The human pathogen Mycobacterium tuberculosis is equipped with physiological and structural traits that enable evasion of immune responses and resistance to antimicrobial compounds. M. tuberculosis possesses an outer envelope that is highly impermeable, allowing it to survive in hostile host environments like macrophages and granulomas. This outer envelope contains the mycomembrane, a membrane composed of complex lipids and mycolic acids, which harbors the abundant and antigenic PE and PPE protein families. PE/PPE proteins perform a variety of roles prior to and during infection, such as membrane maintenance, interaction with host cells, and specialized nutrient uptake in the low-nutrient environment of macrophages. While PE/PPE proteins are encoded by approximately 10% of the M. tuberculosis genome, the protein families remain understudied. This can mostly be explained by difficulties related to working with M. tuberculosis. Most M. tuberculosis PE/PPE proteins form heterodimers and depend on translocation via the Type VII ESX-5 secretion system, which is absent in the non-pathogenic model organism Mycobacterium smegmatis. Interestingly, bioinformatic tools such as AlphaFold fail to predict the majority of PE/PPE protein structures. Particularly the C-terminal functional domains have not been structurally investigated. In this project we have developed the tools to purify PE/PPE proteins by reconstituting the ESX-5 gene cluster in fast-growing M. smegmatis, thereby bypassing constraints related to working with slow-growing M. tuberculosis. Furthermore, we optimized the fractionation and purification of mycobacterial membranes and we were able to identify PE/PPE proteins and ESX-5 proteins with mass spectrometry and immunoblotting. We successfully overexpressed M. tuberculosis pe/ppe genes and solubilized PE/PPE proteins from the M. smegmatis mycomembrane. In the next steps, we will upscale protein production to investigate the structure and function of PE/PPE proteins. For this, we will utilize a combination of biochemical and biophysical techniques such as Cryo-EM.