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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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.
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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