Conalbumin

Supernatant (500 µl) from Transwell-grown MDCK cells infected with PAK for 60 minutes was collected by aspiration; residual bacteria was removed by filtration through a 0.22 micron filter (Millipore) and used immediately. Supernatant sterility was confirmed by plating ∼50 µl on LB agar plates. Exponential phase PAKΔpopB were pelleted by centrifugation (14000 RPM×5 min), re-suspended in 500 µl of filtered supernatant, and inoculated on the apical surface of Transwell-grown MDCK cells (MOI = 100). Cell-associated aggregation was quantified by spinning disk confocal microscopy. Results are normalized to PAK control (100%) and reported for three or more independent experiments.
To further characterize the aggregate-inducing factor, filtered supernatants were subjected to one of several treatments prior to addition of PAK. Heat treatment used 95°C for 30 minutes. Proteinase K (Sigma) was added at a final concentration of 200 µg/ml to supernatants and incubated for 60 minutes at 37°C. A stock solution of conalbumin (Sigma) was prepared in MEM immediately prior to use and added at a final concentration of 20 µg/ml to supernatants. Biofilm formation was normalized to PAK control with untreated filtered supernatant (100%). Results are reported for three or more independent experiments.

The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His₁₀-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min⁻¹ with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M⁻¹cm⁻¹ (StyI) and 48,150 M⁻¹cm⁻¹ (StyJ) as well as molecular weights of 29,836.3 g mol⁻¹ (StyI) and 30,095.6 g mol⁻¹ (StyJ) as predicted by Expasy ProtParam (56 (link)).

The seven-protein mix comprised human serum albumin (HSA), cytochrome C (bovine heart), ovotransferrin (Conalbumin, chicken egg white), myoglobin (equine heart), lysozyme C (chicken egg white), and catalase (bovine liver), all purchased individually from Sigma Aldrich (St. Louis, MO). Creatine kinase Type M (rabbit muscle) was purchased from Roche (Basel, Switzerland). The cross-linker BS³ was purchased from Thermo Scientific Pierce (Rockford, IL).

The molecular masses of the purified proteins in a denatured state were analyzed by SDS-PAGE (53 (link), 54 ). Immunodetection of the His₁₀-tagged proteins was performed via Western blotting (55 (link)) using a nitrocellulose blotting membrane (Amersham Protran 0.45-μm NC, GE Healthcare, Germany) and a Penta-His horseradish peroxidase (HRP) conjugate antibody (Qiagen, Germany).
Native sizes were analyzed via size exclusion chromatography on a HiLoad 16/600 Superdex 75 pg (GE Healthcare, Germany) using 10 mM Tris-HCl, pH 8, with 300 mM NaCl at a flow rate of 1 ml min⁻¹ with an ÄKTA purifier (Amersham Pharmacia Biotech, UK). Calibration was performed with standard proteins of known sizes (RNase A, 13,700 Da; carbonic anhydrase, 29,000 Da; conalbumin, 75,000 Da; aldolase, 158,000 Da; blue dextran; Sigma-Aldrich, Steinheim, Germany) under similar conditions. The resulting standard curve was used for size determination of respective elution peaks of StyI and StyJ. Peak fractions were collected and analyzed by SDS-PAGE and Western blotting.
Final concentrations of pure proteins were calculated from the respective absorptions at 280 nm, applying molar extinction coefficients of 42,860 M⁻¹cm⁻¹ (StyI) and 48,150 M⁻¹cm⁻¹ (StyJ) as well as molecular weights of 29,836.3 g mol⁻¹ (StyI) and 30,095.6 g mol⁻¹ (StyJ) as predicted by Expasy ProtParam (56 (link)).

Ubiquitin (8.6 kDa) from bovine erythrocytes, cytochrome C (12.4 kDa) from equine heart, myoglobin (17.6 kDa) from equine heart, carbonic anhydrase (29 kDa) from bovine erythrocytes, bovine serum albumin (BSA; 66.4 kDa), conalbumin (77 kDa) from chicken egg white, concanavalin A (102 kDa) from Canavalia ensiformis, alcohol dehydrogenase from Saccharomyces cerevisiae (147.5 kDa), trastuzumab monoclonal antibody (148 kDa), beta amylase (β amylase; 223.8 kDa) from sweet potato, pyruvate kinase (232 kDa) from rabbit muscle, apoferritin (480 kDa) from equine spleen, chaperonin 60 (GroEL; ~802 kDa) from Escherichia coli, ammonium acetate, tris acetate, potassium chloride, ethylenediaminetetraacetic acid (EDTA), adenosine‐5′‐triphosphate (ATP), magnesium chloride, ammonium hydroxide, and acetic acid were all purchased from Sigma‐Aldrich (Zwijndrecht, The Netherlands). Pierce™ LTQ ESI positive ion calibration solution (195 to 1522 Da) and cesium iodide (CsI; 392.7 to 11,304 Da) were purchased from Thermo Fisher Scientific, The Netherlands. Glu‐fibropeptide B (1.6 kDa) was obtained from Waters, The Netherlands. Methanol, acetone, isopropanol, and LC–MS grade water were purchased from Biosolve (Valkenswaard, The Netherlands).