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.

Keywords: Expressed Protein Selenoesters; Peptides; Protein Modifications; Protein Semi-Synthesis; Proteins.

Scheme 1

A) Expressed protein ligation (EPL) and desulfurization. B) DSL‐deselenization. C) Expressed protein selenoester ligation (EPSL) methodology.

Scheme 2

A) Conversion of ubiquitin acyl hydrazide 2 to ubiquitin aryl selenoester 3 and one‐pot selenoesterification‐DSL‐ deselenization (EPSL) for the ligation of TMEM43(2–31) to ubiquitin. B) Analytical HPLC of crude acyl hydrazide 2 to selenoester 3 conversion. C) Analytical HPLC of purified ubiquitin aryl selenoester 3. D) ESI‐MS spectrum of 3. E) Analytical HPLC of purified ubiquitin‐TMEM43(2–31) conjugate 5. F) ESI‐MS spectrum of 5. NB: ESI‐MS spectra generated from the entire UV peak from LC‐MS.

Scheme 3

A) Semi‐synthesis of lipidated YPT6 proteins via EPSL. B) HPLC chromatogram for S‐palmitylated YPT6 7. C) ESI‐MS spectrum of S‐palmitylated YPT6 7 (spectrum generated from the entire UV peak from LC‐MS, see Supporting Information for full dataset for 6 and 7).

Scheme 4

A) Semi‐synthesis of phosphorylated variants of Hsp27 using EPSL. B) HPLC chromatogram for Ser176 phosphorylated Hsp27 13. C) ESI‐MS spectrum of Ser176 phosphorylated Hsp27 13 (spectrum generated from the entire UV peak from LC‐MS, see Supporting Information for full data set for 12–14).