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

An anaerobic cell-free protein synthesis platform enables production of an oxygen-independent fluorescent reporter and a [4Fe-4S] enzyme, pyruvate:ferredoxin oxidoreductase (PFOR), with specific activity 5.2 {+/-} 0.3 x 10-4 U/{micro}L extract.

• Why it matters: Anaerobic microbes are hard to study and engineer because maintaining anoxic conditions is labor-intensive and genetic tools for non-model anaerobes are limited.
• What they did: They built anaerobic CFPS using either inert-gas sparging or lyophilization to remove oxygenated water, then tested function by expressing oxygen-independent fluorescent reporters and measuring PFOR activity.
• The result: The formulation supports oxygen-sensitive protein expression in vitro and, being modular with only minor CFPS workflow changes, enables broader study of diverse oxygen-sensitive proteins.

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