Same\nDay Access to Folded Synthetic Proteins

Understanding\nprotein function is a cornerstone of modern biology.\nResearch centers worldwide dedicate significant efforts to prepare\nindividual proteins for study, the isolation and purification of which\ncan take days to months. We developed a workflow that enables same-day\naccess to functional synthetic proteins. Chemical synthesis provides\naccess to crude protein chains in hours, but the removal of the synthetic\nside products is typically a days-long process. We find that chemical\nmodifications on side products lead to significant and unpredictable\nchanges in the folding behavior. Consistent with these findings, we\ndiscovered that approaches based on biophysical properties characteristic\nof the folded protein target can discriminate against chemically similar\nspecies. Confirming our protocol with nine protein targets, we show\nthat appropriate desalting followed by different folding strategies\nenables isolation of functional single-domain proteins in hours instead\nof days. Each target was isolated in under 10 h, including some proteins\nwith post-translational modifications, non-natural amino acids, and\ndisulfide bonds. Rapid biological discovery requires on-demand access\nto proteins, and the folding pipeline described here is uniquely suited\nto enabling these efforts. The folding process presented here was\nnot assessed on complex proteins, and therefore, it may require further\noptimization in those cases.