Aaron R. Goerke

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

We describe an E. coli‐based cell‐free system for the production of proteins with a non‐natural amino acid (nnAA) incorporated site‐specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl‐tRNA synthetase (mTyrRS) and tRNATyr pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200–300 µg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m2/mL. This new E. coli‐based cell‐free procedure produced up to 400 µg/mL of eCAT109pAz, 660 µg/mL of eDHFR10pAz, and 210 µg/mL of mDHFR31pAz with p‐azido‐L‐phenylalanine (pAz) incorporated site‐specifically at the amber nonsense codon. O‐methyl‐L‐tyrosine and p‐acetyl‐L‐phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell‐free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)‐catalyzed (3 + 2) cycloadditions (click chemistry). Biotechnol. Bioeng. 2009;102: 400–416.

Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase.

Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc production using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biology provides the freedom to control both the catalyst and chemical compositions in biological reactions, and we capitalized on this to produce large amounts of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell extract source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metabolism, and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412mug/mL of purified GLuc, relative to 5mug/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2x10(24)photons/s/mol, the highest reported activity for any characterized luciferase.