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Figure 2. Binding of recombinant putative CBD of Cnm protein to immobilized ECM proteins. (A) Strain ZAXF was grown in the presence of 2 x 10–3% arabinose as an inducer, and the crude cell-free extract was prepared as described in the text. Binding of recombinant Cnm protein to immobilized ECM proteins, collagen type I, fibronectin, and laminin was indicated as a function of protein concentration (1–10 µg in 20 µL of PBS with 0.1% BSA) of the extracts applied to the wells. BSA was used as a negative control. (B) The extract contained 10 µg protein from strain ZAXF cells grown in the absence of arabinose (uninduced ZAXF), and those from strains pBAD (as a negative control) and SBP6 (a strain expressing another histidine-tagged protein as a negative control) cells grown in the presence of 2 x 10–3% arabinose were also used as controls for binding of recombinant Cnm protein to collagen type I and laminin. (C) Biotin-labeled strains Z1 and 05A02 whole cells were examined for binding to the ECM proteins as described in the text.
We measured relative binding by monitoring absorbance at 490 nm following the peroxidase reaction for 3 min in the recombinant assays and for 4 min in whole-cell assays with o-phenylenediamine, and H2O2 was terminated with the addition of 2 M H2SO4. All OD490 nm values were corrected for the responses of peroxidase activities with the respective ECM proteins. Data points represent the means of OD490 nm values ± standard deviation from more than 3 independent experiments.
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