Site‐Specific N‐Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide. Issue 52 (13th November 2015)

• Record Type: Journal Article
• Authors: Nguyen, Giang K. T.; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P.
• Is Part Of: Angewandte Chemie international edition. Volume 54:Issue 52(2015)
• Page Start: 15694
• Page End: 15698
• Publication Date: 2015-11-13
• Subjects: butelase 1 -- labelling -- ligation -- thiodepsipeptide; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201506810
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: An efficient ligase with exquisite site‐specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase‐mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N‐terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. Abstract : An irreversible butelase‐mediated ligation using a thiodepsipeptide substrate is described, with a ligation yield of >95 % and a minimal excess of substrate and low catalytic amounts of butelase 1. This method was used to introduce a functional tag to ubiquitin and green fluorescent protein with high yields.