Rapid Production of Native and Mirror-Image Tumor Necrosis Factor-α through the Synergy of Automated Flow Peptide Synthesis and Native Chemical Ligation

Tumor necrosis factor-alpha (TNF-α) plays a central role in immune response regulation. Due to the correlation between elevated TNF-α production and a range of diseases, inhibiting the interaction of this protein with its native receptors as a therapeutic avenue has been thoroughly explored. Despite advancements in the development of lead TNF-α inhibitors, concerns remain regarding immunogenicity and loss of activity in vivo. To facilitate the discovery of stable and less immunogenic therapeutic modalities, we describe a rapid synthesis protocol that capitalizes on the synergy between automated fast-flow peptide synthesis (AFPS) technology, native chemical ligation (NCL), and high-throughput screening of folding conditions to arrive at functional synthetic proteins, native and mirror-image TNF-α. Specifically, an NCL reaction using only two fragments that were readily produced by AFPS afforded synthetic L- and D-TNF-α in milligram quantities (up to 5.5 mg, ~28% yield). Subsequent oxidation and dialysis led to folded TNF-α homotrimers, exhibiting analogous characteristics to the recombinant TNF-α. Overall, this innovative approach can serve as a general protocol for accessing proteins that are intractable by modern protein synthesis methods, therefore enabling the development of novel and stable therapeutics.