Figure 6

Identification of co-purified proteins using a chromosomally-coded His-tagged HerA. (A) Schematic diagram showing the construction of a strain that can encode a N-terminal His-tagged HerA by modification of the chromosomal herA. The MID strategy was used for the construction. (B) Confirmation of the constructed strain using PCR and X-gal staining. herA locus-specific flanking primers were used for PCR amplification. 1, the control strain E233S; 2, the constructed strain pMIDHis-herA-T. (C) Schematic map showing the purification of the proteins. (D) Western blot analyses of in situ N-His-tagged HerA and its co-purified proteins with specific antibodies. The fractions of 8, 9, 10, and 11 ml collected from the gel filtration were analyzed. C, purified protein. The standard molecular mass markers are indicated with arrows. (E) Pull-down assay confirming the interaction between HerA and the ATPase. A total of 50 μg HerA-C-His (or HerA) was mixed with 50 μg ATPase (or ATPase-C-His) at 70°C for 30 min in a buffer containing 25 mM Tris-HCl, pH 9.0, and 200 mM NaCl. The mixture was incubated with nickel-agarose beads. Unbound proteins were washed with the buffer above supplemented with 25 mM imidazole and target proteins were eluted by 250 mM imidazole. The fractions were detected by western blot with protein-specific antibodies. (F) Detection of the interaction between HerA and Hjc by pull-down assay. The procedure was as those used in (E). A total of 42 μg HerA-C-His and 12 μg Hjc were used in the assay.