Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.
Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.
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DOI：
10.1021/acs.bioconjchem.2c00451
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发表时间：
2022-12
影响因子：
4.7
通讯作者：
Amy Ryan;Olivia Shade;Anirban Bardhan;Aleksander Bartnik;A. Deiters
中科院分区：
文献类型：
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作者：
Amy Ryan;Olivia Shade;Anirban Bardhan;Aleksander Bartnik;A. Deiters
尽管有一系列的共价蛋白质修饰，但很少有技术可以量化细胞中的蛋白质生物结合。在这里，我们描述了一种新的方法，通过与HaloTag形成共价键来定量纤维素蛋白的生物结合。这种方法利用非天然氨基酸(UAA)诱变，选择性地在蛋白质表面安装一个具有生物正交性的小手柄。我们利用反电子需求Diels-Alder环加成反应的快速动力学和高选择性来评价四嗪苯丙氨酸(TETF)与应变反式环辛烯-氯代烷(sTCO-CA)和反式-环辛烯赖氨酸(TCOK)与四嗪-氯代烷(Tet-CA)的反应。在生物偶联之后，氯代烷配体暴露于HaloTag酶的标记下，允许通过简单的蛋白质印迹分析直接定量生物偶联。我们展示了这个工具的多功能性，可以快速准确地测定不同的尿酸/氯烷对的生物结合效率，以及不同蛋白质上的不同位置，包括绿色荧光蛋白和雌激素相关受体Errα。
Despite a range of covalent protein modifications, few techniques exist for quantification of protein bioconjugation in cells. Here, we describe a novel method for quantifying in cellulo protein bioconjugation through covalent bond formation with HaloTag. This approach utilizes unnatural amino acid (UAA) mutagenesis to selectively install a small and bioorthogonally reactive handle onto the surface of a protein. We utilized the fast kinetics and high selectivity of inverse electron-demand Diels-Alder cycloadditions to evaluate reactions of tetrazine phenylalanine (TetF) with strained trans-cyclooctene-chloroalkane (sTCO-CA) and trans-cyclooctene lysine (TCOK) with tetrazine-chloroalkane (Tet-CA). Following bioconjugation, the chloroalkane ligand is exposed for labeling by the HaloTag enzyme, allowing for straightforward quantification of bioconjugation via simple western blot analysis. We demonstrate the versatility of this tool for quickly and accurately determining the bioconjugation efficiency of different UAA/chloroalkane pairs and for different sites on different proteins of interest, including EGFP and the estrogen-related receptor ERRα.