Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.
National Cancer Institute, National Institutes of Health, Frederick, MD, USA.
Methods Mol Biol. 2021;2355:83-92. doi: 10.1007/978-1-0716-1617-8_8.
Sortase A is a thiol transpeptidase expressed by Gram-positive bacteria. This enzyme is capable of site-specifically ligating peptides containing the C-terminal recognition motif LPXTG to peptides containing an N-terminal polyglycine sequence, forming a native peptide bond. Here, we describe the preparation and application of sortase A to the ligation of two individually folded disulfide-rich animal venom peptides in order to form a heterodimeric double-knotted peptide with a native peptide linker. This method is mild enough to preserve the structures and disulfide connectivities of the peptides during ligation. We employed a highly efficient sortase A pentamutant (SrtA5°), which brings the reaction to completion within 15 min with a ~50-80% yield of ligated peptide.
Sortase A 是一种由革兰氏阳性菌表达的硫醇转肽酶。这种酶能够将含有 C 末端识别基序 LPXTG 的肽特异性地连接到含有 N 末端聚甘氨酸序列的肽上，形成天然的肽键。在这里，我们描述了 sortase A 的制备及其在连接两个单独折叠的富含二硫键的动物毒液肽中的应用，以形成具有天然肽接头的异二聚体双纽结肽。该方法足够温和，可在连接过程中保持肽的结构和二硫键连接性。我们使用了一种高效的 sortase A 五突变体（SrtA5°），它可以在 15 分钟内使反应完全完成，连接肽的产率约为 50-80%。
