Department of Cancer Biology and Genetics, the Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States.
Department of Cancer Biology and Genetics, the Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States.
Methods Enzymol. 2023;682:319-350. doi: 10.1016/bs.mie.2022.12.004. Epub 2023 Jan 12.
Expressed protein ligation (EPL) allows for the attachment of a synthetic peptide into the N- or C-terminus of a recombinant protein fragment to generate a site-specifically modified protein with substantial yields for biochemical and biophysical studies. In this method, multiple posttranslational modifications (PTMs) can be incorporated into a synthetic peptide containing an N-terminal Cysteine, which selectively reacts with a protein C-terminal thioester to afford an amide bond formation. However, the requirement of a Cysteine at the ligation site can limit EPL's potential applications. Here, we describe a method called enzyme-catalyzed EPL, which uses subtiligase to ligate protein thioesters with Cysteine-free peptides. The procedure includes generating protein C-terminal thioester and peptide, performing the enzymatic EPL reaction, and purifying the protein ligation product. We exemplify this method by generating phospholipid phosphatase PTEN with site-specific phosphorylations installed onto its C-terminal tail for biochemical assays.
表达蛋白连接（EPL）允许将合成肽连接到重组蛋白片段的 N 端或 C 端，以生成具有生物化学和生物物理研究大量产量的特异性修饰蛋白。在该方法中，多种翻译后修饰（PTM）可以被掺入含有 N 端半胱氨酸的合成肽中，该半胱氨酸选择性地与蛋白 C 端硫酯反应以形成酰胺键。然而，在连接位点需要半胱氨酸可以限制 EPL 的潜在应用。在这里，我们描述了一种称为酶催化 EPL 的方法，该方法使用枯草杆菌蛋白酶连接酶将蛋白硫酯与不含半胱氨酸的肽连接。该过程包括生成蛋白 C 端硫酯和肽，进行酶促 EPL 反应，以及纯化蛋白连接产物。我们通过在其 C 端尾部安装特定磷酸化的磷脂磷酸酶 PTEN 来生成该方法的实例，以进行生化测定。
