Chemical Protein Synthesis: Advances, Challenges, and Outlooks

Abstract

Contemporary chemical protein synthesis has been dramatically advanced over the past few decades, which has enabled chemists to reach the landscape of synthetic biomacromolecules. Chemical synthesis can produce synthetic proteins with precisely controlled structures which are difficult or impossible to obtain via gene expression systems. Herein, we summarize the key enabling ligation technologies, major strategic developments, and some selected representative applications of synthetic proteins and provide an outlook for future development.

Advances in chemical probing of protein O-GlcNAc glycosylation: structural role and molecular mechanisms

“…190 Understandably, these synthetic targets can be challenging, especially those glycosylated at a position more than 50 residues from the N-or C-terminus, and require the presence of cysteine sites that otherwise need to be engineered for the ligation step and subsequently converted to the native residue via desulphurisation. Nonetheless, continuous methodological advances in chemical protein synthesis 191,192 are enabling one to take full advantage of the potential of these ligation methods, which are uniquely suited to provide access to sitespecifically and stoichiometrically modified O-GlcNAcylated proteins. These chemically pure glycoproteins can then be utilised for in vitro biochemical and structural biology studies to probe structure-function correlations and mechanisms of O-GlcNAcylation at the molecular level.…”

Serine/threonine ligation-assisted chemical synthesis of HMGA1a protein with site-specific post-translational modifications

“…Specifically, chemical peptide ligation technologies, such as native chemical ligation (NCL) and its variants, KAHA ligation, Serine/Threonine ligation (STL), and cysteine/penicillamine ligation (CPL), can selectively and specifically forge a natural peptidic bond between the C- and N-terminus of two unprotected peptides. Therefore, a protein containing noncanonical elements can be re-constructed by stitching peptides together via several rounds of peptide ligation ( Tan et al., 2020 ).…”

“…Of the ligation technologies developed to date, the C-terminal salicylaldehyde ester-based chemoselective condensation, STL, has been widely used for synthesis of cyclic peptides and proteins. ( Zhang et al., 2013 ; Tan et al., 2020 ). This reaction occurs between the N-terminal serine or threonine or cysteine peptide fragment and the peptide fragment with C-terminal salicylaldehyde ester to afford an N,O/S -benzylidene-acetal-linked intermediate in a pyridine/acetic acid mixture.…”

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode

“…Chemical protein synthesis, powered by chemo-selective peptide ligation reactions, has produced almost hundreds of synthetic proteins which conveniently incorporate non-canonical amino acids, among many other purposes ( Agouridas et al, 2019 ; Tan et al, 2020 ). Although the chemical synthesis of selenoproteins, like SelM and SelW, has been reported ( Dery et al, 2017 ), the synthesis of SelF is, however, supposed to be more challenge as it contains six other Cys residues in the UGGT binding domain, which certainly complicates the refolding process.…”