Fig. 1. Purification of ATR/TEM8 VWA domain. (A) SDS–PAGE analysis of stepwise purification. Samples were boiled in the presence of reducing agent and analyzed via 12% SDS–PAGE, followed by Coomassie staining. Lane 1, Prestained Precision Plus Protein Standards (kDa); lane 2, elution after Glutathione Sepharose 4B purification; lane 3, thrombin digestion of Glutathione Sepharose 4B fractions; lane 4, pool after thrombin cleavage; lane 5, pool of Q-Sepharose-HP column fractions. (B) SEC-FPLC analysis of purified ATR/TEM8 VWA domain. Purified ATR/TEM8 VWA domain was loaded onto a HiLoad 16/60 Superdex 75 pg using a BioRad Duo Separations Module FPLC.

Fig. 2. Q-TOF MS of purified ATR/TEM8 VWA domain. The predominant peak at 22,441.0 Da corresponds to the expressed ATR/TEM8 VWA domain with two extra residues from vector after thrombin cleavage (glycine and serine) at its N-terminus.

Fig. 3. SPR sensorgrams measuring the binding of PA to the recombinant CMG2 VWA domain (protein was generated based on the published protocol [36]) (a), or ATR/TEM18 VWA domain (b) in the presence of 1 mM Mn²⁺, or the binding of PA to the ATR/TEM8 VWA domain in 1 mM Ca²⁺ only (c).

Fig. 4. Intoxication blocking effect of the bacterially derived ATR/TEM8 VWA domain (dashed line) compared with the protein purified from mammalian system (solid line). Isolation of mammalian-produced ATR/TEM8 VWA was performed as described [24].

Purification of ATR/TEM8 A-domain from 4 L of E. coli culture