Figure 3.

Regulation of split protein activity in cells by competition with coexpressed or externally added peptides. (A) Schematic representation of luciferase activity attenuated by a displacer peptide and (B) of attenuation of luciferase reconstitution in HEK293T cells with co-expressed peptide N7. Reconstitution of CC–split luciferase (nLuc:N8 and cLuc:CC) was attenuated by a cognate CC-displacer peptide (N7) that forms stronger CCs. (C) Luciferase activity of HEK293T cells co-transfected with plasmids expressing nLuc:N8 (60 ng) and cLuc tethered with N7, P7A, P7, or P7SN (30 ng) and a plasmid expressing N7 displacer peptide (0, 80, and 155 ng). Luciferase activity was measured 48 h after transfection. (D) Schematic representation of luciferase activity attenuated by a displacer peptide, which was added to split luciferase-expressing cells. (E) Luciferase activity of HEK293T cells expressing the CC-split luciferase treated with N7 peptide (0–10 µM in DOTAP) for 2 h. Forty-eight hours after transfection of HEK293T cells with plasmids expressing nLuc:N8 (60 ng) and cLuc tethered with N7, P7A, P7, or P7SN (30 ng), cells were treated with CC-displacer peptide N7. (F) Schematic representation of luciferase activity attenuated by the addition of a displacer peptide with either matched or mismatched electrostatic motifs. (G) Luciferase activity of HEK293T cells treated with N7 or N5; P7A or P5A; P7 or P5 peptide (1, 2 or 10 µM in DOTAP) for 2 h. Cells were treated 48 h after transfection with plasmids expressing nLuc:N8 (30 ng) and cLuc:P7 (10 ng). The values (C, E, G) represent the means (± s.d.) from four independent cell cultures, individually transfected with the same mixture of plasmids, and are representative of two independent experiments. For the amounts of plasmids, see Table S2. Statistical analyses and the corresponding p-values are listed in Table S3.