Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at the membrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheral membrane proteins remains challenging; mostly because there is no unified purification strategy for these proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane. There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrial ultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of the membrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure of FAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domain proteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structure determination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To our knowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Our results show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* is active because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is a possible avenue to express and purify other challenging BAR domain proteins.
Keywords: BAR protein; Membrane bending; Solubility tag.
Copyright © 2021. Published by Elsevier Inc.