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Protein synthesis and semisynthesis offer immense promise for life science and have impacted pharmaceutical innovation. Nevertheless, 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. Here we introduce the protein activation and ligation with multiple enzymes (PALME) platform designed for the 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 the 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. This methodology's practical utility was demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. Together, the modular PALME platform exhibits unprecedented broad accessibility for the traceless protein synthesis and functionalization and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

Traceless enzymatic protein synthesis without ligation sites constraint