Fig. 1
From: Bacterial encapsulins as orthogonal compartments for mammalian cell engineering
Assembly of encapsulins and targeting of cargo in HEK293T cells. a Schematic of the heterologous expression of surface-modified encapsulin variants loaded with endogenous cargo proteins. b Genetic constructs encoding the shell protein A (light blue) with a FLAG-tag as C-terminal surface modification as well as individual Myc-tagged cargo proteins (red) B, C, and D that can also be combined in a multi-gene expression construct (BCDP2A). c Co-immunoprecipitation of AFLAG and silver-stained SDS-PAGE from cells co-expressing just B, C, or D, or a combination of these three proteins expressed either via a mixture of individual DNA constructs (MycB + MycC + MycD), or by a multi-gene expression construct (BCDP2A). The top panel shows a western blot (WB) against the exterior FLAG-tag in AFLAG. The bottom panel shows the corresponding WB against the Myc epitope. d Coomassie-stained Blue Native PAGE (BN CM) of purified material from HEK293T expressing AFLAG and BCDP2A yielding a band above 1.2 MDa. e Cell viability after 48 h of overexpression of encapsulins (AFLAG) with or without cargo (BCDP2A) assessed by an LDH release assay. A construct expressing the fluorescent protein mEos4b served as a control. The bars represent the mean ± SEM (p = 0.1965, Kruskal–Wallis, n = 4; no significant (ns) differences at α = 0.05 were found in Dunn’s multiple comparisons test between mEos4b and AFLAG expressed without or with BCDP2A)
