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Journal of Biomolecular NMR

, Volume 51, Issue 4, pp 449–456 | Cite as

A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility

  • Alvar D. Gossert
  • Alexandra Hinniger
  • Sascha Gutmann
  • Wolfgang Jahnke
  • André Strauss
  • César Fernández
Article

Abstract

An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract, similar protein amounts as with full media are obtained, without compromising on isotope incorporation. In single and dual amino acid labeling experiments incorporation ratios are consistently ≥90% for all amino acids tested. This enables NMR studies of eukaryotic proteins and their interactions even for proteins with low expression levels. We show applications with human kinases, where protein–ligand interactions are characterized by 2D [15N, 1H]- and [13C, 1H]-HSQC spectra.

Keywords

Isotope labeling Amino acid selective Insect cells Baculovirus Kinase NMR 

Notes

Acknowledgments

We thank Sylvie Antz and Sébastien Rieffel for maintaining cell stocks, Sihame Haddad and Arun Bhat for helpful feedback on the experimental protocol and René Knecht for the analysis of the amino acid content of yeast extract.

Supplementary material

10858_2011_9570_MOESM1_ESM.pdf (539 kb)
Supplementary material 1 (PDF 540 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Alvar D. Gossert
    • 1
  • Alexandra Hinniger
    • 1
  • Sascha Gutmann
    • 1
  • Wolfgang Jahnke
    • 1
  • André Strauss
    • 1
  • César Fernández
    • 1
  1. 1.Novartis Institutes for BioMedical ResearchBaselSwitzerland

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