Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana .

Autor: Morris DA; James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.; Center for Predictive Medicine, University of Louisville School of Medicine, Louisville, KY, USA., Reeves MA; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA., Royal JM; James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.; Center for Predictive Medicine, University of Louisville School of Medicine, Louisville, KY, USA.; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA., Hamorsky KT; James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.; Center for Predictive Medicine, University of Louisville School of Medicine, Louisville, KY, USA.; Department of Medicine, University of Louisville School of Medicine, Louisville, KY., Matoba N; James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY, USA.; Center for Predictive Medicine, University of Louisville School of Medicine, Louisville, KY, USA.; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA.
Jazyk: angličtina
Zdroj: Process biochemistry (Barking, London, England) [Process Biochem] 2021 Feb; Vol. 101, pp. 42-49. Date of Electronic Publication: 2020 Nov 04.
DOI: 10.1016/j.procbio.2020.10.018
Abstrakt: Here we describe refined methods for the isolation and detection of a KDEL-tagged, plant-produced recombinant cholera toxin B subunit (CTB) that exhibits unique mucosal wound healing activity. The protein was transiently overexpressed in Nicotiana benthamiana , which generates some C-terminal KDEL truncated molecular species that are deficient in epithelial repair activity. With a new CHT chromatographical method described herein, these product-derived impurities were successfully separated from CTB with the intact KDEL sequence, as confirmed by mass spectrometry. In addition, an immunoassay capable of specifically detecting GM1 ganglioside-binding CTB with intact KDEL sequences was developed. Coupled together, these methods will aid in the quality control of KDEL-attached CTB produced in plant-based manufacturing systems towards a novel topical biotherapeutic for the treatment of acute and chronic mucosal inflammation.
Competing Interests: Declaration of Interest None.
Databáze: MEDLINE
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