Rapid Total Synthesis of DARPin pE59 and RNase B. a

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.
MIT Faculty Start-up Fund
Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)
Deshpande Center for Technological Innovation
Damon Runyon-Rachleff (Innovation Award)
Sontag Foundation (Distinguished Scientist Award)
AstraZeneca (Firm) (Distinguished Graduate Student Fellowship)
Daniel S. Kemp Summer Fellowship
National Institutes of Health (U.S.). Biotechnology Training Program (5T32GM008334-25)