FIG. 3.
Amino acid sequence analysis of peptides encoded by yopN1-12 secretion signals. (A) YopN1-12-Npt, synthesized from wild-type (WT) yopN1-12 secretion signal, was purified from bacterial extracts generated via French pressure cell by chromatography on a MonoQ column. The crude lysate (L, for load) and eluate (M, for MonoQ) were subjected to SDS-PAGE and electrotransfer to PVDF membrane. The membranes were analyzed by immunoblotting with αNPT or were stained with Coomassie brilliant blue. The arrow points to the species that was subjected to Edman degradation. (B) Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample generated in panel A. High concentrations of glycine that eluted during each cycle were interpreted as sample contamination by electrotransfer buffer. (C) YopN1-12-Npt, synthesized from the yopN1-12 wobble variant (see Fig. 2), was purified from bacterial extracts generated via French pressure cell by chromatography on a MonoQ column. The crude lysate (L) and eluate (M) were subjected to SDS-PAGE and electrotransfer to PVDF membrane. The membranes were analyzed by immunoblotting with αNPT or were stained with Coomassie brilliant blue. The arrow points to the species that was subjected to Edman degradation. (D) Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample depicted in panel C. (E) YopN was purified by reversed-phase high-performance liquid chromatography (HPLC) on a C18 column from the supernatant of centrifuged Y. enterocolitica strain 8081 cultures, separated on SDS-PAGE, and stained with Coomassie Brilliant Blue. The arrow points to the species that was subjected to Edman degradation. Molecular size markers (Marker) were included for size measurements. (F) Edman degradation profile reveals the elution of phenylthiohydantoin residues per sequencing cycle of the sample shown in panel E. MW, molecular size.