Figure 5.

In vitro cargo loading of T4GALA. A) Schematic of T4GALA in vitro cargo loading including protein cage disassembly, in vitro loading of targeting peptide-fused cargo (right) and T4GALA reassembly resulting in detectable fluorescence from newly encapsulated mNeon cargo. Conversely, the same procedure is carried out with mNeon lacking the targeting peptide, which fails to result in cargo loading (left) and results in no detectable fluorescence after reassembly. B) NativePAGE analysis showing high molecular weight bands for assembled T4GALA via Coomassie blue staining (top) and fluorescence analysis of mNeon (bottom). C) Size-exclusion chromatography analysis showing in vitro cargo loading of targeting peptide-fused mNeon (mNeonTP; absorbance at 506 nm, solid green line) via co-elution with T4GALA as compared to a relative lack of mNeon cargo loading when lacking targeting peptide (dashed lines). D) SDS-PAGE analysis (left) of size-exclusion results for T4GALA with and without mNeon (lanes 2 and 4, respectively; 8 mL fractions from SEC above) and respective excess unencapsulated mNeon (lanes 3 and 5, respectively; 17 mL fractions from SEC above). Corresponding TEM analysis (right) of mNeonTP-loaded T4GALA from preceding SEC (8 mL fraction; lane 2 from SDS-PAGE gel on left). Scale bar: 50 nm.