Traceless enzymatic protein synthesis without ligation sites constraint.

Ruifeng Li,Jing Feng,Yu’e Tian,Tong Zhu,Xinyu Yang,Marcel Schmidt,Yinglu Cui,Timo Nuijens,Bian Wu

doi:10.1093/nsr/nwab158

Abstract

Protein synthesis and semisynthesis offer immense promise for life sciences and have impacted pharmaceutical innovation. The absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets, however. Here we introduce the PALME (protein activation and ligation with multiple enzymes) platform designed for sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization and protein N/C-terminal or internal functionalization. The practical utility of this methodology is demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. The modular PALME platform exhibits unprecedentedly broad accessibility for traceless protein synthesis and functionalization, and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

Highlights

Breaking away from the central dogma, protein total synthesis and semisynthesis are powerful strategies for generating and functionalizing naturally inaccessible proteins, which have enabled groundbreaking applications driving life science advances and impacted the industrial production of biomolecular therapeutics[1,2,3]
The thioester functionality of the acyl donor is initially masked in the form of a C-terminal hydrazide and sequentially retrieved via a combination of nitrite oxidation and thiolysis. We envisioned that this strategy might be adapted for Peptiligase-catalyzed ligation, while it was unclear if there is an appropriate alcohol reagent for peptide acyl shifting
The whole process was conducted in one pot in 3 h with only trace amounts of enzymes, exhibiting excellent catalytic efficiency, chemoselectivity, and regioselectivity in the presence of a multitude of side-chain reactive functionalities. These results demonstrated that all catalytic modules exhibited broad substrate spectrum and functioned well in series, and the protein activation and ligation with multiple enzymes (PALME) platform was ready for further investigation

Summary

Introduction

Breaking away from the central dogma, protein total synthesis and semisynthesis are powerful strategies for generating and functionalizing naturally inaccessible proteins, which have enabled groundbreaking applications driving life science advances and impacted the industrial production of biomolecular therapeutics[1,2,3]. Revisiting the fascinating principle of nature and possessing the Peptiligase family for peptide ligation, we envisioned that sequence-independent assembly of native peptides might be feasible through iterative activation and ligation processes using multiple enzymes that present both strict regioselectivity and broad substrate specificity. To this end, we sought to design a multienzyme cooperative activation and ligation strategy (Fig. 1d) for traceless protein synthesis and functionalization, and we present this platform, termed protein activation and ligation with multiple enzymes (PALME). Our results highlight that enzymes with diverse functions can be rationally harnessed to offer traceless protein synthesis and functionalization with remarkable flexibility in choice of the ligation sites and peptide substrates, providing unprecedentedly broad application potential

Results And Discussion

Conclusion

Conflicts of interest