Figure 1
Optimization of the N-terminal APEX2(NA)/C-terminal APEX2(CA) complementation pair in cells. (A) Schematic of protein-fragment complementation (PFC) of APEX2. The interaction between proteins 1 (P1) and 2 (P2) brings inactive NA and CA into close proximity to reconstitute the peroxidase activity. This enzyme can biotin-label neighboring proteins within 20 nm for MS and catalyze the polymer of DAB for EM imaging. (B) The initial PFCs fused with FRB-flag and myc-FKBP12 to generate incremental truncation libraries. (C) Structure of wild-type soybean ascorbate peroxidase (1OAG). The NA and CA fragments are highlighted in green and yellow, respectively. (D) DAB staining for the rapamycin-induced FRB/FKBP12 association in live cells. HEK-293 cells were transfected with FRB-flag-NA and CA-myc-FKBP12, followed by treatment with rapamycin or DMSO as indicated. DAB staining was performed as described. Scale bars, 10 μm. (E) Biotin labeling was tested using the confocal image. HEK-293 cells were transfected with different plasmids as indicated and treated with rapamycin or DMSO. Biotin labeling was initiated with H2O2 following biotin-phenol incubation. Scale bars, 10 μm. (F) Western blot to test biotin labeling in the live cells. Cells were treated as in (E). The whole lysates were resolved by 10% SDS-PAGE and tested by streptavidin-HRP for biotinylated proteins, anti-flag antibody for FRB-NA and anti-myc antibody for CA-FKBP12. Equal amount of protein in each lane was loaded as quantified by β-actin. The original blots were presented in the Supplementary Fig. S3.
