Fig. 2.

Specific encapsulation of two cargo proteins. (A) Schematic of T_{1-spyt-snpt-6xHis} depicting the position of the linkers 1 to 3, the SpyTag, and the SnoopTag. (B) Schematic of a HO-shell wiffleball with T_{1-spyt-snpt-6xHis} T₂ and T₃ and conjugated cargo mTurquoise2_SnpC (blue) and _SCmVenus (yellow). (C) Schematic of a HO-shell minimal wiffleball with only T_{1-spyt-snpt-6xHis} and conjugated cargo mTurquoise2_SnpC (blue) and _SCmVenus (yellow). (D) NiNTA purification of wiffleballs coexpressed with mTurquoise2_SnpC and _SCmVenus. Lane 1: BMC-H and T₁; lane 2: BMC-H and T_{1-spyt-snpt-6xHis}; lane 3: BMC-H, T₁, T₂ and T₃; lane 4: BMC-H, BMC- T_{1-spyt-snpt-6xHis}, T₂ and T₃. (Left) Coomassie stained SDS/PAGE gel. H T_{1-spyt-snpt-6xHis} and H T_{1-spyt-snpt-6xHis} T₂T₃ architectures show conjugations between T_{1-spyt-snpt-6xHis}, mTurquoise2_SnpC and _SCmVenus, either individually (∼80 kD) or together (∼120 kDa). (Right) α6xHis Western blot. T_{1-spyt-snpt-6xHis} contains a 6xHis tag used for detection. Conjugation bands between T_{1-spyt-snpt-6xHis} and the fluorescent proteins can be observed. (E) Fluorescense emission spectrum after mVenus excitation at 490 nm. The emission spectra were normalized to mVenus emission intensity. (F) Fluorescense emission spectrum using mTurquoise2 excitation at 425 nm avoiding mVenus excitation. mVenus emission can be observed in the loaded wiffleballs due to FRET, while this isn’t present when only mVenus and mTurpuoise2 are expressed by themselves without being loaded into the wiffleballs (mVenus+mTurquoise2). Emission spectra were normalized to mTuriquise2 emission intensity. (G) TEM image of H T_{1-spyt-snpt-6xHis} minimal wiffleballs. (Scale bar, 200 nm.) (H) TEM image H T_{1-spyt-snpt-6xHis}T₂T₃ full wiffleballs. (Scale bar, 200 nm.)