FIGURE 1.

Tandem affinity purification. Lane A, tandem affinity purification with TAP-tagged MoeB. N-terminal TAP-tagged MoeB was expressed from pAF41 in strain CL100(DE3) (ΔiscS). Extracts were purified as described under “Experimental Procedures.” Eluted protein complexes were analyzed by 12% SDS-PAGE. The indicated bands were excised from the gel, digested with trypsin, and analyzed by MALDI peptide mapping. The 47 kDa band was identified as YnjE, the 26 kDa band was identified as MoeB, and the 31 kDa band was identified as calmodulin-binding peptide CB-MoeB (indicated by arrows). Lanes B–D, tandem affinity purification with N-terminal TAP-tagged YnjEΔ1–21. N-terminal TAP-tagged YnjEΔ1–21 was expressed from pAU1 in strain JLD42301(DE3) (ΔynjE). Extracts were purified as described under “Experimental Procedures.” Eluted protein complexes were analyzed by SDS-PAGE followed by Coomassie Blue staining (B) and immunodetection after transfer to a PVDF membrane (C and D). Lane B, 12% SDS-polyacrylamide gel of the eluted proteins after IgG-Sepharose and calmodulin-Sepharose. The stained bands were excised from the gel, digested with trypsin, analyzed by MALDI peptide mapping, and confirmed by MS/MS analyses. The 47 kDa band was identified as TAP-tagged YnjEΔ1–21, and the 26 kDa band was identified as MoeB (indicated by arrows). Lane C, TAP-tagged YnjEΔ1–21 detected with polyclonal YnjE antiserum. Lane D, MoeB detected with a polyclonal MoeB antiserum.