Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry and Microbiology, Rhodes University, Makhanda, South Africa.
Methods Mol Biol. 2023;2693:113-123. doi: 10.1007/978-1-0716-3342-7_10.
Many molecular chaperones act as holdases by binding hydrophobic regions of substrates to prevent aggregation. Therefore, measuring holdase activity is an amenable method to determine chaperone activity. The holdase function is reliably and easily achieved by monitoring the suppression of heat-induced aggregation of well-characterized model protein substrates. However, the standard assay format requires large amounts of protein and hence is not applicable to all proteins. Using DnaK from Escherichia coli and heat-induced aggregation of malate dehydrogenase, we describe a protocol for absorbance and fluorescence-based miniaturized versions of the standard aggregation suppression assay that are affordable and have wide application for low abundance holdases. The assay can be used for both fundamental characterization of holdase function in proteins and screening of inhibitors of holdase activity.
许多分子伴侣通过结合底物的疏水区域来防止聚集，从而充当持留伴侣。因此，测量持留伴侣活性是一种可行的方法来确定伴侣活性。通过监测热诱导的模型蛋白底物的聚集抑制，可以可靠且容易地实现持留伴侣功能。然而，标准测定方法需要大量的蛋白质，因此不适用于所有蛋白质。本文使用大肠杆菌中的 DnaK 和苹果酸脱氢酶的热诱导聚集，描述了一种基于吸光度和荧光的标准聚集抑制测定的微型化版本的协议，该协议经济实惠，适用于低丰度持留伴侣的广泛应用。该测定可用于蛋白质中持留伴侣功能的基础表征和持留伴侣活性抑制剂的筛选。
