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.

中文翻译：

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无连接位点限制的无痕酶蛋白合成

蛋白质合成和半合成为生命科学提供了巨大的希望，并影响了药物创新。然而，缺乏一种普遍适用的、具有灵活选择连接位点的无痕肽缀合方法仍然是获取许多重要合成目标的瓶颈。在这里，我们介绍了蛋白质激活和多酶连接 (PALME) 平台，该平台设计用于生物大分子的无序列限制合成和修饰。上游激活模块接受和处理容易获得的合成肽和重组蛋白，避免了与激活肽底物的制备和操作相关的挑战。协同下游偶联模块为序列肽缩合、环化、和蛋白质 N/C 末端或内部功能化。通过合成从药物成分到具有综合挑战性的蛋白质的一系列生物活性靶标，证明了该方法的实用性。总之，模块化 PALME 平台对无痕蛋白质合成和功能化展示了前所未有的广泛可及性，并具有扩展蛋白质化学和合成生物学范围的巨大潜力。