Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with 11C

Abstract

Selenocysteine (Sec; U in one-letter code) is the twenty-first naturally occurring amino acid, with a selenium atom that gives this cysteine (Cys) homolog unique biochemical properties, including a high nucleophilicity and significant reactivity with electrophilic agents. This can be used in biotechnological Sec-dependent applications. Here, we describe how Sec can be introduced into a carboxy-terminal tetrapeptide motif (-Gly-Cys-Sec-Gly-COOH, known as a Sel-tag) for recombinant proteins by tailoring the encoding gene to become compatible with the Escherichia coli selenoprotein synthesis machinery. We also describe how the Sel-tag can be used as a basis for efficient one-step protein purification, rapid Sec-targeting protein labeling with electrophilic compounds, or radiolabeling with the positron emitter 11C.

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Figure 1: Construction of a bacterial-type SECIS element for production of a Sel-tagged protein.
Figure 2: Oxidized and reduced Sel-tag.
Figure 3: Scheme for the strategy of primer design for construction of a Sel-tagged protein.
Figure 4: One-step Sel-tagged protein purification over PAO-Sepharose.
Figure 5: Biologically controlled in situ 75Se-labeling of a Sel-tagged protein.
Figure 6: Fluorescence labeling of Sel-tagged proteins.
Figure 7: Radiolabeling of Sel-tagged proteins with 11C.

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Acknowledgements

The authors are grateful to all of our collaborators in the original studies as cited herein. The work in the laboratories of the authors is supported by Karolinska Institutet, the Swedish Cancer Society, the Swedish Research Council and the European Union.

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Correspondence to Elias S J Arnér.

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Cheng, Q., Stone-Elander, S. & Arnér, E. Tagging recombinant proteins with a Sel-tag for purification, labeling with electrophilic compounds or radiolabeling with 11C. Nat Protoc 1, 604–613 (2006). https://doi.org/10.1038/nprot.2006.87

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