Improving the throughput and scalability of expressed protein ligation <em>via</em> flow chemistry — ASN Events
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  3. Improving the throughput and scalability of expressed protein ligation via flow chemistry

Improving the throughput and scalability of expressed protein ligation via flow chemistry (#269)

Lucas Kambanis 1 2 , Anthony Ayoub 1 2 , Peter H.G Eguland 3 , Lucien Lambrechts 1 2 , Charlotte Franck 1 2 , Timothy S Chisholm 1 2 , Richard J Payne 1 2
  1. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, NSW
  2. School of Chemistry, The University of Sydney, New South Wales, Australia
  3. Novo Nordisk A/S, CMC API Development, Smørmosevej 17-19, DK-2880 Bagsværd, Denmark

The development and application of chemoselective peptide ligation chemistry, namely the gold standard native chemical ligation (NCL), has transformed the field of chemical protein synthesis.1-3 We recently reported the development of a flow-based NCL-photodesulfurization method to synthesise therapeutic peptides.4 Importantly, this method leverages the superior mixing, heat transfer and interaction with specific wavelengths of light that flow chemistry provides, thus leading to rapid reaction rates. Capitalising on this work, we herein report our findings towards the application of flow methodology to access site-selectively modified expressed proteins. The power and utility of this approach was exemplified through the synthesis of a sulfated variants of the tick-derived chemokine binding protein ACA-01. ACA-01 contains 8 native cysteine residues, which can prove problematic with ligations in flow due to the formation of unproductive thioesters. We have developed conditions that are specifically suited to cysteine rich expressed protein ligations with remarkable rate enhancements compared to analogous batch ligations.5

Prior to this work, flow ligations had only been applied to small peptides, the expansion to larger more complex protein examples showcases the versatility and utility of NCL in flow and provides potential avenues toward industrial applications in the future.  

  1. Kent, S. B. H., Total chemical synthesis of proteins. Chem. Soc. Rev. 2009, 38 (2), 338-351.
  2. Bondalapati, S.; Jbara, M.; Brik, A., Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nat. Chem. 2016, 8 (5), 407-418.
  3. Kulkarni, S. S.; Sayers, J.; Premdjee, B.; Payne, R. J., Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nat. Rev. Chem. 2018, 2 (4), 1-17.
  4. Chisholm, T. S., Clayton, D., Dowman, L. J., Sayers, J. and Payne, R. J., Native chemical ligation–photodesulfurization in flow. J. Am. Chem. Soc. 2018, 140 (29), 9020-9024.
  5. Franck, C., Foster, S. R., Johansen-Leete, J., Chowdhury, S., Cielesh, M., Bhusal, R. P., Mackay, J. P., Larance, M., Stone, M. J. and Payne, R. J., Semisynthesis of an evasin from tick saliva reveals a critical role of tyrosine sulfation for chemokine binding and inhibition. PNAS, 2020, 117(23), 12657-12664.
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