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. Author manuscript; available in PMC: 2016 Jul 1.
Published in final edited form as: Virology. 2015 Mar 17;481:166–178. doi: 10.1016/j.virol.2015.02.022

Figure 2. CpGV-MMP expression, purification and enzymatic activity.

Figure 2

(A) E. coli expression and purification of CpGV-MMP. His-tagged CpGV-MMP protein was produced in E. coli and purified using imidazole and a cobalt-charged resin. Indicated fractions (20 μl) were analyzed by immunoblotting using anti-His antibody. The migration of CpGV-MMP is indicated with an arrow. Sup: supernatant. Sup. after beads represents the supernatant collected after incubation with metal affinity resin beads

(B) MMP activity assays using E. coli-purified CpGV-MMP. CpGV-MMP, CpGV-MMP-Mut, induced pET32a empty vector (30 ng total protein) or control human MMP-9 (30 ng) were incubated at 37° C with 50 μl of generic fluorescent MMP substrate. MMP inhibitor, 400 μM GM6001 or 50 μM EDTA, was added to the CpGV-MMP or MMP-9 protein-substrate mixture. Fluorescence measurements were performed every 5 min at λex= 485 nm and λem= 535 nm. The error bars represent standard deviations for two independent protein purifications and assay repeats.