FIG. 2.

Use of protein pull-down assay for identification of S. marcescens proteins interacting with pirin_Sm. Glutathione-Sepharose 4B beads were used to capture protein complexes comprising GST-pirin_Sm fusion protein and the potential pirin_Sm interactors in CH-1. After elution, SDS-PAGE analysis was performed for protein separation. The cell lysate of E. coli DH5α containing either pSC10 (GST-pirin_Sm fusion protein was oversynthesized) (lane 1), pSC11 (GST-NlpB_Sm [28] fusion protein was oversynthesized) (lane 2), or pGEX (GST Tag only) (lane 3) was mixed with CH-1 lysate at a volume ratio of 1:1. Lane 4, GST-pirin_Sm fusion protein; lane 5, GST-NlpB_Sm fusion protein; lane 6, GST Tag protein; lane M, protein markers. Identification of the separated proteins was achieved by amino acid sequence analysis using electrospray ionization-MS/MS and comparison of the partial amino acid sequences with nonredundant protein databases in the SEQUEST Browser (http://fields.scripps.edu/sequest/). The three proteins with highest identity scores were E. coli PDH subunit E1 (band a, 99 kDa), E. coli PDH subunit E2 (band b, 76 kDa), and E. coli succinate dehydrogenase subunit E2 (band c, 50 kDa).