Structural and functional characterization of the Curli adaptor protein CsgF(1)
Untangling a Repetitive Amyloid Sequence: Correlating Biofilm‐Derived and Segmentally Labeled Curli Fimbriae by Solid‐State NMR Spectroscopy (2)
Curli are fibrillar cell adhesion proteins produced by Enterobacteriaceae such as Escherichia coli. They are involved in biofilm formation and attachment to surfaces. Amyloid fibrils are formed by the major Curli subunit CsgA and its nucleator CsgB. In CsgF deletion mutants, CsgA is not attached to the cell, polymerizes only inefficiently and accumulates in the extracellular medium.
Protein expression and purification
Recombinant CsgF was overexpressed in E. coli BL 21 (DE3). Cultures were grown in LB or minimal medium with 100 ug/mL Ampicillin at 37 °C to an optical density of ~ 1. Then, the temperature was reduced to 16 °C and the expression was induced by the addition of IPTG to a concentration of 0.5 mM. The cultures were incubated with agitation for 16 h and harvested by centrifugation (20 min, 6000 g). CsgF was purified under denaturing conditions. The entire
cells were incubated for 24 h in denaturing buffer (8 M guanidine hydrochloride, 150 mM NaCl, 100 mM potassium phosphate, pH 7.2), sonicated, and then centrifuged (45 min, 37 000 g). The supernatant was incubated with pre-equilibrated Ni2+ – Sepharose and passed through a gravity flow column. The beads were washed with a denaturing buffer including up to 20 mM Imidazole. After that CsgF was refolded on the column by stepwise reduction of Guanidine-HCl concentrations (8-6-4-2 M) and finally washing with 50 mM CHES pH 10, 100 mM NaCl, 10% glycerol. Folded CsgF was eluted in 50 mM CHES pH 10, 100 mM NaCl, 300 mM Imidazole. The protein purity was evaluated by SDS/PAGE and mass spectrometry. CsgF was dialyzed against 1% acetic acid, lyophilized, and stored dry until further use.
Recombinant CsgA was overexpressed in E. coli BL 21 (DE3). The culture was harvested by centrifugation (15 min, 6000 x g) and the cell pellets were resuspended in denaturing buffer A (8 M GuHCl, 50 mM K-phosphate, 150 mM NaCl, pH 7.2) and stirred for 48 hours. Sonication (30 min., 1s on / 4s off, amplitude 70%) was applied to the cell suspension to affirm the complete dissociation and to disrupt the DNA. The cell debris was removed by centrifugation (30 min, 36000 x g) and the supernatant incubated (≥ 30 min.) with equilibrated Ni2+-Sepharose. Subsequently, the solid phase was packed into a column and washed threefold with 25 column volumes of buffer A containing 5, 10 and 20 mM imidazole. Finally proteins were eluted with buffer A containing 300 mM (3 x 5 CV) and 500 mM (5 CV) imidazole. The purity was determined by SDS-PAGE after TCA precipitation. Elution fractions of high yield and purity were combined and filtered through an Amicon spin filter, 30000 MWCO.