An anaerobic cell-free protein synthesis formulation for the in vitro study of oxygen sensitive proteins

Anaerobic microbes harbor unique enzymes and metabolic pathways with potential biotechnological applications, yet these microbes remain difficult to study and engineer. Research is limited by the labor-intensive workflows required to maintain anoxic environments and the lack of genetic tools available for non-model anaerobes. Here, we present an anaerobic cell-free protein synthesis (CFPS) platform for the in vitro study of oxygen-sensitive enzymes. This study establishes two methods for conducting anaerobic CFPS, achieving an anoxic environment by (1) sparging with inert gas or (2) lyophilizing to remove oxygenated water. We show the functionality of our anaerobic CFPS formulation by producing oxygen-independent fluorescent protein reporters and a [4Fe-4S] cluster pyruvate:ferredoxin oxidoreductase (PFOR) with a specific activity of 5.2 {+/-} 0.3 x 10-4 U/{micro}L cell-free extract. This highly modular platform will allow study of diverse oxygen-sensitive proteins and requires only minor modifications to commonly used CFPS workflows.