Conjugation of the prokaryotic ubiquitin-like protein (Pup) to cellular proteins tags these proteins for degradation by a proteasome in actinobacteria. To study the Pup-proteasome system in in vitro biochemical assays, Pup-tagged (i.e., pupylated) proteins are often used. However, the purification of a homogeneous preparation of pupylated proteins often suffers from poor yields and limitations in terms of selecting the target protein and its site of pupylation. Here, we report on the development of a biochemical methodology we term Pup-Click for the generation of pupylated protein mimics in vitro. Pup-Click relies on a natural pupylation reaction combined with the use of a synthetic peptide and genetic code expansion via the use of unnatural amino acids and Click chemistry. In principle, this approach allows for conjugation of Pup to any selected target at potentially any desired position. Importantly, pupylated protein mimics generated by Pup-Click are recognized and processed by enzymes of the Pup-proteasome system. As such, Pup-Click can serve as a powerful tool for studying this protein degradation pathway.

中文翻译：

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Pup-Click-一种新的化学酶方法，用于生成单一的Pupylated目标。

原核泛素样蛋白（Pup）与细胞蛋白的缀合将这些蛋白标记为由放线菌中的蛋白酶体降解。为了在体外生化分析中研究Pup-蛋白酶体系统，经常使用Pup标记（即pupylated）蛋白。但是，纯化的均一化的pupylated蛋白制剂经常遭受收率低和在选择目标蛋白及其pupylation位点方面的限制。在这里，我们报告了一种称为Pup-Click的生化方法的发展，该方法可用于体外生成pupylated蛋白模拟物。Pup-Click依赖于天然pupylation反应，结合使用合成肽和通过使用非天然氨基酸和Click化学来扩展遗传密码。原则，该方法允许将Pup与可能在任何期望位置的任何选择的靶标缀合。重要的是，由Pup-Click生成的pupylated蛋白模拟物可被Pup-蛋白酶体系统的酶识别和加工。因此，Pup-Click可以用作研究此蛋白降解途径的强大工具。