Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates.
Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates.
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DOI：
10.3390/pharmaceutics14122826
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发表时间：
2022-12-16
期刊：
影响因子：
5.4
通讯作者：
Young DD
中科院分区：
文献类型：
--
作者：
Williard AC;Switzer HJ;Howard CA;Yin R;Russell BL;Sanyal R;Yu S;Myers TM;Flood BM;Kerscher O;Young DD
用非典型氨基酸（ncAA）修饰蛋白质代表了提供具有位点特异性修饰的生物缀合物的均质样品的有用技术。这种技术可以直接应用于异常SUMO化模式的检测，这往往是疾病状态的指示。修饰的SUMO捕获蛋白，由与麦芽糖结合蛋白MBP融合的无催化活性的ULP 1片段（UTAG）组成，是用于SUMO化蛋白的结合和标记的有用试剂。评估了该UTAG融合蛋白的突变以促进用于ncAA遗传掺入的琥珀抑制技术，从而为修饰提供功能性手柄。最终，麦芽糖结合蛋白（MBP）融合中的两个位点被鉴定为理想的掺入和生物缀合，而不干扰UTAG蛋白的SUMO捕获能力。该功能随后被用于标记HeLa细胞中的SUMO化蛋白，并证明它们在细胞核中的富集。这种修饰的UTAG-MBP-ncAA蛋白在诊断和治疗方面具有深远的应用。
Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.
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影响因子：
2.7
作者：
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通讯作者：
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影响因子：
21.3
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影响因子：
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作者：
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影响因子：
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作者：
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