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Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana.
- Source :
-
Process Biochemistry . Feb2021, Vol. 101, p42-49. 8p. - Publication Year :
- 2021
-
Abstract
- • A novel mucosal wound-healing protein (CTBSEKDEL) was produced in plants. • C-terminally truncated byproducts were removed by ceramic hydroxyapatite chromatography. • An immunoassay was developed as a surrogate potency assay for CTBSEKDEL. 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 13595113
- Volume :
- 101
- Database :
- Academic Search Index
- Journal :
- Process Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 148450306
- Full Text :
- https://doi.org/10.1016/j.procbio.2020.10.018