Fig. 2
The expression of antibody fragments in S. cerevisiae. a Schematic of CPOTud-based vector for expression of antibody fragments in S. cerevisiae. VhH/VH, variable heavy chain; VL, variable light chain; CH1, the first constant domain of the heavy chain; CL, constant light chain; FLAG, FLAG-tag at the C-terminus of genes; his6, 6xHis-tag at the C terminus of genes; α-pre and α-pro, the components of α-factor leader, which is a native secretion leader; 2A, 2A peptide; Yap3-TA57, a synthetic secretion leader; POT1, selective marker from Schizosaccharomyces pombe to complement the lack of the TPI1 gene in the host; 2 μm, 2 micron origin. b SDS-PAGE gel for the detection of antibody fragments in the HA host strain. 1, HA.CPOTud; 2, HA.Nan; 3, HA.Ran; 4, HA.Ran (non-reducing); 5, HA.Pex; M, protein ladder; 6, HA.CPOTud; 7, HA.Nan; 8, HA.CPOTud; 9, HA.Ran; 10, HA.CPOTud (non-reducing condition); 11, HA.Ran (non-reducing condition); 12, HA.CPOTud; 13, HA.Pex; 1–5, non-concentrated supernatant; 6–13, supernatants concentrated 25-, 50- or 270-fold using 10 K MW Pierce Concentrator PES. Arrows indicate expected proteins. MW, protein ladder. Western blots of cell supernatant from recombinant strains based on the HA host strain to detected proteins Nan (c), Pex (d) and Ran (e). Secreted antibody fragments were obtained after cultivation in SD-2xSCAA without BSA for 72 h. Nan, Pex and Ran-H were detected using an anti-6x-His-tag monoclonal antibody. Ran-L was detected using an anti-FLAG-tag monoclonal antibody. Nan and Pex were analyzed under reducing conditions. Ran was analyzed under reducing (R, left lane in each panel) and nonreducing (NR, right lane of each panel) conditions. The original images of the western blots are shown in Additional file 1: Fig. S1