Figure 3

(A) Size fractionation of purified Inv197 and Inv497. A 200 g aliquot of Inv497 and Inv197 proteins cleaved from MBP–Inv derivative by factor Xa and purified from MBP was size-separated using a Superose 6 column. The elution profile of both proteins is shown, monitored by their absorbance at 280 nm. Marker proteins are as described in the Materials and methods. The peak fractions of these marker proteins and the void sample (Blue Dextran 2000) are indicated by arrows. (B) In vitro cross-linking of purified Inv497. A 2 g aliquot of purified Inv497 was cross-linked for 30 min at room temperature using increasing amounts of formaldehyde or Sulfo-EGS. Samples were added to SDS sample buffer, and not heated unless otherwise indicated, before loading on a 12% SDS–polyacrylamide gel. Shown is an immunoblot using anti-invasin mAb3A2. Sample heated to 100°C (lane 1), incubated in the absence of heating without cross-linker (lanes 2 and 6) and with 0.4% (lane 3) or 0.8% (lane 4) formaldehyde; treated with 0.8% formaladehyde, then boiled for 20 min (lane 5), treated with Sulfo-EGS at 1 mM (lane 7) and 2 mM (lane 8) final concentration. The molecular mass standard is shown on the left, cross-linked complexes are indicated by closed arrows, and the position of Inv497 and Inv197 is indicated by an open arrow.