Figure 5.

In vitro Fc fusion by PTS (a) Schematic drawing of the ligation of model proteins (SUMO and GB1) with the C-terminal Fc domain by PTS. (b) SDS-PAGE analysis of the N- and C-terminal precursor proteins (H₆-GB1-Int_N, Int_C-CPPC-Fc, and H₆-SUMO-Int_N) independently purified by IMAC. (c) Time course of the ligation reaction between H₆-GB1-Int_N and Int_C-CPPC-Fc in the absence (left panel) or presence (right panel) of 0.5 mM TCEP. Samples were taken after 0, 1, 2, 3, and 20 h of incubation, and analyzed by SDS-PAGE. (d) The effect of 5 mM DTT instead of 0.5 mM TCEP was tested for the reaction mixture of H₆-GB1-Int_N and Int_C-CPPC-Fc. Samples were analyzed by SDS-PAGE after 0, 2, and 7 h of incubation. (e–g) Comparison between the three Fc constructs (Int_C-CPPC-Fc (e), Int_C-Fc (f), and Int_C+1S-Fc (g)). Protein ligation with H₆-SUMO-Int_N was analyzed with and without 0.5 mM TCEP by SDS-PAGEs. Incubation times in hours (h) are indicated above the lanes of SDS-PAGEs.