Effects of Kifunensine on Production and N -Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

The production and -glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was perfo...

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Veröffentlicht in:International journal of molecular sciences 2020-09, Vol.21 (18), p.6896
Hauptverfasser: Macharoen, Kantharakorn, Li, Qiongyu, Márquez-Escobar, Veronica A, Corbin, Jasmine M, Lebrilla, Carlito B, Nandi, Somen, McDonald, Karen A
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Sprache:eng
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Zusammenfassung:The production and -glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW) or 7.5 ± 0.4 mg L in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different -glycoforms. -Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.
ISSN:1422-0067
1422-0067
DOI:10.3390/ijms21186896