Proteoliposome Preparation and Functional Reconstitution of Human Mitochondrial Transporters Via Wheat Germ Cell-Free...

Mitochondrial membrane proteins play a crucial role in the transport of metabolites essential to cellular metabolism, and their dysfunction is linked to a wide range of metabolic diseases. Here, we present a method for the expression, reconstitution, and analysis of members of the solute carrier family 25 (SLC25), the largest family of human mitochondrial transport proteins, using the wheat germ cell-free protein synthesis (WG-CFPS) system. This approach enables the generation of proteoliposomes containing a defined and highly purified protein species. This workflow enables efficient production of active membrane proteins without the limitations of a cell-based expression system, such as aggregation into inclusion bodies, the need for purification, and the cytotoxic effects of membrane protein overexpression. SLC25A11 and SLC25A21 proteins, as examples of human mitochondrial transporters, are directly translated into asolectin liposomes in the presence of the non-ionic detergent Brij-35. Later, to form proteoliposomes suitable for downstream transport assays, the proteins were reconstituted into a substrate-loaded liposome. The article provides detailed procedures for template preparation, cell-free expression, proteoliposome assembly, and validation of incorporated protein transport activity. This method, by reducing the time required to express, purify, and reconstitute membrane proteins, provides a platform for studying human mitochondrial metabolite transport activity. Once the reagents and plasmid are prepared, the complete work, from protein expression to functional analysis, can be completed within 3 days. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Plasmid design and development Basic Protocol 2: Cell-free protein expression Basic Protocol 3: Active reconstitution and evaluation of transport activity.