title: A redox trap to augment the intein toolbox.
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.
authors.affiliation: Department of Biological Sciences, University at Albany, Life Sciences Building 2061, 1400 Washington Avenue, Albany, New York 12222, USA. callahan@binghamton.edu
authors.forename: Brian P
authors.initials: BP
authors.lastname: Callahan
authors.forename: Matthew
authors.initials: M
authors.lastname: Stanger
authors.forename: Marlene
authors.initials: M
authors.lastname: Belfort
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods*; D002853:Chromatography, Liquid; D004258:DNA Polymerase III / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D047668:Inteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D019154:Protein Splicing; D012264:Ribonucleotide Reductases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012264:Ribonucleotide Reductases; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III