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.