Glutarate and N -acetyl-L-glutamate buffers for cell-free synthesis of selectively 15 N-labelled proteins

Abstract

Cell-free protein synthesis provides rapid and economical access to selectively 15N-labelled proteins, greatly facilitating the assignment of 15N-HSQC spectra. While the best yields are usually obtained with buffers containing high concentrations of potassium L-glutamate, preparation of selectively 15N-Glu labelled samples requires non-standard conditions. Among many compounds tested to replace the L-Glu buffer, potassium N-acetyl-L-glutamate and potassium glutarate were found to perform best, delivering high yields for all proteins tested, with preserved selectivity of 15N- Glu labelling. Assessment of amino-transferase activity by combinatorial 15N-labelling revealed that glutarate and N-acetyl-L-glutamate suppress the transfer of the 15N- α-amino groups between amino acids less well than the conventional L-Glu buffer. On balance, the glutarate buffer appears most suitable for the preparation of samples containing 15N-L-Glu while the conventional L-Glu buffer is advantageous for all other samples. Show more