Expressed Protein Selenoester Ligation

Abstract

Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one‐pot semi‐synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi‐synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane‐associated GTPase YPT6, and site‐specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine

“…118 and 119). Acetylacetone (acac)-based activation and treatment with selenocystamine then provided ubiquitin diselenide dimer 27 (via initial selenoester formation and a rapid Se-to-N acyl shift 89 , Supplementary Fig. 120).…”

The semisynthesis of nucleolar human selenoprotein H

“…S3 and S4†). Indeed, following expression, we saw that conversion to thioester via hydrazide 38,39 was much faster than the wild-type (fully completed within 4 h, Fig. 3).…”

The semisynthesis of nucleolar human selenoprotein H

“…28 As a result, we chose to mutate Ser43 to Ala or Thr in order to improve thiolysis efficiency (Figures S3-S4). Indeed, following expression, we saw that conversion to thioester via hydrazide 29,30 was much faster than the wild-type (fully completed within 4 h, Figure 3). The purified SELENOH(44-122) was then ligated with both S43A and S43T variants of SUMO-SELENOH(2-43)-COSR.…”