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

02 February 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


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.


synthetic proteins
flow chemistry
native chemical ligation
mirror-image proteins

Supplementary materials

Supporting Information
Materials, methods, and analytical characterization.


Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.