Figure 2.

Construct design and purification of EcFeoC. A. Because of poor native expression, EcFeoC was expressed as a maltose-binding protein (MBP; salmon) fusion (MBP-EcFeoC). On the N-terminus is encoded an additional (His)₆ tag (purple) for orthogonal purification. Preceding the EcFeoC portion of the polypeptide (green) is an encoded TEV protease cleavage site. B. Cleaved, purified EcFeoC is monomeric (≈ 9000 g/mol, 9 kDa) based on its gel-filtration retention volume on Superdex 75. The compared standards (K_av versus log MW, linearity R²=0.97) are: blue dextran (void), alcohol dehydrogenase (150000 g/mol, 150 kDa), bovine serum albumin (66000 g/mol, 66 kDa), carbonic anhydrase (29000 g/mol, 29 kDa), cytochrome c (12000 g/mol, 12 kDa), and aprotinin (6500 g/mol, 6.5 kDa). C. SDS-PAGE analysis (acrylamide mass fraction of 15 %, left panel) and Tris-tricine gel analysis (gradient of acrylamide mass fraction from 10 % to 20 %, right panel), demonstrating EcFeoC purity after cleavage and SEC. Black arrows indicate the location of the purified EcFeoC. A small amount of dimeric EcFeoC (≈ 18000 g/mol, 18 kDa) is observed in the Tris-tricine analysis at high protein concentration, but this dimeric species is only observed after freeze-thawing of the protein and cannot be dissociated by sample boiling.