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

Abstract

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 ${}^{13}$C-labelled amino acids from inexpensive ${}^{13}$C-labelled precursors. The system is demonstrated with selective ${}^{13}$C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis-trans isomerase B. Starting from 3-${}^{13}$C-pyruvate, ${}^{13}$C-HSQC cross-peaks are obtained devoid of one-bond ${}^{13}$C-${}^{13}$C scalar couplings. Starting from 2-${}^{13}$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.