Site-Specific Generation of Protein-Protein Conjugates Using Native Amino Acids
Site-Specific Generation of Protein-Protein Conjugates Using Native Amino Acids
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DOI：
10.26434/chemrxiv.9941948.v1
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发表时间：
2019-10
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通讯作者：
Marco J. Lobba;Christof Fellmann;Alan M Marmelstein;Johnathan C. Maza;E. Kissman;S. A. Robinson;Brett T. Staahl;Cole Urnes;Rachel J. Lew;J. Doudna;M. Francis;Casey S Mogilevsky
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文献类型：
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作者：
Marco J. Lobba;Christof Fellmann;Alan M Marmelstein;Johnathan C. Maza;E. Kissman;S. A. Robinson;Brett T. Staahl;Cole Urnes;Rachel J. Lew;J. Doudna;M. Francis;Casey S Mogilevsky
嵌合蛋白质-蛋白质缀合物为免疫治疗、靶向药物递送和疫苗开发提供了平台。然而，许多理想的构建体不能通过直接表达产生，并且两种蛋白质的靶向偶联在化学上具有挑战性。在这里，我们提出了一种新的方法，使用天然氨基酸的蛋白质的快速和位点特异性偶联。酪氨酸酶氧化多肽上暴露的酪氨酸残基，产生邻醌，邻醌与其他蛋白质中战略性放置的半胱氨酸残基快速反应。这种方法用于用小分子、肽甚至完整蛋白质修饰CRISPR-Cas9和其他底物。细胞穿透肽与CRISPR-Cas9的缀合显示相对于未修饰的Cas9将细胞基因组编辑效率提高20倍。该技术代表了生物分子偶联的新范式，并为前所未有的多功能生物缀合物铺平了道路。
Chimeric protein-protein conjugates provide platforms for immunotherapy, targeted drug delivery, and vaccine development. However, many desirable constructs cannot be produced through direct expression, and the targeted coupling of two proteins is chemically challenging. Here we present a new approach for the rapid and site-specific coupling of proteins using native amino acids. Tyrosinase oxidizes exposed tyrosine residues on polypeptides, generating ortho-quinones that react rapidly with strategically placed cysteine residues in other proteins. This approach was used to modify CRISPR-Cas9 and other substrates with small molecules, peptides and even intact proteins. The conjugation of cell penetrating peptides to CRISPR-Cas9 was shown to increase cellular genome editing efficiency by 20-fold relative to unmodified Cas9. This technology represents a new paradigm for biomolecular coupling, and paves the way to an unprecedented range of multifunctional bioconjugates.