Extended Data Fig. 1: “ABC” design and purification and characterization of “ABC” tricistronic and “AB” bicistronic constructs.

a, ΔddG filter metric. b, ΔScore metric. Dark red points correspond to the single mutation P114F. The bright red point corresponds to the double mutant P114F/F131V. The red dotted boxes represent cutoffs used to select mutants for testing. c, Recovery of trimer geometry was assayed by assembling double mutant I53-50A trimers in clarified E. coli lysates with purified I53-50B pentamer and evaluating the presence or absence of I53-50 nanocages by native PAGE. Black wedges indicate increasing pentamer concentration in each series of assembly reactions. For gel source data, see Supplementary Fig. 2. d, The ABC heterotrimer was purified by IMAC with a step elution followed by e, StrepTrap purification. The A chain contained a hexa-histidine and SUMO tag, the B chain contained a Strep tag, and the C chain contained sfGFP and avi tags. The eluate of this two-step purification method should therefore only contain trimers that include both the A and B chains. An optimal result would be equimolar amounts of the A, B, and C chains. f, SDS-PAGE of the StrepTrap purification revealed that the eluate contained an excess of the A and B chains and less of the C chain. For gel source data, see Supplementary Fig. 3. To test the ability of the A and B chains only to assemble into heterotrimers, we expressed an AB bicistronic gene and g, purified the resulting proteins by IMAC with a gradient elution. Two broad and overlapping peaks were observed. The leading half of the first peak and trailing half of the second peak were collected and h, further purified by SEC. Peak 2 has a lower retention volume than peak 1, suggesting a difference in molecular weight. These results are consistent with assembly of an ABB heterotrimer (earlier IMAC elution, later SEC elution) and an AAB heterotrimer (later IMAC elution, earlier SEC elution). We confirmed this interpretation by native mass spectrometry (Fig. 1g).