研究顺式剪接内含子结构和催化活性的方法。
Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins.
机构信息
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
Department of Chemistry, Georgia State University, Atlanta, GA, USA.
出版信息
Methods Mol Biol. 2020;2133:55-73. doi: 10.1007/978-1-0716-0434-2_4.
The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.
蛋白质剪接的自催化过程由内含子（intein）促成，内含子中断了称为外显子（extein）的侧翼多肽。通过在大肠杆菌中外源表达内含子融合蛋白与非天然外显子的方法，对蛋白质剪接的机制进行了研究。内含子可用于生成反应性α-硫酯，以及具有 N 端半胱氨酸残基的蛋白质，以促进表达蛋白的连接。因此，更详细地了解内含子的功能可以对生物技术应用产生重大影响。在这里，我们提供了生化方法来研究剪接活性，以及 NMR 方法来研究内含子结构和催化机制。
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