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Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method

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Abstract

Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus.

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Abbreviations

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

INSET:

Isotopically invisible solubility/stability enhancement tag

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Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research and the National Project on “Targeted Proteins Research Program” from the Ministry of Education, Science and Culture of Japan.

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Authors and Affiliations

  1. Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan

    Yoshihiro Kobashigawa, Hiroyuki Kumeta, Kenji Ogura & Fuyuhiko Inagaki

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  1. Yoshihiro Kobashigawa
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  2. Hiroyuki Kumeta
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  3. Kenji Ogura
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  4. Fuyuhiko Inagaki
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Corresponding author

Correspondence to Fuyuhiko Inagaki.

Electronic supplementary material

Experimental materials and methods, including that for protein expression, purification and conjugation as well as SDS-PAGE analysis of protein ligation are available. Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 432 kb)

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Kobashigawa, Y., Kumeta, H., Ogura, K. et al. Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method. J Biomol NMR 43, 145–150 (2009). https://doi.org/10.1007/s10858-008-9296-5

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  • DOI: https://doi.org/10.1007/s10858-008-9296-5

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