Figure 6.
Detection of 15 sequential peptide affinity (SPA)-tagged small open reading frame-encoded proteins (SEPs) in Sinorhizobium meliloti crude lysates. (A) Schematic representation of the empty plasmid pSW2 (contains no promoter and no ribosome-binding site upstream of the linker [L] and SPA-encoding sequence) and a pSW2-SEP plasmid for the analysis of sORF translation. The constitutive PsinI promoter (hatched box), the corresponding TSS (flexed arrow), the sORF coding sequence with its −15-nt-long region, the SPA-tag (with its molecular size indicated) preceded by a linker (L) (gray boxes), and the Trrn terminator (hairpin) are depicted. (B)to (F) Western blot analysis of crude lysates (upper panels) and the corresponding Coomassie-stained gels, and (G)corresponding Ponceau-stained membrane for selected SEPs. Monoclonal FLAG-directed antibodies were used. Migration of marker proteins (in kDa) is shown on the left side. *Unspecific signal. Above the panels, the numbers of the analyzed SEP protein (Table S7), the presence (+) or absence (−) of a predicted TMH, and the molecular size (in kDa) of the SEP without the SPA tag are given. M: protein marker. C: empty vector control, lysate from a strain containing pSW2.