RIS ID
38876
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-alpha-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.
Included in
Life Sciences Commons, Physical Sciences and Mathematics Commons, Social and Behavioral Sciences Commons
Publication Details
Jia, X., Ozawa, K., Loscha, K. & Otting, G. (2009). Glutarate and N-acetyl-L-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins. Journal of Biomolecular NMR, 44 (2), 59-67.