Exploiting the 21st amino acid—purifying and labeling proteins by selenolate targeting

Abstract

Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pKa and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either γ-emitting 75Se or short-lived positron emitters such as 11C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.

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Figure 1: Features of the Sel-tag as compared to a His-tag.
Figure 2: Protein purification using the Sel-tag.
Figure 3: Fluorescence labeling of Sel-tagged proteins.
Figure 4: Radiolabeling of Sel-tagged proteins with 11C.

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Acknowledgements

We thank C. Gitler and E. Kalef, Weizmann Institute of Science, Rehovot, Israel, for helpful advice regarding PAO-Sepharose synthesis; M. Engberg for initial assistance in cloning work; E. Refai, Department of Medical Biochemistry and Biophysics, Karolinska Institute, for assistance and providing native VIP; and H. Grönlund, Department of Medicine, Clinical Immunology and Allergy, Karolinska Institute, for providing the Der p 2-6 His clone. This work was supported by the Karolinska Institute, the Swedish Cancer Society (projects 4056 and 4722), the Swedish Research Council for Medicine (projects 14527 and 14528), the Swedish Asthma and Allergy Associations Research Foundation, Åke Wibergs Foundation, Lars Hiertas Foundation, Konsul Th. C. Bergs Foundation, Magnus Bergvalls Foundation, Hesselmans Foundation and the Swedish Cancer and Asthma fund.

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Correspondence to Elias S J Arnér.

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Johansson, L., Chen, C., Thorell, J. et al. Exploiting the 21st amino acid—purifying and labeling proteins by selenolate targeting. Nat Methods 1, 61–66 (2004). https://doi.org/10.1038/nmeth707

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