SG13009 Escherichia coli harboring pREP-4 plasmid and pQE-30 expression vector containing the fiber knob DNA sequence were cultured in 20 ml of LB broth with ampicillin (100 μg/ml) and kanamycin (50 μg/ml) overnight from glycerol stocks made in previous studies (54). One liter of Terrific Broth (modified, Sigma-Aldrich) containing ampicillin (100 μg/ml) and kanamycin (50 μg/ml) was inoculated with the overnight E. coli culture and incubated at 37°C until they reached an optical density of 0.6. Isopropyl-β-d-thiogalactopyranoside was then added to a final concentration of 0.5 mM, and the culture was incubated at 37°C for 4 hours. Cells were then harvested by centrifugation at 3000g, resuspended in lysis buffer [50 mM tris (pH 8.0), 300 mM NaCl, 1% (v/v) NP-40, lysozyme (1 mg/ml), and 1 mM β-mercaptoethanol], and incubated at room temperature for 30 min. Lysate was clarified by centrifugation at 30,000g for 30 min and filtered through a 0.22-μm syringe filter (Millipore, Abingdon, UK). Clarified lysate was then loaded onto a 5-ml HisTrap FF nickel affinity chromatography column (GE Healthcare) at 2.0 ml/min and washed with 5 column volumes into elution buffer A [50 mM tris (pH 8.0), 300 mM NaCl, and 1 mM β-mercaptoethanol]. Protein was eluted by 30-min gradient elution from buffer A to B (buffer A + 400 mM imidazole). Fractions were analyzed by reducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE), and fiber knob-containing fractions were further purified using a Superdex 200 10/300 size exclusion chromatography column (GE) in crystallization buffer [10 mM tris (pH 8.0) and 30 mM NaCl]. Fractions were analyzed by SDS-PAGE and pure fractions were concentrated by centrifugation in VivaSpin (10,000 molecular weight cutoff) (Sartorius, Goettingen, Germany) preceding crystallization.

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