Proteolytic sensitivity of FliC(XynA) in the polymeric and monomeric form.

<p>(A) Copolymers of flagellin and FliC(XynA) obtained by 0.6 M AS at 1∶1 mixing ratio (w/w) were incubated with trypsin at a 30∶1 (w/w) ratio. (B) Proteolysis of monomeric FliC(XynA) at a protein to protease ratio of 300∶1 (w/w). At the indicated time points, portions were removed from the reaction mixtures, mixed with electrophoresis sample buffer and boiled for 3 min. Experiments were done in 20 mM Tris–HCl, 150 mM NaCl (pH 7.8) at room temperature at a 1 mg/ml protein concentration.</p

Design of the FliC(XynA) fusion construct.

<p>The hypervariable D3 domain of Salmonella flagellin (residues 190–284) was replaced by xylanase A from <i>B. subtilis</i>. The C_α backbone trace of flagellin and XynA is rainbow-colored from blue to red representing the sequence from the NH₂- to COOH terminus.</p

Products of the polymerization of FliC(XynA) subunits.

<p>Dark-field light micrographs of (A) FliC(XynA) filaments (0.6 M AS) and (B) copolymers of flagellin and FliC(XynA) at 1∶1 (w∶w) mixing ratio (0.4 M AS). Polymerization experiments were done in 20 mM Tris-HCl (pH 7.8) containing 150 mM NaCl at 1 to 1.5 mg/ml protein concentration, and 4 M AS was added to a final concentration indicated in parentheses to initiate filament formation. (C) SDS/PAGE analysis of copolymer samples polymerized at FliC(XynA) to flagellin ratios of 1∶1 and 1∶2. After AS-induced (0.6 M) polymerization, samples were centrifuged and the pellet was dissolved in 20 mM Tris-HCl (pH 7.8) buffer containing 150 mM NaCl. The molecular masses of flagellin and FliC(XynA) are 51.5 kDa and 64 kDa, respectively. Band intensities determined by densitometry were {49.3 (1∶1); 50.7 (1∶2)} and {49.6 (1∶1) and 34.2 (1∶2)} for FliC and FliC(XynA), respectively. (D) Copolymers observed by atomic force microscopy.</p

Catalytic activity of recombinant xylanase A and the FliC(XynA) fusion protein in the monomeric and polymeric forms.

<p>Measurements were done at 1.2 µM enzyme (catalytic unit) concentration in 50 mM phosphate buffer (pH 6.0) at 37°C. The amount of FliC(XynA) subunits incorporated into the copolymers was estimated by densitometric analysis of SDS-PA gels of filament samples.</p