Visualization of the antibody–antigen interaction was achieved us

Visualization of the antibody–antigen interaction was achieved using the enhanced chemiluminescent reaction (Agilent Technologies). The affinity-purified antibodies showed cross-reactions with other as yet unidentified polypeptides in E. coli extracts. Blue-native (BN)-PAGE was performed with 5–15% gradient gels according to Schägger & von Jagow (1991). Far-UV CD spectra were recorded on a Jasco J710 spectropolarimeter. Spectra of purified FocA (0.3 mg mL−1) were recorded in 20 mM Tris-HCl, 150 mM NaCl, 0.2 mM EDTA,

pH 8, at 20 °C in a 0.5-cm cuvette. Ixazomib clinical trial Before recording spectra, FocA was centrifuged at 100 000 g for 1 h. The α-helical content of FocA based on the CD spectrum was determined using the program cdnn (Böhm et al., 1992). The β-galactosidase see more activity was determined and calculated according to Miller (1972). Each experiment was performed three times independently, and the activities for each sample were determined in triplicate. The activities are presented with SDs. Plasmids for the overproduction of N- (FocAStrep–N) and C-terminally (FocAStrep–C) Strep-tagged FocA protein were constructed as described (see Materials and methods).

In order to assess whether FocAStrep–N and FocAStrep–C were functional in vivo, plasmids pASK-IBA5focA and pASK-IBA3focA were introduced into E. coli strain RM201 (ΔfocA-pflB) containing a single-copy fdhF∷lacZ transcriptional fusion to monitor alterations in the intracellular formate concentration. RM201 cannot generate formate endogenously (Sawers & Böck, 1989) and therefore the fdhF promoter cannot be activated by formate-dependent FhlA unless formate is supplied exogenously (Rossmann et al., 1991). When grown anaerobically with glucose, but

in the absence of exogenous formate, RM201 λfdhF∷lacZ showed a basal β-galactosidase activity of approximately 5-Fluoracil purchase 30–35 U, regardless of which plasmid was transformed into the strain (Table 2). Inclusion of 50 mM formate in the anaerobic growth medium resulted in a β-galactosidase activity of approximately 1000 U for the strain transformed with the empty vectors pASK-IBA5 and pASK-IBA3 (Table 2). This high β-galactosidase enzyme activity indicates that formate was transported into the cells in the absence of FocA, representing the transport of formate by an as yet unidentified system(s) and diffusion of undissociated formic acid (see also Suppmann & Sawers, 1994). Introduction of the tagged FocA derivatives into the RM201 λfdhF∷lacZ increased β-galactosidase activity roughly 2–2.5-fold (Table 2). This increase indicates that the intracellular formate levels increased in the presence of both FocAStrep–N and FocAStrep–C, and demonstrates that both proteins were active in importing exogenous formate into anaerobic E. coli cells.

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