Identification of substrates of LarA-activated Lon protease and Identification of Lon protease interactome

With this analysis we aim to identify proteins that are substrates of LarA-activated Lon protease in Caulobacter crescentus. We will compare samples of wild type cells either harboring an empty vector (vector control, VC) or overexpressing 3xFLAG-tagged LarA (F-LarA). Based on our previous data we hypothesize that proteins affected by LarA-overexpression which results in Lon protease activation will decrease in levels compared to the vector control sample. The aim of this analysis is the identification of the interactome of the Lon protease in Caulobacter crescentus. It is a follow-up analysis of a project in which we were looking for novel substrates of the Lon protease in Caulobacter crescentus. To narrow down the list of potential substrates we obtained through the first analysis, we have purified the protease (Lon WT) as well an inactive TRAP mutant and interacting proteins using a Twin-Strep-tag (Iba lifesciences). As a control, a cell lysate without Strep-tag containing protein was included in the purification set-up. The aim is to identify the proteins that specifically interact with the Lon protease by mass spectrometry.