Figure 3.

Antibody reconstitution mediated by PTS using split intein Npu DnaE analyzed by SDS-PAGE. (a) Antibody fragments Fab Int^N and huFc Int^C were mixed in a ratio 2:1 and incubated for 2 h at 37°C in the presence of 0.5 mM reducing agent TCEP. Samples were taken after every 10 min and analyzed by SDS-PAGE (Lane 1–7). Lane 1 shows antibody fragments Fab-Int^N (38.3 kDa) and huFc-Int^C (31.7 kDa) under reduced conditions at 0 min. The new band at 49.8 kDa in Lanes 2–7 indicates the reconstituted heavy chains (HCR) of Fab and huFc under reduced conditions. Lane 7 shows complete depletion of antibody fragments Fab-Int^N and huFc-Int^C after 2 h. (b) Lane 1 illustrates antibody reconstitution with a surplus of Fab-Int^N over huFc-Int^C (3:1 ratio). Non-reconstituted antibody fragments were still present after 2 h (Lane 2). Non-reconstituted antibody fragments were purified by addition of Ni²⁺ beads after incubation of 1 h at RT, leaving only the reconstituted antibody (Lane 3, S). Non-reconstituted antibodies were captured by Ni²⁺ beads (Lane 4, E: Elution with 500 mM imidazole). Final antibody reconstitution was achieved after re-oxidation with DHAA for 2 h at 37°C (Lane 5, Ox.).