Journal: Toxins

Article Title: Purification and Characterization of His-Tagged Recombinant Bacteroides fragilis Toxin-2 Variants In Vitro and In Vivo

doi: 10.3390/toxins18040189

Figure Lengend Snippet: Purification and structural characterization of His-tagged BFT variants: ( a ) Silver-stained SDS-PAGE analysis of IMAC purification. Lane 1: His-rNTBF culture supernatant. Lane 2: Purified and concentrated I-hBFT. Lane 3: His-rETBF culture supernatant. Lane 4: Purified and concentrated A-hBFT. Purified hBFT samples show enrichment to near homogeneity with predominant bands at ~19 kDa. ( b ) Western blot analysis of culture duration effects on His-tagged BFT expression at 24 h and 48 h for I-hBFT (lanes 1–2) and A-hBFT (lanes 3–4). ( c ) AlphaFold3-predicted structures of mature I-hBFT and A-hBFT. The E349A mutation in I-hBFT disrupts the zinc-binding motif, highlighting structural basis for catalytic inactivation. Mutated alanine residue (A349) shown in red; original glutamic acid residue (E349) shown in green.

Article Snippet: Alternative purification was performed using the Native IMAC Buffer Kit (#6200239, Bio-Rad, Hercules, CA, USA) with gravity-flow glass columns.

Techniques: Purification, Staining, SDS Page, Western Blot, Expressing, Mutagenesis, Binding Assay, Residue