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Selective backbone labeling of proteins using {1,2-13C2}-pyruvate as carbon source

Journal of Biomolecular NMR volume 44, pages 167–173 (2009)Cite this article

Abstract

A simple isotope labeling approach for selective 13C/15N backbone labeling of proteins is described. Using {1,2-13C2}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of 13Cα-13CO spin-pairs into the backbones of protein molecules with medium-to-high levels of 13C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-13C2}-pyruvate based protein production.

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Abbreviations

MSG:

Malate synthase G

GB1:

Immunoglobulin binding domain B1 of streptococcal protein G

HSQC:

Heteronuclear single-quantum coherence

TROSY:

Transverse relaxation optimized spectroscopy

3-PG:

3-Phosphoglycerate

TCA:

Tricarboxylic acid cycle

PEP:

Phosphoenolpyruvate

OA:

Oxaloacetate

AKG:

α-Ketoglutarate

R5P:

Ribose-5-phosphate

TPP:

Thiamine pyrophosphate

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Acknowledgments

This work was supported in part by the Nano-Biotechnology Award to VT (University of Maryland). The authors are grateful to Dr. Patrik Lundström (University of Toronto) for helpful discussions related to pyruvate-based isotope labeling of proteins.

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  1. Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, 20742, USA

    Chenyun Guo, Chun Geng & Vitali Tugarinov

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  1. Chenyun Guo
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  2. Chun Geng
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  3. Vitali Tugarinov
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Correspondence to Vitali Tugarinov.

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Guo, C., Geng, C. & Tugarinov, V. Selective backbone labeling of proteins using {1,2-13C2}-pyruvate as carbon source. J Biomol NMR 44, 167–173 (2009). https://doi.org/10.1007/s10858-009-9326-y

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  • DOI: https://doi.org/10.1007/s10858-009-9326-y

Keywords

  • Selective isotope labeling
  • {1,2-13C2}-Pyruvate
  • Protein backbone
  • MSG