Cell-free protein production and labeling protocol for NMR-based structural proteomics

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Abstract
Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods. To illustrate this platform, we describe its application to a specific target (At3g01050.1) from Arabidopsis thaliana. After cloning the target gene into a specialized plasmid, we carry out a small-scale (50 μl) in vitro sequential transcription and translation trial to ascertain the level of protein production and solubility. Next, we prepare mRNA for use in a 4-ml semicontinuous cell-free translation reaction to incorporate 15N-labeled amino acids into a protein sample that we purify and test for suitability for NMR structural analysis. We then repeat the cell-free approach with 13C,15N-labeled amino acids to prepare a doubly labeled sample. The three-dimensional (3D) structure of At3g01050.1 shows that this protein is an unusual member of the β-grasp protein family.