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Stable isotope labeling of protein by Kluyveromyces lactis for NMR study

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Abstract

Stable isotope labeling for proteins of interest is an important technique in structural analyses of proteins by NMR spectroscopy. Escherichia coli is one of the most useful protein expression systems for stable isotope labeling because of its high-level protein expression and low costs for isotope-labeling. However, for the expression of proteins with numerous disulfide-bonds and/or post-translational modifications, E. coli systems are not necessarily appropriate. Instead, eukaryotic cells, such as yeast Pichia pastoris, have great potential for successful production of these proteins. The hemiascomycete yeast Kluyveromyces lactis is superior to the methylotrophic yeast P. pastoris in some respects: simple and rapid transformation, good reproducibility of protein expression induction and easy scale-up of culture. In the present study, we established a protein expression system using K. lactis, which enabled the preparation of labeled proteins using glucose and ammonium chloride as a stable isotope source.

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Fig. 1
Fig. 2

Abbreviations

K. lactis :

Kluyveromyces lactis

MBP:

Maltose-binding protein

YNB:

Yeast nitrogen base

MALDI-TOF:

Matrix-assisted laser desorption ionization-time of flight

HSQC:

Heteronuclear single quantum coherence

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Acknowledgements

This work was supported by the Ministry of Economy, Trade and Industry (METI) and the New Energy and Industrial Technology Development Organization (NEDO). We would like to thank to Mr. Osamu Ichikawa (Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan) for assistance with the P. pastoris protein expression system. In addition, we are especially grateful to Naoki Yoshida, Ph.D., Hitoshi Tainaka, Ph.D. (New England Biolabs Japan, Inc.), and C. H. Taron, Ph.D. (New England Biolabs, Inc.), for technical support regarding the K. lactis protein expression system.

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Authors and Affiliations

  1. Japan Biological Informatics Consortium (JBiC), Aomi, Koto-ku, Tokyo, 135-0064, Japan

    Toshihiko Sugiki

  2. Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), Aomi, Koto-ku, Tokyo, 135-0064, Japan

    Toshihiko Sugiki, Ichio Shimada & Hideo Takahashi

  3. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Ichio Shimada

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  1. Toshihiko Sugiki
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  2. Ichio Shimada
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  3. Hideo Takahashi
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Correspondence to Ichio Shimada or Hideo Takahashi.

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Sugiki, T., Shimada, I. & Takahashi, H. Stable isotope labeling of protein by Kluyveromyces lactis for NMR study. J Biomol NMR 42, 159–162 (2008). https://doi.org/10.1007/s10858-008-9276-9

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