Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana
| Autor: | Somen Nandi, Raymond L. Rodriguez, Jasmine M. Corbin, Kalimuthu Karuppanan, Carlito B. Lebrilla, Karen A. McDonald, Salem Alkanaimsh, Muchena J. Kailemia |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Tris 0303 health sciences Environmental Engineering Downstream processing biology Biomedical Engineering Nicotiana benthamiana Bioengineering Serine hydrolase biology.organism_classification 01 natural sciences 03 medical and health sciences chemistry.chemical_compound Protein sequencing Affinity chromatography chemistry N-linked glycosylation Biochemistry 010608 biotechnology Butyrylcholinesterase 030304 developmental biology Biotechnology |
| Zdroj: | Biochemical Engineering Journal. 142:58-67 |
| ISSN: | 1369-703X |
| DOI: | 10.1016/j.bej.2018.11.004 |
| Popis: | Butyrylcholinesterase (BChE) is a glycosylated serine hydrolase found in human serum that has been shown to protect against various cholinesterase-inhibiting organophosphate nerve agents. The supply of plasma-derived butyrylcholinesterase (hBChE) is constrained by the availability of human blood and a complex purification process and its high cost. This constraints necessitates the development of expression platforms capable of large-scale, low-cost production of an active recombinant BChE (rBChE). Traditionally, procainamide affinity chromatography has been used to purify BChE. Recently, an effective affinity chromatography resin based on a potent cholinesterase inhibitor (tacrine-huperzine A hybrid; huperine X termed as Hupresin®) was developed. Here, we describe a purification scheme of rBChE from Nicotiana benthamiana plants. Different extraction buffers were screened for their ability to extract rBChE and native plant proteins. Citrate buffer at pH 4 was selected to minimize extraction of host plant proteins and showed a 4.5-fold enhancement in rBChE specific activity compared to Tris buffer, pH 8. DEAE-Sepharose chromatography increased the purity of rBChE by 70% by removing major host plant protein impurities. The rBChE was then adsorbed to Hupresin® and purified to homogeneity for an overall process yield of 34%. The purification process represents a threefold higher product yield over the established process. Mass spectrometry confirmed the protein sequence and site-specific N-glycosylation analysis was performed. |
| Databáze: | OpenAIRE |
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