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

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive (13)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 (13)C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [(13)C,(1)H]-HSQC cross-peaks free of one-bond (13)C–(13)C couplings. Selective (13)C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10858-023-00420-9.