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.
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Abbreviations
- CI-BABP:
-
Chicken ileum bile-acid binding protein
- PpiB:
-
E. coli peptidyl-prolyl cis–trans isomerase b
- HSQC:
-
Heteronuclear single quantum coherence
- hCypA:
-
Human cyclophilin A
- WNVpro:
-
West Nile virus NS2B/NS3 protease
- DENpro:
-
Dengue virus type 2 NS2B/NS3 protease
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Acknowledgments
We thank a referee for pointing out the possibility of using a 1:1 buffer of glutarate and N-acetyl-l-Glu. The CI-BABP vector was kindly provided by Ms. Serena Zanzoni and Prof. Henriette Molinari. Financial support by the Australian Research Council is gratefully acknowledged.
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Jia, X., Ozawa, K., Loscha, K. et al. Glutarate and N-acetyl-l-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins. J Biomol NMR 44, 59–67 (2009). https://doi.org/10.1007/s10858-009-9315-1
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DOI: https://doi.org/10.1007/s10858-009-9315-1