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. 2009 Jun 24;83(17):8759–8770. doi: 10.1128/JVI.01777-08

FIG. 4.

FIG. 4.

Expression, purification, and size characterization of FlPV-1 E6. (A) Expression and purification. The MBP-FlPV-1 E6 fusion was overexpressed in bacteria (lane 1). The expression pellet was resuspended and sonicated, and the cleared supernatant was loaded onto MBP-specific amylose resin (lane 2, cleared supernatant input; lane 3, MBP-E6 eluate) and subjected to TEV digestion, yielding MBP and FlPV-1 E6 proteins (lane 4). FlPV-1 E6 was then separated from MBP and TEV by gel filtration (lane 5). (B) Analytical gel filtration. Purified FlPV-1 E6 was loaded onto an analytical Superdex 75 10/30 gel filtration column. The elution peak of FlPV-1 E6 (9.2 kDa) is indicated, together with those of myoglobin (17.6 kDa) and HPV-16 E6C (8.9 kDa), utilized as size markers. mAU, milli-absorbance units. (C) Analytical ultracentrifugation. The molecular weight distribution of purified FlPV-1 E6 was derived from sedimentation velocity experiments. The distribution corresponds to a protein of approximately 10 kDa, and no larger oligomeric form is detected.