Improved spectral resolution of [¹³C,¹H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive ¹³C-labelled precursors

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive ¹³C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of ¹³C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [¹³C,¹H]-HSQC cross-peaks free of one-bond ¹³C–¹³C couplings. Selective ¹³C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.