Improvement of an Existing Part

Protein splicing is a unique post-translational phenomenon where an internal peptide sequence, known as the intein, removes itself from a non-functional precursor protein while concurrently ligating the flanking precursor ends (i.e., exteins) with a peptide bond to restore the reassembled precursor gene’s function. In the iGEM2021, team Moscow_City has demonstrated a constitutive protein splicing device using N_Intein (BBa_K4088892) and C_intein (BBa_K4088893).

In our iGEM2022 season, some efforts were made to make this protein splicing device controllable by blue light. To make this happen, we fused a light-sensitive lov2 domain on the C_intein. In the dark state, the flavin-binding LOV2 domain associates with its carboxyl-terminal helical extension (Jα) in a tightly docked conformation, presumably inhibiting the C_intein function.  Photoactivation of LOV2 leads to the unwinding and undocks the Jα helix, thereby allowing C_intein-mediated protein splicing (Figure 1).

However, by fusing the Lov2 domain directly to (BBa_K4088893), we observed no inhibition of the C_intein function under dark conditions. Since the truncation of the first few amino acids from the N-terminus of C_intein has been reported to improve Lov2-mediated light responsiveness, we hereby present C_Intein_t3 (BBa_K4414011), a 3-amino-acid truncated version of (BBa_K4088893) as an improvement to allow Lov2-mediated blue-light control of protein splicing.

Figure1. Schematic representation of Blue light control protein splicing system

We evaluated whether the improved C_intein can mediate the lov2-based blue light responsiveness by assaying the intein-mediated reconstitution of split-EGFP. Hence, we first fused the lov2 domain (BBa_K4016004) and the EGFP_71-239 domain on the N-terminus and the C-terminus of C_intein_t3 respectively. A similar construct using the original C_intein was also constructed as a head-to-head control. Also, another plasmid expressing the fusion protein containing the N_Intein (BBa_K4088892) and the EGFP_1-70 domain was obtained.

As a head-to-head comparison, HEK-293T cells were co-transfected with the lov2-C_intein_t3-EGFP_71-239;expressing and the EGFP_1-70-N_intein expressing plasmids, cells co-transfected with lov2-C_intein-EGFP_71-239;expressing and the EGFP_1-70-N_intein were used as control. Cells were either exposed to blue light (with ON [4 s]-OFF [56 s] cycles) or maintained under dark conditions since the 6th hour post-transfection. Cells were imaged or harvested for Western Blotting 48 h post-light illumination.

The fluorescent imaging of the cells expressing both lov2-C_intein-EGFP_71-239 and EGFP_1-70-N_intein proteins showed strong EGFP signal in both the blue-light-illuminated group and control group kept under dark conditions. Suggesting that lov2 is not capable of inhibiting the function of the original C_intein (BBa_K4088893) under dark conditions. Intriguingly, cells expressing both EGFP_1-70-N_intein and the improved lov2-C_intein_t3-EGFP_71-239 proteins showed nearly no fluorescent signal in control cells that were not illuminated by blue light, and a strong GFP signal in blue light treated group (Figure 1a). The reconstitution of EGFP was also validated via Western Blotting, which showed a significantly stronger EGFP signal in blue-light treated cells compared to the untreated cells (Figure 1b). These results demonstrated that the improvement from BBa_K4088893 to BBa_K4414011, enabled lov2-mediated light-inducible protein splicing in mammalian cells.

Figure2. Reconstitution of split-EGFP mediated by blue light-stimulated protein trans-splicing. (a) Top: the lov2-C_intein-EGFP_71-239 expressing and the EGFP_1-70-N_intein expressing plasmids in HEK-293 cells exposed in dark(left) or in blue light(right); Button: the lov2-C_intein_t3-EGFP_71-239 expressing and the EGFP_1-70-N_intein expressing plasmids in HEK-293 cells exposed in dark(left) or in blue light. (b) Immunoblotting of the indicated proteins in the lov2-C_intein_t3-EGFP_71-239 expressing and the EGFP_1-70-N_intein expressing cells w/wo blue light treatment.