Abstract
Incorporating synthetic probes site-specifically into proteins is of central interest in several areas of biotechnology and protein chemistry. Bioconjugation techniques provide a simple and effective means of chemically modifying a protein. In particular, covalent chemical modifications of cysteine residues belong to one of the most important reactions due to the unique reactivity of its thiol moiety and the relatively low abundance of this amino acid in proteins. However, such types of modifications cannot be performed in a regioselective fashion when one or more additional cysteines are present. To address this limitation, we have developed an approach where a short cysteine-containing tag (Cys-Tag) fused to one part of a split intein and modified at its sulfhydryl group can be used to label proteins by trans-splicing with a protein of interest (POI) fused to the other half of the split intein. In this way, it is possible to selectively label a protein containing multiple cysteines. The artificially split Mycobacterium xenopi GyrA intein and the Synechocystis sp. DnaB intein were highly suitable for this purpose and were successfully used for the labeling of several proteins. This approach enables a simple route for labeling proteins by site-specific cysteine bioconjugation with any one of several commercially available cysteine-modifying probes.
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Acknowledgments
The author’s would like to thank all members of the Mootz lab for helpful discussions. T.D. acknowledges a Ph.D. stipend from the International Max Planck Research School in Chemical Biology. Funding for this work was provided by the DFG and the Fonds der Chemischen Industrie.
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Dhar, T., Kurpiers, T., Mootz, H.D. (2011). Extending the Scope of Site-Specific Cysteine Bioconjugation by Appending a Prelabeled Cysteine Tag to Proteins Using Protein Trans-Splicing. In: Mark, S. (eds) Bioconjugation Protocols. Methods in Molecular Biology, vol 751. Humana Press. https://doi.org/10.1007/978-1-61779-151-2_9
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DOI: https://doi.org/10.1007/978-1-61779-151-2_9
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