Figure. 5
Sortase A-mediated covalent coupling of RBD and BLS. (A) SEC-HPLC profiles correspond to Gly-Gly-Gly-BLS (black), RBD (red), the control reaction mixture without Sortase A (violet) and the product of the reaction catalyzed by Sortase A, (green, lower panel). An equivalent volume of a 10 mM EDTA, 10 mM CaCl2 solution was loaded as a control (magenta, lower pane). Decameric BLS has a molecular weight of approximately 170 kDa, and each RBD subunit adds approximately 26 kDa or 40 kDa (excluding or including glycosylation, respectively). The observed multiplicity was ~ 6.6 (RBD6.6/BLS10) with a yield of ~ 30% relative to the initial RBD mass. The RBD-BLS oligomer eluted at ~ 25 min. The peak observed around 38–43 min corresponds to EDTA-Ca2+. Since Sortase A requires 10 mM CaCl2 for the activation of its catalytic activity, 10 mM EDTA was added to stop the reaction, which was carried out during 240 min at 4 °C. (B) SDS-PAGE analysis of Sortase A-mediated RBD-BLS coupling. To facilitate the detection of the Sortase A reaction products, the protein samples were treated or not with endoglycosidase H. Lanes 1–2: RBD + BLS without Sortase A, deglycosylated (lane 1), or not (lane 2), Lanes 3–4: RBD + BLS in the presence of Sortase A, deglycosylated (lane 3) or not (lane 4). Full Image of the SDS-PAGE is in Supplementary Material, Figure S5.
