Cell-Free Protein Synthesis of Particulate Methane Monooxygenase into Nanodiscs

Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an Escherichia coli lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs. We used a SUMO fusion construct to generate the native PmoB subunit and showed that the SUMO protease (Ulp1) cleaves the protein in the reaction mixture. Using an affinity tag to isolate the complete pMMO complex, we demonstrated that the complex forms without the need for exogenous translocon machinery or chaperones, confirmed by negative stain electron microscopy. This work demonstrates the potential for using CFPS to express multi-subunit membrane-bound metalloenzymes directly into lipid bilayers.