Same Day Access to Folded Synthetic Proteins

Understanding protein function is a cornerstone of modern biology. Research centers worldwide dedicate significant efforts to prepare individual proteins for study, the isolation and purification of which can take days to months. We developed a workflow that enables same-day access to functional synthetic proteins. Chemical synthesis provides access to crude protein chains in hours, but the removal of the synthetic side products is typically a days-long process. We find that chemical modifications on side products lead to significant and unpredictable changes in the folding behavior. Consistent with these findings, we discovered that approaches based on biophysical properties characteristic of the folded protein target can discriminate against chemically similar species. Confirming our protocol with nine protein targets, we show that appropriate desalting followed by different folding strategies enables isolation of functional single-domain proteins in hours instead of days. Each target was isolated in under 10 h, including some proteins with post-translational modifications, non-natural amino acids, and disulfide bonds. Rapid biological discovery requires on-demand access to proteins, and the folding pipeline described here is uniquely suited to enabling these efforts. The folding process presented here was not assessed on complex proteins, and therefore, it may require further optimization in those cases.