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Journal of Biomolecular NMR

, Volume 44, Issue 2, pp 59–67 | Cite as

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

  • Xinying Jia
  • Kiyoshi Ozawa
  • Karin Loscha
  • Gottfried Otting
Article

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.

Keywords

Cell-free protein synthesis Combinatorial 15N-labelling 15N-HSQC Potassium glutarate Potassium N-acetyl-l-glutamate Selective 15N-Glu labelling 

Abbreviations

CI-BABP

Chicken ileum bile-acid binding protein

PpiB

E. coli peptidyl-prolyl cistrans 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

Notes

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.

Supplementary material

10858_2009_9315_MOESM1_ESM.pdf (982 kb)
(PDF 982 kb)

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Xinying Jia
    • 1
  • Kiyoshi Ozawa
    • 1
  • Karin Loscha
    • 1
  • Gottfried Otting
    • 1
  1. 1.Research School of ChemistryThe Australian National UniversityCanberraAustralia

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