Fig. 4

In vitro assembly and MMC purification of a functionalized γPFD protein filament. a Strategy for the assembly of functionalized protein filaments by combining bacterial lysates containing different protein components. The bars indicate the relative volumes of lysates a, b c, and d (which correspond to γPFD-SpyT, CFP-SpyC, YFP-SpyC, and SpyC, respectively) used in the different mixtures. Increasing the ratio of SpyC in the lysate mixture interspaces the fluorescent proteins CFP-SpyC and YFP-SpyC attached to γPFD-SpyT. b SDS-PAGE showing recombinant protein in bacterial lysates and after MMC. The large γPFD-SpyT filaments (lane a) pass through the resin whereas the small protein components are retained in the resin. c Lysate mixtures before and after MMC. Lysate mixtures demonstrate the conjugation of varying ratios of SpyC and the fluorescent proteins CFP-SpyC and YFP-SpyC to filaments and subsequent purification using MMC. d Improved purification of functionalized filaments using a larger volume of CC700 resin followed by TX-114 phase separation. e Fluorescence emission spectra of each functionalized filament preparation. Decreasing the amount of SpyC increases the likelihood that CFP-SpyC and YFP-SpyC are located close to each other on the γPFD-SpyT filament, increasing FRET (indicated by the higher emission at 527 nm relative to 476 nm). f The FRET emission of each filament preparation exhibits a strong correlation with the amount of SpyT used as a spacer between the fluorescent proteins. Data points represent the average of two experiments