Cell-free synthesis of proteins with selectively (13)C-labelled methyl groups from inexpensive precursors

The novel eCell system maintains the activity of the entire repertoire of metabolic Escherichia coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively [Formula: see text] C-labelled amino acids from inexpensive [Formula: see text] C-labelled precursors. The system is demonstrated with selective [Formula: see text] C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis–trans isomerase B. Starting from 3- [Formula: see text] C-pyruvate, [Formula: see text] C-HSQC cross-peaks are obtained devoid of one-bond [Formula: see text] C– [Formula: see text] C scalar couplings. Starting from 2- [Formula: see text] C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows us to produce perdeuterated proteins with protonated methyl groups in a residue-selective manner. The system uses the isotope-labelled precursors sparingly and is readily scalable.