Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.
Nat Protoc. 2013 Sep;8(9):1787-99. doi: 10.1038/nprot.2013.101. Epub 2013 Aug 29.
Methods for site-specific modification of proteins should be quantitative and versatile with respect to the nature and size of the biological or chemical targets involved. They should require minimal modification of the target, and the underlying reactions should be completed in a reasonable amount of time under physiological conditions. Sortase-mediated transpeptidation reactions meet these criteria and are compatible with other labeling methods. Here we describe the expression and purification conditions for two sortase A enzymes that have different recognition sequences. We also provide a protocol that allows the functionalization of any given protein at its C terminus, or, for select proteins, at an internal site. The target protein is engineered with a sortase-recognition motif (LPXTG) at the place where modification is desired. Upon recognition, sortase cleaves the protein between the threonine and glycine residues, facilitating the attachment of an exogenously added oligoglycine peptide modified with the functional group of choice (e.g., fluorophore, biotin, protein or lipid). Expression and purification of sortase takes ∼3 d, and sortase-mediated reactions take only a few minutes, but reaction times can be extended to increase yields.
用于蛋白质定点修饰的方法应该在涉及的生物或化学靶标的性质和大小方面具有定量性和多功能性。它们应该对靶标进行最小的修饰，并且基础反应应该在生理条件下在合理的时间内完成。连接酶介导的转肽反应符合这些标准，并且与其他标记方法兼容。在这里，我们描述了两种具有不同识别序列的 sortase A 酶的表达和纯化条件。我们还提供了一种方案，允许在给定蛋白质的 C 末端或某些蛋白质的内部位置进行功能化。目标蛋白在所需修饰的位置设计有连接酶识别基序（LPXTG）。连接酶识别后，在苏氨酸和甘氨酸残基之间切割蛋白质，促进外加的带有所选功能基团（例如，荧光团、生物素、蛋白质或脂质）的寡甘氨酸肽的附着。连接酶的表达和纯化需要约 3 天，连接酶介导的反应只需要几分钟，但可以延长反应时间以提高产量。
