Bogyo Matthew, Baruch Amos, Jeffery Douglas A, Greenbaum Doron, Borodovsky Anna, Ovaa Huib, Kessler Benedikt
Stanford University, Stanford, California.
Celera Genomics, South San Francisco, California.
Curr Protoc Protein Sci. 2004 Sep;Chapter 21:21.17.1-21.17.35. doi: 10.1002/0471140864.ps2117s36.
Recent genome sequencing projects have identified new peptidases in multiple organisms, many with unknown functions, suggesting the need for new tools to study these enzymes. This unit outlines selection and use of small-molecule and protein-based probes to covalently modify peptidases in complex cellular environments. These activity-based probes (ABPs) have been designed based on well characterized peptidase inhibitor scaffolds, but make use of new techniques to greatly enhance their utility for studying families of related peptidases. In particular, ABPs can be used to track activity of peptidases in crude cell extracts, intact cells, and in vivo, allowing rapid purification and identification of labeled targets. They can be used with libraries of small molecules to rapidly assess potency and selectivity of compounds in complex, physiologically relevant samples. Probe selection, probe tagging using reporters, labeling of recombinant targets, crude protein extracts, and peptidase targets in cell culture systems, affinity purification of targets, and inhibitor screening using affinity probes are outlined.
最近的基因组测序项目在多种生物体中鉴定出了新的肽酶，其中许多功能未知，这表明需要新的工具来研究这些酶。本单元概述了在复杂细胞环境中用于共价修饰肽酶的小分子和基于蛋白质的探针的选择和使用。这些基于活性的探针（ABP）是基于特征明确的肽酶抑制剂支架设计的，但利用了新技术来大大提高其用于研究相关肽酶家族的效用。特别是，ABP可用于追踪粗细胞提取物、完整细胞和体内肽酶的活性，从而快速纯化和鉴定标记的靶标。它们可与小分子文库一起使用，以快速评估复杂生理相关样品中化合物的效力和选择性。文中概述了探针选择、使用报告分子进行探针标记、重组靶标的标记、粗蛋白提取物和细胞培养系统中的肽酶靶标、靶标的亲和纯化以及使用亲和探针进行抑制剂筛选。
