Fig. 4
From: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering
Multi-component processes and enzymatic reactions can be targeted to encapsulins in mammalian cells. a Overview schematic of sets of cargo molecules for bimolecular fluorescence and enzyme complementation inside the nanocompartment. Targeting of foreign cargo proteins can be achieved either via a minimal C-terminal encapsulation signal (EncSig) or via C‑terminal fusions to the native cargo proteins B, C, or D. b Silver-stained SDS-PAGE from a co-immunoprecipitation (Co-IP) of AFLAG co-expressed with photoactivatable mCherry1 with EncSig (PAmCherry1-EncSig) or with either one of the halves of split PAmCherry1 fused to C or B, or a combination of both (C-PA-s1 + B-PA-s2). Fluorescence originating from complemented split PAmCherry1 inside the encapsulins was detected on BN-PAGE loaded with whole cell lysates of cells expressing AFLAG and C-PA-s1 + B-PA-s2 after 2 min of photoactivation (PA) on an UV imager. c Live cell confocal microscopy images (scale bar represents 20 µm) of HEK293T cells expressing B-PAs1 and C-PAs2 with or without the shell-protein AFLAG before and after 60 s of photoactivation (PA) with 405 nm (upper panel) demonstrating efficient bimolecular fluorescence complementation inside encapsulin compartments. Fluorescence of photoactivated split PAmCherry1 was excited using a 561 nm laser. Fluorescence signals of the sample without and with AFLAG were quantified by calculating the ratio of the mean signal after PA divided by the signal before PA. The bars in the lower panel represent the mean fluorescence intensity ratios averaged over independent transfection experiments ± SEM (p = 0.0123, unpaired t-test, n = 3). d Luminescence signal from BN-PAGE incubated with luciferase substrate and loaded with whole cell lysates of HEK293T co-expressing split luciferase fragments fused to either B or C (B-SmBit, C-LgBit) and AFLAG (left panel). The luminescent band corresponds to the complemented split luciferase inside the assembled nanocompartment. The bar graph (right panel) shows the corresponding total luminescence signals from the cell lysates expressing B-SmBit and C-LgBit with or without AFLAG, (mean ± SEM across three independent transfection experiments, in each experiment three technical replicates were averaged, p < 0.0001, unpaired t-test, n = 3)
