Fig. 2: Chimeric Lpp-OmpA-eMA SNAP enables controllable antigen loading on OMVs.

a Dose-response curve generated by loading biotin-GFP (b-GFP) or unmodified GFP (GFP) on SNAP-OMVs isolated from hypervesiculating E. coli strain KPM404 ΔnlpI expressing the Lpp-OmpA-eMA construct from plasmid pTrham (induced with 0.5 mM L-rhamnose). Blank OMVs were isolated from plasmid-free KPM404 ΔnlpI cells. Binding activity was determined by ELISA in which Lpp-OmpA-eMA SNAP-OMVs were immobilized on plates and subjected to varying amounts of biotin-GFP, after which plates were extensively washed prior to detection of bound biotin-GFP using anti-polyhistidine antibody to detect C-terminal 6xHis tag on GFP. Data were normalized to the maximum binding signal corresponding to Lpp-OmpA-eMA SNAP-OMVs in the presence of 3.3 nM biotin-GFP. Data are the mean ± SD with n = 2 biologically independent experiments. b Same OMVs as in (a) but dose-response was generated by first incubating OMVs with biotin-GFP or unmodified GFP in solution, washing to remove unbound protein, and determining GFP levels by ELISA-based detection using a standard curve with known amounts of GFP mixed with OMVs. Data are the mean ± SD with n = 3 biologically independent experiments. c Comparison of GFP levels on Lpp-OmpA-eMA SNAP-OMVs versus ClyA-GFP OMVs. ClyA-GFP OMVs were isolated from KPM404 ΔnlpI cells expressing ClyA-GFP fusion construct from plasmid pBAD18 as described in Kim et al.¹⁸. Data are the mean ± SD with n = 6 biologically independent experiments for all cases except for ClyA-GFP OMVs where n = 3 biologically independent experiments. d Transmission electron micrograph of Lpp-OmpA-eMA SNAP-OMVs alone or following incubation with unmodified GFP or biotin-GFP as indicated. The scale bar represents 200 nm. The Z-average diameter of each formulation was measured by DLS and reported below each corresponding micrograph. Micrographs representative of two independently repeated experiments with similar results. Source data are provided as a Source Data file.