Large-scale growth of C. elegans and isolation of membrane protein complexes

Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in similar to 9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.

Automated summary

This study presents a detailed protocol for isolating membrane protein complexes from transgenic Caenorhabditis elegans. The genetically modified worms are grown abundantly using standard laboratory equipment, and purification conditions are optimized on a small scale using fluorescence-detection size-exclusion chromatography. The optimized conditions are then applied to a large-scale preparation, allowing for the purification of sufficient quantities of target proteins for structural, biochemical, and biophysical studies. The utility of this approach is demonstrated by successfully isolating membrane protein complexes from transgenic C. elegans.