Fig. 6
From: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering
Efficient iron loading of eukaryotically expressed encapsulin nanospheres a Schematic of a dual-promoter construct used for generation of a stable cell line expressing AFLAG and all native cargos B, C, and D. Also depicted is a construct encoding the iron-transporter MmZip14FLAG (Zip14) used to transport additional amounts of iron into the cell. b Co-immunoprecipitation (Co-IP) against the FLAG epitope from a whole cell lysate of a stable HEK293T clone expressing AFLAG together with B, C, and D analyzed by silver-stained SDS PAGE and the corresponding WB against the FLAG epitope (left panel). The pair of Blue Native (BN) gels visualizes proteins from whole cell lysates via Coomassie staining (CM) (left panel) and iron content via treatment with DAB enhanced Prussian Blue (DAB PB) (right panel) from the same stable cell line. Robust iron loading of the assembled nanocompartments was achieved by transient co-expression of MmZip14FLAG-IRES-ZsGreen1 in which case 0.25 mM ferrous ammonium sulfate (FAS) for 48 h was sufficient to see strong iron loading. c BN gel stained with CM or DAB PB loaded with whole cell lysates of HEK293T cells transiently expressing AFLAG + BCDP2A and Zip14FLAG supplemented with different concentrations of FAS (0–3 mM) for 48 h (upper panel). The strong bands, which correspond to the assembled nanoshell, indicate high expression levels of encapsulins and efficient, dose-dependent iron loading. The lower panel shows a CM and DAB PB-stained BN gel from whole cell lysates of HEK293T cells expressing Zip14FLAG and different combinations of native cargo molecules: MycB, MycC, and MycD alone, or all three (BCDP2A) with or without AFLAG. The robust DAB PB stains show that the ferritin-like cargo proteins B or C are sufficient for iron loading into encapsulins. FAS was supplemented at 2.5 mM for 48 h
