Improved spectral resolution of [C,H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [C,H]-HSQC cross-peaks free of one-bond C-C couplings. Selective C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.