A cell-free system for target discovery of small membrane proteins

Abstract

Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility and productivity. Furthermore, AcrZ produced in this system was used successfully for in vitro co-immunoprecipitations to identify interaction partners. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life. ImportanceSmall membrane proteins are shown to be involved in various biological processes in all domains of life and "can no longer be ignored". Due to their small size and hydrophobicity, functional investigation of small membrane proteins is challenging. In this work, we present a simple, versatile, cell-free approach for synthesizing small membrane proteins in vitro. We show that the small membrane proteins produced with our system are functional and in sufficient amounts for downstream target discoveries. Furthermore, our approach may uncover additional regulatory functions of small membrane proteins studied with conventional methods. Our work provides a robust alternative workflow for functional studies, which opens up new possibilities to advance our understanding of small membrane protein biology.