Production and Purification of PODS Proteins

All PODS proteins were synthesized as previously described [22 (link), 23 (link)]. All constructs were fused to the H1 immobilization tag [24 (link)]. Briefly, baculovirus (BV) DNA and transfer DNA were cotransfected into standard Spodoptera frugiperda 9 (Sf9) cells using TransIT-Insect (Mirus Bio). Resulting infective BV was harvested, and a plaque purification was then performed to isolate a single recombinant BV. Isolated plaques were first screened, and positive BV was then harvested, expanded, and finally used to infect large scale Sf9 cell cultures to produce PODS crystals. Subsequently, crystals were harvested and purified by lysing Sf9 cells using successive rounds of sonication and PBS washes. Finally, purified PODS were sterility tested and lyophilized prior to use in experiments. Although equivalence depends on context, $1.5\times {10}^4$ PODS crystals are approximately functionally equivalent to 1 ng of many standard growth factors, cytokines, or chemokines in terms of bioactivity [23 (link)].