A redox trap to augment the intein toolbox

Authors

  • Brian P. Callahan,

    Corresponding author
    1. Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222; telephone: 607-777-3089; fax: 607-777-4478
    Current affiliation:
    1. Department of Chemistry, Binghamton University, 4400 Vestal Parkway East, Vestal, NY 13850.
    • Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222; telephone: 607-777-3089; fax: 607-777-4478.
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    • Some of the research was performed while the authors were at Wadsworth Center, NY State Department of Health.

  • Matthew Stanger,

    1. Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222; telephone: 607-777-3089; fax: 607-777-4478
    Search for more papers by this author
    • Some of the research was performed while the authors were at Wadsworth Center, NY State Department of Health.

  • Marlene Belfort

    Corresponding author
    1. Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222; telephone: 607-777-3089; fax: 607-777-4478
    • Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222; telephone: 607-777-3089; fax: 607-777-4478.
    Search for more papers by this author
    • Some of the research was performed while the authors were at Wadsworth Center, NY State Department of Health.


  • Author contributions: BPC designed and performed all experiments with technical assistance from MS and under the guidance of MB. MS prepared the figures. BPC and MB wrote the manuscript.

Abstract

The unregulated activity of inteins during expression and consequent side reactions during work-up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism-based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein's catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein-mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide-trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild-type cells. Biotechnol. Bioeng. 2013; 110: 1565–1573. © 2012 Wiley Periodicals, Inc.

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