Extended Data Fig. 4: Separate expression and reconstitution via annealing for a previously designed heterodimer (DHD) and its specificity when mixed with DHT03.
From: De novo design of obligate ABC-type heterotrimeric proteins
a Three chains for DHT03_3arm_A21/B21/C82 were separately expressed and individually purified, mixed together at an equimolar ratio (10uM each), annealed, and run through SEC. A peak corresponding to the heterotrimer is observed. b Two chains for DHD131 were separately expressed and individually purified (each with noticeably low solubility), mixed together at an equimolar ratio (10uM each), annealed, and run through SEC. A monodisperse peak corresponding to the heterodimer is observed. c All five chains for DHT03_3arm and DHD131 were mixed together at an equimolar ratio (10uM each), annealed, and run through SEC with two peaks observed matching the original controls in a and b, respectively. The main fraction from each peak was analyzed by native mass spec, which determined the correct reconstitution of the DHT03 heterotrimer and DHD131 heterodimer, without the presence of a chain from the other hetero-oligomer being tested.
