Figure 1

The bidirectional degron approach enhances the TEV protease induced protein instability (TIPI) system. (A) Schematic illustration of the bidirectional degrons. The degrons consist of an unstructured domain (orange) with cODC sequences (8 or 14 amino acids containing the cysteine-alanine (CA) motif), the TEV protease recognition site (ENLYFQ-F) and an N-degron fused to the affinity domain SF3b155^381-424 (blue). The affinity domain binds to the human protein p14, which was fused to the TEV protease. Usage of these affinity domains enhances cleavage of the degradation tags. (B) Regulation of target protein abundance by the TEV protease induced protein instability (TIPI) system using the bidirectional degron fused to the C-terminus of the target. Details can be found in the text. (C) Sequence of the GFP-TDegF tag with cODC insertions. Amino acids derived from yeast enhanced GFP [64] are shown in black - cODC1 (red) or cODC2 (blue) - spacer sequence (orange) - TEV protease recognition site (green). The cysteine-alanine motif is marked by bold letters; the cysteine in the cODC1 construct, which was mutated to alanine to verify proteasomal degradation is underlined; the TEV protease cleavage site is indicated (|); sequences of the N-degron and SF3b155^381-424 can be found in Figure S3 (see Additional file 1) or [25]. (D) Control of single domain stability by internal tagging of a two domain target protein. The TIPI system offers modules with stabilizing or destabilizing sequences flanking the TEV protease recognition site. The target protein is tagged between two domains (D1, D2). Depending on the modules used for modification of the target, the domains are destabilized or remain stable after cleavage. Phenylalanine and cODC1 confer destabilization, whereas methionine and unmodified TDeg should not influence protein stability. All possible combinations are shown; features of the different constructs are summarized in Table 1.