proISG15 (NM_0011286469) was codon optimized for expression in E. coli, and internal restriction endonuclease recognition sequences were altered by silent mutagenesis for cloning purposes (Huang et al., 2009Huang et al., 2009 ). This optimized gene was synthesized with an amino terminal 3X FLAG-Tag and 10X His-Tag, as well as NcoI and BamHI sites for cloning purposes at the 5’ and 3’ ends, respectively (GenScript). This synthetic gene was subcloned into pUC57 and subsequently moved to pET26b (EMD Millipore) for bacterial expression and purification.

All assays were performed in duplicate in Buffer E (100 mM NaCl, 50 mM HEPES [pH 7.5], 0.01 mg/mL bovine serum albumin (BSA), 5 mM DTT) using a Corning Costar half-volume black 96-well plate with a reaction volume of 50 μL. The rates of the reactions were observed using an Infinite M1000 series plate reader (Tecan, Inc.). Specifically, the increase in fluorescence (excitation λ, 360 nm; emission, 460 nm) of 7-amino-4-methylcourmarin (AMC) upon cleavage from hUb-AMC, hISG15-AMC, (Boston Biochem, MA) and ZRLRGG-AMC (Bachem) substrates was monitored for JXwn06 and MLV vOTU (GenScript). To calculate turnover rates for 1 μM hISG15-AMC, 1 μM hUb-AMC, and 50 μM ZRLRGG-AMC, 1 μM, 4 nM, and 4 μM of enzyme were used against hISG15-AMC, hUb-AMC, and ZRLRGG-AMC, respectively.
The turnover rates for poly-Ub fluorescence resonance energy transfer (FRET) linkage substrates K11, K48, and K63 (Boston Biochem, MA) at 1 μM were determined by monitoring the increase in fluorescence (excitation λ, 544 nm; emission, 572 nm) resulting by the separation of a FRET TAMRA/QXL pair. Cleavage of three commercially available FRET TAMRA/QXL pair configurations per K48 and K63 poly-Ub linkage FRET substrates were assessed. Each di-Ub FRET substrate at 1 μM was evaluated against an enzyme concentration of 500 nM.