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


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.


  1. Kryukov, G.V. et al. Characterization of mammalian selenoproteomes. Science 300, 1439–1443 (2003).

    Article  CAS  Google Scholar 

  2. Stadtman, T.C. Selenocysteine. Annu. Rev. Biochem. 65, 83–100 (1996).

    Article  CAS  Google Scholar 

  3. Böck, A. et al. Selenocysteine: the 21st amino acid. Mol. Microbiol. 5, 515–520 (1991).

    Article  Google Scholar 

  4. Gieselman, M.D., Zhu, Y., Zhou, H., Galonic, D. & van der Donk, W.A. Selenocysteine derivatives for chemoselective ligations. Chembiochem 3, 709–716 (2002).

    Article  CAS  Google Scholar 

  5. Hondal, R.J. & Raines, R.T. Semisynthesis of proteins containing selenocysteine. Methods Enzymol. 347, 70–83 (2002).

    Article  CAS  Google Scholar 

  6. Lian, G. et al. Preparation and properties of a selenium-containing catalytic antibody as type I deiodinase mimic. J. Biol. Chem. 276, 28037–28041 (2001).

    Article  CAS  Google Scholar 

  7. Müller, S. et al. The formation of diselenide bridges in proteins by incorporation of selenocysteine residues: biosynthesis and characterization of (Se)2-thioredoxin. Biochemistry 33, 3404–3412 (1994).

    Article  Google Scholar 

  8. Arnér, E.S.J., Sarioglu, H., Lottspeich, F., Holmgren, A. & Böck, A. High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes. J. Mol. Biol. 292, 1003–1016 (1999).

    Article  Google Scholar 

  9. Gladyshev, V.N., Jeang, K-T. & Stadtman, T.C. Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene. Proc. Natl Acad. Sci. USA 93, 6146–6151 (1996).

    Article  CAS  Google Scholar 

  10. Zhong, L., Arnér, E.S.J., Ljung, J., Åslund, F. & Holmgren, A. Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue. J. Biol. Chem. 273, 8581–8591 (1998).

    Article  CAS  Google Scholar 

  11. Zhong, L., Arnér, E.S.J. & Holmgren, A. Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc. Natl. Acad. Sci. USA 97, 5854–5859 (2000).

    Article  CAS  Google Scholar 

  12. Nordberg, J., Zhong, L., Holmgren, A. & Arnér, E.S.J. Mammalian thioredoxin reductase is irreversibly inhibited by dinitrohalobenzenes by alkylation of both the redox active selenocysteine and its neighboring cysteine residue. J. Biol. Chem. 273, 10835–10842 (1998).

    Article  CAS  Google Scholar 

  13. Mutt, V. Vasoactive intestinal polypeptide and related peptides. Isolation and chemistry. Ann. NY Acad. Sci. 527, 1–19 (1988).

    Article  CAS  Google Scholar 

  14. Simoncsits, A. et al. Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide. Eur. J. Biochem. 178, 343–350 (1988).

    Article  CAS  Google Scholar 

  15. Heymann, P.W., Chapman, M.D., Aalberse, R.C., Fox, J.W. & Platts-Mills, T.A. Antigenic and structural analysis of group II allergens (Der f II and Der p II) from house dust mites (Dermatophagoides spp). J. Allergy Clin. Immunol. 83, 1055–1067 (1989).

    Article  CAS  Google Scholar 

  16. Kalef, E., Walfish, P.G. & Gitler, C. Arsenical-based affinity chromatography of vicinal dithiol-containing proteins: purification of L1210 leukemia cytoplasmic proteins and the recombinant rat c-erb Aβ1 T3 receptor. Anal. Biochem. 212, 325–334 (1993).

    Article  CAS  Google Scholar 

  17. Zhou, G.Y., Jauhiainen, M., Stevenson, K. & Dolphin, P.J. Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification. J. Chromatogr. 568, 69–83 (1991).

    Article  CAS  Google Scholar 

  18. Hoffman, R.D. & Lane, M.D. Iodophenylarsine oxide and arsenical affinity chromatography: new probes for dithiol proteins. Application to tubulins and to components of the insulin receptor-glucose transporter signal transduction pathway. J. Biol. Chem. 267, 14005–14011 (1992).

    CAS  PubMed  Google Scholar 

  19. Müller, S., Heider, J. & Böck, A. The path of unspecific incorporation of selenium in Escherichia coli. Arch. Microbiol. 168, 421–427 (1997).

    Article  Google Scholar 

  20. Gespach, C., Bawab, W., de Cremoux, P. & Calvo, F. Pharmacology, molecular identification and functional characteristics of vasoactive intestinal peptide receptors in human breast cancer cells. Cancer Res. 48, 5079–5083 (1988).

    CAS  PubMed  Google Scholar 

  21. Gromer, S., Arscott, L.D., Williams, C.H., Jr, Schirmer, R.H. & Becker, K. Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. J. Biol. Chem. 273, 20096–20101 (1998).

    Article  CAS  Google Scholar 

  22. Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 60, 523–533 (2003).

    Article  CAS  Google Scholar 

  23. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  24. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  25. Arnér, E.S.J. Recombinant expression of mammalian selenocysteine-containing thioredoxin reductase and other selenoproteins in Escherichia coli. Methods Enzymol. 347, 226–235 (2002).

    Article  Google Scholar 

  26. Ma, S., Caprioli, R.M., Hill, K.E. & Burk, R.F. Loss of selenium from selenoproteins: conversion of selenocysteine to dehydroalanine in vitro. J. Am. Soc. Mass Spectrom. 14, 593–600 (2003).

    Article  CAS  Google Scholar 

  27. Fichna, J. & Janecka, A. Synthesis of target-specific radiolabeled peptides for diagnostic imaging. Bioconjug. Chem. 14, 3–17 (2003).

    Article  CAS  Google Scholar 

  28. Okarvi, S.M. Recent progress in fluorine-18 labelled peptide radiopharmaceuticals. Eur. J. Nucl. Med. 28, 929–938 (2001).

    Article  CAS  Google Scholar 

  29. Waibel, R. et al. Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat. Biotechnol. 17, 897–901 (1999).

    Article  CAS  Google Scholar 

  30. Kapanidis, A.N., Ebright, Y.W. & Ebright, R.H. Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni2+:nitrilotriacetic acid (n)-fluorochrome conjugates. J. Am. Chem. Soc. 123, 12123–12125 (2001).

    Article  CAS  Google Scholar 

  31. Chua, K.Y. et al. Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay. Int. Arch. Allergy Appl. Immunol. 91, 118–123 (1990).

    Article  CAS  Google Scholar 

  32. Arnér, E.S.J., Zhong, L. & Holmgren, A. Preparation and assay of mammalian thioredoxin and thioredoxin reductase. Methods Enzymol. 300, 226–239 (1999).

    Article  Google Scholar 

  33. Said, S.I. & Mutt, V. Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur. J. Biochem. 28, 199–204 (1972).

    Article  CAS  Google Scholar 

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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, JO. et al. Exploiting the 21st amino acid—purifying and labeling proteins by selenolate targeting. Nat Methods 1, 61–66 (2004).

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