Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry and Microbiology, Rhodes University, Makhanda, South Africa.
Biomedical Research and Drug Discovery Research Group, Faculty of Health Sciences, Higher Colleges of Technology, Sharjah, United Arab Emirates.
Methods Mol Biol. 2023;2693:105-111. doi: 10.1007/978-1-0716-3342-7_9.
The development of mutant microorganisms lacking J domain proteins (JDPs; formerly called Hsp40s) has enabled the development of complementation assays for testing the co-chaperone function of JDPs. In these assays, an exogenously expressed novel JDP is tested for its ability to functionally substitute for a non-expressed or nonfunctional endogenous JDP(s) by reversing a stress phenotype. For example, the in vivo functionality of prokaryotic JDPs can be tested on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant strain of the bacterium Escherichia coli (OD259). Similarly, the in vivo functionality of eukaryotic JDPs can be assessed in a thermosensitive ydj1 mutant strain of the yeast Saccharomyces cerevisiae (JJ160). Here we outline the use of these thermosensitive microorganisms in complementation assays to functionally characterize a JDP from the bacterium, Agrobacterium tumefaciens (AgtDnaJ), and a JDP from the trypanosomal parasite, Trypanosoma cruzi (TcJ2).
缺乏 J 结构域蛋白（JDPs；以前称为 Hsp40s）的突变微生物的发展，使人们能够开发互补测定法来测试 JDPs 的共伴侣功能。在这些测定中，通过逆转应激表型，测试外源性表达的新型 JDP 是否能够替代非表达或无功能的内源性 JDP（s）来发挥功能。例如，可以根据原核 JDPs 逆转细菌大肠杆菌（OD259）的 dnaJ cbpA 突变株的热敏性的能力来测试其体内功能。同样，可以在酵母酿酒酵母（JJ160）的热敏性 ydj1 突变株中评估真核 JDPs 的体内功能。在这里，我们概述了使用这些热敏微生物在互补测定中，以功能表征来自细菌根癌农杆菌（AgtDnaJ）的 JDP 和来自锥虫寄生虫克氏锥虫（TcJ2）的 JDP。
