Fig. 2
From: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering
Combined encapsulin:cargo construct and secreted encapsulin variant. a Scheme of a P2A bicistronic expression construct encoding StrepTagII-tagged (STII) nanocompartments containing Myc-tagged C as cargo protein (MycC-IntP2A-ASTII) as well as a variant with an N-terminal BM40 secretion peptide and StrepTagII (STII). b Cryo-electron microscopy image of material from HEK293T cells expressing MycC-IntP2A-ASTII purified via Strep-tag II/Strep-Tactin XT affinity chromatography showed the assembled nanospheres of ~32 nm diameter. Scale bar is 100 nm. c The corresponding BN-PAGE analysis of the identical material revealed a single band larger than 1.2 MDa. The accompanying silver-stained SDS-PAGE showed the coprecipitation of the cargo MycC with the StrepTagII-modified nanoshell. d Luciferase-based cell viability assay after 48 h of overexpression of MycC-IntP2A-ASTII and AFLAG with or without cargo BCDP2A. Cells overexpressing the fluorescent protein EYFP as well as untransfected HEK293T cells served as negative controls. To induce toxicity as positive control, untransfected HEK293T cells were treated with 1 mM H2O2 24 h prior to the assay. The bars represent the mean ± SEM (p = 0.442 excluding the positive control, Kruskal–Wallis, n = 6; no significant (ns) differences at α = 0.05 were found in Dunn’s multiple comparisons test between any of the encapsulin:cargo conditions and either EYFP or Control). e BN CM loaded with cell culture supernatant of HEK293T cells expressing ASTII with an N-terminal BM40 secretion signal showed a single band >1.2 MDa
