Poster Presentation International Peptide Symposium 2023

New semi-synthetic technologies for accessing post-translationally modified proteins (#279)

Sameer S. Kulkarni 1 2 , Emma Watson 1 2 , Joshua Maxwell 1 2 , Gerhard Niederacher 3 , Jason Johansen-Leete 1 2 , Alexander Norman 1 2 , Christian Becker 3 , Richard J. Payne 1 2
  1. The University of Sydney, Sydney, NSW, Australia
  2. Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, NSW, Australia
  3. Faculty of Chemistry, Institute of Biological Chemistry, University of Vienna, Vienna, Austria

Native chemical ligation (NCL)1 has enabled the chemical synthesis of hundreds of modified proteins, however, the generation of larger proteins >180 residues via this methodology is a significant undertaking. Muir and co-workers developed a semi-synthetic variant of NCL, labelled expressed protein ligation (EPL), that enables the generation of larger modified protein targets by fusing a recombinant protein to a synthetic modified peptide fragment.2 Unfortunately, EPL typically requires millimolar concentrations of substrates for the reaction to proceed, restricting its applicability for accessing poorly soluble or aggregation-prone proteins. To solve this problem, we recently developed a novel selenium-mediated semi-synthetic methodology, dubbed the expressed protein selenoester ligation (EPSL).3 EPSL offers several key advantages over NCL-based methods, including: 1) the ability to perform ligations at µM-nM concentrations and 2) the ability to chemoselectively deselenize post ligation to preserve native Cys residues in the structure. The power and utility of EPSL will be presented here by highlighting its use in the efficient semi-synthesis of differentially phosphorylated variants of the oligomeric chaperone protein Hsp27 and lipidated analogues of YPT6.

  1. 1. P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. H. Kent, Science 1994, 266, 776
  2. 2. T. W. Muir, D. Sondhi, P. A. Cole, Proc. Natl. Acad. Sci. USA 1998, 95, 6705–6710
  3. 3. S. S. Kulkarni, E. E. Watson, et al., Angew. Chem. Int. Ed. 2022, 61(20), e202200163