FIGURE 3: Limited proteolysis and glycosylation of secreted C-terminally tagged recombinant RBD. (A) Schematic overview and expected mass of the secreted RBD variants with swapped C-terminal peptide tags. Variant 2 with an N-terminal 3xHA tag instead of the N-terminal signal peptide served as a cytosolic control. Variant 4 also contains an SP20 sequence for extensive O-glycosylation. (B) Comparative Western blot analysis with antibodies against the RBD domain (left) or the 8xHis tag (right) in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures. An induced culture without plasmid served as negative control (n.c.) and recombinant His-tagged RBD with an expected size of 35 kDa as positive control (p.c.). The asterisk labels a band that was caused by a contamination of the supernatant fraction in the experiment shown. Both membranes were stripped and redecorated with an antibody against the HA tag. The calculated masses from panel A are indicated.