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

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Isolation and detection of a KDEL-tagged recombinant cholera toxin B subunit from Nicotiana benthamiana
Process Biochemistry ( IF 3.7 ) Pub Date : 2021-02-01 , DOI: 10.1016/j.procbio.2020.10.018
David A Morris _{1,

2} , Micaela A Reeves ₃ , Joshua M Royal _{1,

2,

3} , Krystal T Hamorsky _{1,

2,

4} , Nobuyuki Matoba _{1,

2,

3}

Affiliation

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.

中文翻译：

从本氏烟草中分离和检测 KDEL 标记的重组霍乱毒素 B 亚基

在这里，我们描述了用于分离和检测 KDEL 标记的植物产生的重组霍乱毒素 B 亚基 (CTB) 的改进方法，该亚基表现出独特的粘膜伤口愈合活性。该蛋白在本氏烟草中瞬时过表达，产生一些缺乏上皮修复活性的 C 末端 KDEL 截短分子种类。使用本文所述的新 CHT 色谱方法，这些源自产物的杂质成功地从具有完整 KDEL 序列的 CTB 中分离出来，如质谱所证实的那样。此外，开发了一种能够特异性检测具有完整 KDEL 序列的 GM1 神经节苷脂结合 CTB 的免疫测定法。结合在一起，

更新日期：2021-02-01

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