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. Author manuscript; available in PMC: 2020 Aug 14.
Published in final edited form as: Cell Host Microbe. 2019 Aug 14;26(2):203–216.e6. doi: 10.1016/j.chom.2019.07.007

Figure 5. Producing soluble multi-hexamer/pentamer assemblies.

Figure 5.

(A) A schematic of di-hexamer assembly (left) and SDS PAGE analysis of the CASpyCat-CASpyTag reaction (right). The SpyTag/SpyCatcher reaction proceeded essentially to completion. (B) Hexamer-SpyCatcher/Tag assembly and purification. CA and CA-Spy fusions are mixed at an appropriate ratio to assemble hexamers (top), purified by anion-exchange chromatography (middle), and analyzed by non-reducing SDS-PAGE (bottom). Hexameric species containing specific amounts of CA-SpyTag/Catcher fusion molecules were efficiently separated. (C) Schematics of producing Tri-hexamer (top) and Hepta-hexamer (bottom right) assemblies. (D) Schematics of pentamer-hexamer assemblies. (E-H) SEC (SEC-MALS in (E)), non-reducing SDS-PAGE, and negative-stain EM analysis of assembled Di-hexamers (E), Tri-hexamers (F), Hepta-hexamers (G), and Pentamer-hexamers (H). Sample micrographs are shown, as indicated, with associated 2-D class averages. In (E) and (F), the 2-D class averages of constructs containing 184A/185A mutant interfaces show non-contacting hexamer subunits. See also Figure S5.