FL1-BP and FL2-BP from B. infantis JCM 1222T were expressed as both nontagged and histidine (His)–tagged fusions. The genes encoding the mature proteins of FL1-BP (amino acid residues 32 to 469) and FL2-BP (amino acid residues 26 to 460) were amplified by PCR and inserted into the Nde I and Xho I sites of pCDF23 (33) using primers enlisted in table S3 (nos. 30 and 31). The resulting sequence-verified plasmids were introduced into E. coli BL21 (DE3) ΔlacZ carrying pRARE2 (19). Transformants were grown in LB medium supplemented with Sp and Cm to OD600 = 0.5, and thereafter, the protein expression was induced with isopropyl β-d-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM. Following overnight incubation at 18°C, cells were harvested, resuspended in 50 mM sodium phosphate buffer (pH 7.0), and disrupted by sonication. The clear lysate obtained by centrifugation was 80% saturated by ammonium sulfate, and the resulting supernatant (>80% fraction) was dialyzed against 20 mM tris-HCl (pH 8.0). Retentates were concentrated with an Amicon Ultra 10 K centrifugal device (Merck Millipore, Burlington, MA, USA) and loaded onto a Mono Q 5/50 GL column (GE Healthcare, Chicago, IL, USA), and proteins were eluted by a linear gradient of 0 to 1 M NaCl in the same buffer. The eluted fractions containing FL-BPs were combined, further purified by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column (GE Healthcare), and used for ITC analysis.

His-tagged proteins were prepared as follows: The genes encoding FL1-BP (amino acid residues 29 to 469) and FL2-BP (amino acid residues 24 to 460) were PCR-amplified and inserted into Nco I and Eco RI sites of pETM11 [a gift from G. Stier, EMBL (European Molecular Biology Laboratory), Center for Biochemistry, Heidelberg, Germany]. The primers used are listed in table S3 (nos. 32 and 33). The recombinant proteins were produced in E. coli BL21 (DE3) as an N-terminal fusion with a TEV (tobacco etch virus nuclear inclusion a endopeptidase)–cleavable His-tag separated by a three–amino acid insertion (Gly-Ala-Met). Protein production was performed by growing the cells in LB medium containing kanamycin at 37°C to OD600 = 0.5; thereafter, the temperature was reduced to 21°C, expression was induced by addition of IPTG to 0.1 mM, and growth was continued for 16 hours. Cells were harvested by centrifugation, resuspended in binding buffer [10 mM Hepes (pH 7.4), 500 mM NaCl, 10 mM imidazole, 10% glycerol, and 0.5 mM dithiothreitol], and lysed by a single passage through a high-pressure homogenizer. After centrifugation, clarified lysates were applied onto a 5-ml HisTrap HP column (GE Healthcare) and purified as recommended by the manufacturer. Eluted pure fractions were pooled, concentrated as described above, applied to a HiLoad Superdex G75 26/60 gel filtration column (GE Healthcare), and eluted with 10 mM MES buffer (pH 6.5) at 1 ml/min. His-tags were cleaved using TEV protease using a standard protocol. Cleaved proteins were recovered after passing through a HisTrap HP column (1 ml) pre-equilibrated with the binding buffer, concentrated as described above, and stored at 4°C until further use. These protein preparations were used for SPR analysis and crystallization. Purity was assessed by SDS–polyacrylamide gel electrophoresis. The protein concentration was determined spectrophotometrically at 280 nm using a theoretical absorption coefficient of 77,810 M−1 cm−1 for FL1-BP and 81,820 M−1 cm−1 for FL2-BP, calculated based on the amino acid sequence.

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