Ready display of antigenic peptides in a protein ‘mimogen’

Abstract

Given the dependence of much modern biology upon the use of antibodies as tools and reagents, their variability and the often associated lack-of-detail about function and identity creates experimental errors. Here we describe the proof-of-principle for a potentially general, versatile method for the display of antigens in a soluble yet standard format on a lateral protein scaffold that mimics normal epitopes in a protein antigen (a 'mimogen') and confirm their utility in phosphorylation-dependent recognition by specific antibodies.

Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step

“…Dha based chemistry is a promising strategy for the preparation of protein conjugates and proteins bearing PTM mimics. [30][31][32] Being the Michael acceptor for the 1,4-addition reaction with various thiols, Dha has become a useful synthetic precursor to obtain a variety of modified proteins. For example, mimics of acetylated histone H3 were prepared and shown to behave as substrates for histone deacetylases.…”

Linker-free incorporation of carbohydrates into in vitro displayed macrocyclic peptides

“…Thiol addition to Dha has previously been employed to modify proteins with thiophosphate, thiol derivatives of carbohydrates, lipids, alkyl groups, 24 and short peptides, 38 as well as a more in depth study of alkyl, aryl and charged small thiols. 43 The substrate scope for modification of in vitro translated peptides was anticipated to be similarly broad, and this was investigated by performing test additions using the following compounds: BME, thio-Glc, 1-thio-β- d - N -acetylglucosamine (as the disulfide, with pre-reduction; ‘thio-GlcNAc’), N -biotinylated cysteine, glutathione, thiophenol, 4-carboxybenzyl thiol, thiophosphate, and dodecane thiol ( Fig.…”

“…DBAA has been extensively applied to studying post-translational modification of proteins, including ubiquitination, 27 – 29 phosphorylation, 30 – 32 glycosylation, 33 and histone acetylation and methylation, 34 to identification of catalytic residues, 35 and to the display of chemically synthesized peptides including epitopes for antibody generation. 36 – 38 It is not, however, obvious if this sequence of reactions is compatible with in vitro translation of peptides or with bioorthogonal reactions, due to the presence of many protein factors and enzymes, nucleotides and other small molecules, including reducing agents, β-mercaptoethanol (BME) and dithiothreitol (DTT). An investigation is thus presented here on the feasibility of DBAA-mediated elimination of cysteine followed by conjugate addition of an exogenous thiol to in vitro translated macrocyclic peptides generated by the FIT system, leading to a strategy to allow the in vitro selection of novel glycopeptide leads.…”

Chemical methods for protein site-specific ubiquitination

“…Dha-based chemistry has been proved as a versatile strategy for the preparation of proteins bearing PTM mimetics. [119][120][121][122] In 2016, Meledin et al reported a chemical protein ubiquitination method via a thiol dehydroalanine Michael addition reaction (Fig. 14B).…”