Application Note 36

Cell-Free Protein Synthesis with ²H/¹⁵N/¹³C-Labeled Amino Acids in H₂O for the Production of Perdeuterated Proteins with ¹H in the Exchangeable Positions

Thomas Carruthers, Choy-Theng Loh, and Gottfried Otting

Research School of Chemistry, Australian National University, Canberra, ACT 0200 Australia

Perdeuteration is essential for NMR studies of big and immobile proteins (> 40 kDa) to slow down the relaxation rates of the remaining ¹H NMR signals. Uniformly ²H/¹⁵N/¹³C‑labeled proteins are typically produced by growing E. coli in minimal medium with ¹³C₆, D₇-glucose as the only carbon source, ¹⁵N-ammonium salts as the only nitrogen source, and perdeuteration achieved simply by making up the medium with D₂O instead of water. As the resulting protein product is uniformly labeled with ²H, observation of the backbone amides by ¹H NMR requires prior ²H-¹H back-exchange of the amide hydrogens. This poses a dilemma for proteins that do not tolerate ²H-¹H back-exchange. For example, the hydrogen-exchange of amide deuterons buried in the interior of the protein depends on local (for some amides even global) unfolding of the protein to expose the amides to the solvent. Proteins for which back-exchange is difficult include the large class of proteins that cannot be reversibly denatured (usually leading to precipitation) and proteins of limited stability that don’t tolerate long incubation times. Here we illustrate the finding that back-exchange is redundant if the protein of interest is produced by cell-freesynthesis from perdeuterated amino acids in H₂O. Importantly, the costs associated with cell-free synthesis compare favorably with those of in vivo expression.

A Brief Description of Cell-Free Protein Synthesis

Cell-free protein synthesis uses a cell extract rather than live cells to produce the protein of interest in a coupled transcription-translation reaction. Using cell extracts has numerous advantages: (i) cell extracts are depleted of the DNA from the original organism so the synthesis machinery only produces the target protein encoded by the DNA that is supplied to the reaction mixture; (ii) the process of preparing the cell extract inactivates a number of enzymes that perform chemical transformations between amino acids in vivo, therefore metabolic conversions between different amino acids are suppressed; (iii) the chemical environment in which the protein synthesis proceeds can readily be controlled and modified; and (iv) proteins can be produced from linear DNA amplified by PCR making it very easy to introduce mutations by site-directed mutagenesis.¹ Cell-free protein synthesis became an important tool in NMR spectroscopy in 1999 when Kigawa and co-workers established conditions for high-yield protein expression.² Cell extracts can readily be prepared from E. coli by breaking the cells, centrifugation and collection of the supernatant. In our hands, homemade S30 extracts routinely sustain the production of about 1 mg of protein per mL of reaction mixture. The protocol involves the use of a dialysis system in which the reaction mixture containing the S30 extract is placed in a dialysis bag and immersed in an outer buffer that supplies low-molecular-weight compounds to sustain the protein synthesis reaction, such as amino acids, nucleotides, and ATP. The target DNA is added to the reaction mixture together with any other macromolecules that aid in protein production, such as tRNA and RNA polymerase. The DNA can be the same plasmid DNA that directs the protein expression in conventional in vivo systems or it can be PCR-amplified DNA. Detailed protocols for the preparation of S30 extracts and cell-free reactions that we have established in our laboratory have been published.³

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