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Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana
- Source :
- Biochemical Engineering Journal. 142:58-67
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
Abstract
- 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.
- Subjects :
- 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
Subjects
Details
- ISSN :
- 1369703X
- Volume :
- 142
- Database :
- OpenAIRE
- Journal :
- Biochemical Engineering Journal
- Accession number :
- edsair.doi...........b519f08c21fe41a25009a014234d9419