Extended Data Figure 3. EPN-01 constructs are released within membrane vesicles.
a, Detergent-dependent antibody accessibility of EPN-01 protein released from mammalian cells. Western blots of EPN-01 harvested from mammalian cell supernatants (Panels 1 and 2) or non-enveloped EPN-01* nanocages purified from E. coli (Panels 3 and 4) are shown. Samples were treated with 0.1% (Panels 1 and 3) or 0.5% CHAPS (Panel 2 and 4). Lane 1 shows input protein, and lanes 2–5 show bound and unbound fractions from immunoprecipitations with anti-Myc-Agarose (lanes 2 and 3) or control anti-rabbit-IgG-Agarose (lanes 4 and 5). b, Detergent-dependent aldolase substrate accessibility of EPN-01* protein released from mammalian cells. EPN-01* produced in 293F cells (top) or E. coli (bottom) were monitored for KPDG aldolase activity in the presence or absence of detergent. Note that the protein produced in mammalian cells only shows enzymatic activity in the presence of detergent, whereas the bacterially produced protein is equally active in both conditions. c, Size distribution of EPNs released into the culture supernatants of 293T cells expressing EPN-01*. n = 142 EPNs total, mean size = 107 ± 44. d, EPN-01* and associated cellular proteins are released from 293T cells. Western blots showing levels of I3-01-Myc, EPN-01*, ALIX, and actin expressed in 293T cells (left) and released into the culture supernatant (right). e, Numbers of EPN-01* protein nanocages encapsulated within six different EPN-01* vesicles of varying sizes. Nanocage numbers were determined by visual counting of reconstructed EPNs from cryo-tomographic tilt series. An average EPN vesicle of diameter 110 nm has a volume of 7 × 105 nm3 and each nanocage has an estimated volume of ~8 × 103 nm3. A 110 nm diameter vesicle could therefore theoretically contain as many as 90 close-packed nanocages, but they actually contain, on average, ~14 nanocages.