Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana
[Display omitted] •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 isol...
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Veröffentlicht in: | Process biochemistry (1991) 2021-02, Vol.101, p.42-49 |
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Sprache: | eng |
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•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. |
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ISSN: | 1359-5113 1873-3298 |
DOI: | 10.1016/j.procbio.2020.10.018 |