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Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS

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Book cover Selenoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1661))

Abstract

Selenoprotein K (SELENOK) and Selenoprotein S (SELENOS) are the members of the endoplasmic-reticulum-associated degradation (ERAD) complex, which is responsible for translocating misfolded proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Besides its involvement in the ERAD, SELENOK was shown to bind and stabilize the palmitoyl transferase DHHC6, and thus contributes to palmitoylation. SELENOK and SELENOS reside in the ER membrane by the way of a single transmembrane helix. Both contain an intrinsically disordered region with a selenocysteine (Sec) located one or two residues away from the C-terminus. Here, we describe the preparation of the Sec-containing forms of SELENOS and SELENOK. SELENOK, which contains no native cysteines, was prepared in an E. coli cysteine auxotroph strain by exploiting the codon and the insertion machinery of Cys for the incorporation of Sec. In contrast, the preparation of SELENOS, which contains functionally important cysteine residues, relied on E. coli’s native Sec incorporation mechanism.

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Acknowledgments

We thank Prof. E. S. J. Arner from the Karolinska Institutet for the generous donation of the pSUABC plasmid and Dr. Marie-Paule Strub at the NIH laboratory of molecular biophysics for the donation of BL21(DE3)cys cells. The Delaware COBRE program supported this research with grants from the National Institute of General Medical Sciences under awards P30 GM110758-02 and P20 GM104316. We acknowledge the use of instrumentation made accessible through the National Science Foundation under Grant No. CHE-1337449. This work was primarily supported by the National Science Foundation under Grant No. MCB-1054447 “CAREER: Reactivity of Selenoproteins” and Grant No. MCB-1616178.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Delaware, 136 Brown Laboratory, Newark, DE, 19716, USA

    Zhengqi Zhang, Jun Liu & Sharon Rozovsky

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  1. Zhengqi Zhang
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  2. Jun Liu
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  3. Sharon Rozovsky
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Correspondence to Sharon Rozovsky .

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  1. Centre International de Recherche en Infectiologie, CIRI, Lyon, France

    Laurent Chavatte

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Zhang, Z., Liu, J., Rozovsky, S. (2018). Preparation of Selenocysteine-Containing Forms of Human SELENOK and SELENOS. In: Chavatte, L. (eds) Selenoproteins. Methods in Molecular Biology, vol 1661. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7258-6_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7258-6_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7257-9

  • Online ISBN: 978-1-4939-7258-6

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