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Functional unit of the PR PREDATOR, expressing GFPnano-hIgG1-Fc and HA tag-Trim21 fusion protein (Part: BBa_K2653016). These two proteins would form a ternary protein degradation complex with GFP and therefore lead its degradation. This part achieves the direct degradation of target protein GFP.
Our improved part is a Rapamycin-induced Predator Pro system (Part: BBa_K3396010). This improved part is designed to achieve an external signal-controlled target protein degradation, we replaced the Trim21 PRYSPRY domain and its interacting partner IgG Fc domain in original part with the FRB domain and FKBP domain, respectively. This replacement enables rapamycin-induced dimerization of truncated-Trim21-FRB and GFPnano-FKBP, leading to the formation of ternary protein degradation complex with GFP. Once rapamycin is added, the degradation of GFP would be triggered.
Figure 1. Schematic representation of Predator and RiPrePro.
This composite part can achieve rapamycin-induced degradation of target protein. To assess its degradation ability, several in vitro experiments including fluorescent imaging, dual-luciferase assay were therefore performed.
BBa_K3396010-containing plasmid and EGFP-expressing plasmid were co-transfected into 293T cells as experimental group. While the cells in control group were co-transfected with EGFP-expressing plasmid and empty plasmid. Cells were cultured for 24h before adding 2ng/ul mM rapamycin. Fluorescent imaging was performed 24h post rapamycin stimulation to quantify the GFP fluorescent intensity.
To test whether the GFP levels can be tuned and continuously regulated by rapamycin, HEK-293T cells were co-transfected with GFP expressing plasmid and RiPrePro1.0 plasmid/empty vector. Fluorescent imaging showed a decrease of GFP fluorescence in RiPrePro1.0 expressing group comparing to the control group (Fig 2A and 2B) under 1 ng/μL rapamycin induction. In addition, the degradation effect could be significantly improved with the increasing concentration of rapamycin (Fig 2C), demonstrating that the degradation manner of RiPrePro1.0 is controllable and tunable.
Figure 2. Rapamycin-induced Predator Pro system based on the FRB-FKBP interaction module. (A) Fluorescence images of the RiPrePro transfected group and its negative control transfected with an empty vector. HEK293T cells in both groups were transfected with GFP-expression plasmid. (B) Intensity quantification of the RiPrePro transfected group and its negative control transfected with an empty vector pcDNA3.1. HEK293T cells in each group was transfected with GFP-expression plasmid. (C) Heatmap of rapamycin concentration on the degradation effect of RiPrePro.