FIG. 1.

Purification of TrwK. Aliquots of pooled fractions corresponding to the different stages of the purification were resolved in sodium dodecyl sulfate polyacrylamide gels (10%) and stained with Coomassie blue. Lane a, cell lysates; lane b, SP-Sepharose flowthrough; lane c, Q-Sepharose elution; lane d, second Q-Sepharose elution; lane e, isocratic Superdex-200 elution. Both bands in lane e were excised manually from the gel and subjected to in-gel digestion with trypsin and analyzed by LC-MS/MS. Peptide fragment fingerprinting identified the larger band as TrwK and the smaller band as a TrwK N-terminal deletion (see the supplemental material).