Glutarate and N-acetyl-L-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins.
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.
Jia
Xinying
X
Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia.
Ozawa
Kiyoshi
K
Loscha
Karin
K
Otting
Gottfried
G
D000596:Amino Acids / Q000378:metabolism; D002021:Buffers; D002474:Cell-Free System; D005971:Glutamates / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000378:metabolism*; D000637:Transaminases / Q000378:metabolism
D000596:Amino Acids; D002021:Buffers; D005971:Glutamates; D005977:Glutarates; D009587:Nitrogen Isotopes; D011506:Proteins; D000637:Transaminases; C016195:N-acetylglutamic acid