Protein expression and purification

CCD fragments were expressed in E. coli C41 cells in LB broth and purified using Ni-NTA affinity chromatography. Lipoyl-CCD fusion constructs used for analytical gel filtration experiments were at this stage purified by size-exclusion chromatography. For MALS and crystallography, CCD fragments were cleaved and purified from their His-lipoyl tags using TEV protease followed by reverse Ni-NTA affinity and size-exclusion chromatography. Human and fly MBP-PB3 domains were expressed in E. coli B834 cells in LB broth and proteins were purified using Ni-NTA affinity, proteolytic (3C) cleavage, reverse Ni-NTA affinity and size exclusion. DmPB3 eluted in a single monomeric peak (by MALS analysis), while HsPB3 eluted as a single dimeric peak. To prepare the HsPB3:STIL^726-750 complex for crystallography, purified STIL^726-750 was added to purified HsPB3 in an ≳fourfold molar excess to ensure saturation. The resultant mixture was concentrated, then subjected to size exclusion in order to separate the complex from free STIL^726-750.