Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step.
Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step.
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DOI：
10.1021/ac402871k
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发表时间：
2013-11-19
影响因子：
7.4
通讯作者：
Tsourkas, Andrew
中科院分区：
文献类型：
--
作者：
Warden-Rothman, Robert;Caturegli, Ilaria;Popik, Vladimir;Tsourkas, Andrew
用各种货物(药物、显像剂、纳米颗粒等)有效地标记基于蛋白质的靶向配体对于分子成像和靶向治疗领域是必不可少的。然而，许多常见的生物偶联技术效率低下，非化学计量，不具有位点特异性，和/或与某些类型的蛋白质支架不兼容。此外，这些技术可能会导致共轭和非共轭产物的混合，这通常很难分离。在这项研究中，利用细菌索酸酶将靶向配体的纯化和C末端的定点连接浓缩为一步。建立了一个模型，以确定高共轭纯度和高效利用货物的最佳反应条件。作为原理的证明，使用排序酶标签表达蛋白连接(STEPL)技术来制备具有荧光标记和/或叠氮修饰的高纯度(>；95%)的肿瘤特异性亲和配体，从而不需要去除未结合的杂质。然后使用点击化学将叠氮修饰的靶向配体高效和定点连接到纳米颗粒上。
Efficient labeling of protein-based targeting ligands with various cargos (drugs, imaging agents, nanoparticles, etc.) is essential to the fields of molecular imaging and targeted therapeutics. Many common bioconjugation techniques, however, are inefficient, non-stoichiometric, not site-specific, and/or incompatible with certain classes of protein scaffolds. Additionally, these techniques can result in a mixture of conjugated and unconjugated products, which are often difficult to separate. In this study, a bacterial sortase enzyme was utilized to condense targeting ligand purification and site-specific conjugation at the C-terminus into a single step. A model was produced to determine optimal reaction conditions for high conjugate purity and efficient utilization of cargo. As proof-of-principle, the sortase-tag expressed protein ligation (STEPL) technique was used to generate tumor-specific affinity ligands with fluorescent labels and/or azide modifications at high purity (>95%) such that is was not necessary to remove unconjugated impurities. Click chemistry was then used for the highly efficient and site-specific attachment of the azide-modified targeting ligands onto nanoparticles.
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