Vypal2: A Versatile Peptide Ligase for Precision Tailoring of Proteins.

Dingpeng Zhang,James P Tam,Side Hu,Julien Lescar,Zhen Wang,Chuan-Fa Liu

doi:10.3390/ijms23010458

Abstract

The last two decades have seen an increasing demand for new protein-modification methods from the biotech industry and biomedical research communities. Owing to their mild aqueous reaction conditions, enzymatic methods based on the use of peptide ligases are particularly desirable. In this regard, the recently discovered peptidyl Asx-specific ligases (PALs) have emerged as powerful biotechnological tools in recent years. However, as a new class of peptide ligases, their scope and application remain underexplored. Herein, we report the use of a new PAL, VyPAL2, for a diverse range of protein modifications. We successfully showed that VyPAL2 was an efficient biocatalyst for protein labelling, inter-protein ligation, and protein cyclization. The labelled or cyclized protein ligands remained functionally active in binding to their target receptors. We also demonstrated on-cell labelling of protein ligands pre-bound to cellular receptors and cell-surface engineering via modifying a covalently anchored peptide substrate pre-installed on cell-surface glycans. Together, these examples firmly establish Asx-specific ligases, such as VyPAL2, as the biocatalysts of the future for site-specific protein modification, with a myriad of applications in basic research and drug discovery.

Highlights

Academic Editor: Peptide ligases are enzymes of relatively rare occurrences when compared to proteases, their ubiquitous counterparts
Using substrates designed to carry short recognition motifs of the ligase, we have demonstrated the versatility of VyPAL2 in a number of applications, including protein labeling, inter-protein ligation, and backbone circularization, as well as
To show that VyPAL2 can ligate proteins as efficiently as model peptides [25,35], we first tested it for the labeling of several model proteins of various sizes: ubiquitin, a DAR

Summary

Introduction

Academic Editor: Peptide ligases are enzymes of relatively rare occurrences when compared to proteases, their ubiquitous counterparts. Peptide ligases catalyze the formation of new peptide bonds [1,2,3,4,5,6,7]. Clitoria ternatea, is the first plant-derived peptidyl Asx-specific ligase (PAL) [13]. It catalyzes peptide bond formation at Asn/Asp residues in two steps—the cysteinyl thiol at the enzyme’s active site first attacks an Asx-Xaa peptide bond in the acyl donor substrate to form an acyl-enzyme thioester intermediate, which undergoes aminolysis by the

Methods

Results

Conclusion