Figure 3
Identification of putative B-cell epitopes to the serum AgSAA immunodetection. (a) Alignment of mature AgSAA and hSAA1 (UniProtKB: P0DJI8) with four hSAA1 derived peptides (peptides 1–4)64. A. gueldenstaedtii homologues to these peptides were named p26-AgSAA (aa 26–43), p58-AgSAA (aa 58–71), p67-AgSAA (aa 67–83) and p88-AgSAA (aa 88–103). Black bars show putative AgSAA B-cell epitopes predicted in silico (Bepipred algorithm). (b) AgSAA structure model built with mSAA (PDB: 4Q5G) as template (SWISS-MODEL). Peptides p26-AgSAA (pink), p58-AgSAA (blue), p67-AgSAA (green) and p88-AgSAA (red) are indicated. Note that p58-AgSAA and p88-AgSAA are exposed at opposite sides of the protein. (c) Expression and purification of rAgSAA in E. coli. Coomassie-stained SDS-PAGE-TRICINE corresponding the total fraction of an uninduced culture (lane 1), the total and soluble fractions of the IPTG-induced culture (lanes 2 and 3), the affinity-purified His-MBP-AgSAA (lane 4), TEV-digested His-MBP-AgSAA (lane 5) and pure rAgSAA obtained after negative purification by IMAC followed by amylose affinity chromatography (lane 6). The band corresponding to His-MBP-AgSAA (54.5 kDa, white head arrow), His-MBP (43.1 kDa, black head arrow), TEVSH (white arrow) and the band corresponding to rAgSAA (11.4 kDa, black arrow) are indicated. Samples were run on three separated gels. The regions of interest in each gel were cropped, aligned using the Mw marker as a reference. The original gels are shown in Supplementary Figure S13. (d) Detection of rAgSAA and native AgSAA by Western blot. rAgSAA, sturgeon serum and prestained Mw marker were run on SDS-PAGE gels and transferred to PVDF membranes. For blotting, membranes were cut and incubated separately with anti-p58-AgSAA-Ig (dilution of 1:4000, 150 s exposure time) or anti-p88-AgSAA-Ig (dilution 1:1500, 150 s exposure time). Figure shows the merge image of chemiluminescent and the prestained Mw marker images. Black head arrows indicate the signal band corresponding to AgSAA.
