Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana

Salem Alkanaimsh, Jasmine M. Corbin, Muchena J. Kailemia, Kalimuthu Karuppanan, Raymond L. Rodriguez, Carlito B Lebrilla, Karen A. McDonald, Somen Nandi

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)58-67
Number of pages10
JournalBiochemical Engineering Journal
Volume142
DOIs
StatePublished - Feb 15 2019

Fingerprint

Glycosylation
Butyrylcholinesterase
Plant Proteins
Tobacco
Purification
Proteins
Affinity chromatography
Affinity Chromatography
Buffers
Procainamide
Tacrine
Costs and Cost Analysis
Hydrolases
Agarose Chromatography
Tromethamine
Organophosphates
Cholinesterase Inhibitors
Cholinesterases
Chromatography
Citric Acid

Keywords

  • Biopharmaceutical
  • butyrylcholinesterase
  • downstream processing
  • Hupresin
  • Nicotiana benthamiana

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Environmental Engineering
  • Biomedical Engineering

Cite this

Alkanaimsh, S., Corbin, J. M., Kailemia, M. J., Karuppanan, K., Rodriguez, R. L., Lebrilla, C. B., ... Nandi, S. (2019). Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana. Biochemical Engineering Journal, 142, 58-67. https://doi.org/10.1016/j.bej.2018.11.004

Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana. / Alkanaimsh, Salem; Corbin, Jasmine M.; Kailemia, Muchena J.; Karuppanan, Kalimuthu; Rodriguez, Raymond L.; Lebrilla, Carlito B; McDonald, Karen A.; Nandi, Somen.

In: Biochemical Engineering Journal, Vol. 142, 15.02.2019, p. 58-67.

Research output: Contribution to journalArticle

Alkanaimsh, S, Corbin, JM, Kailemia, MJ, Karuppanan, K, Rodriguez, RL, Lebrilla, CB, McDonald, KA & Nandi, S 2019, 'Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana' Biochemical Engineering Journal, vol. 142, pp. 58-67. https://doi.org/10.1016/j.bej.2018.11.004
Alkanaimsh, Salem ; Corbin, Jasmine M. ; Kailemia, Muchena J. ; Karuppanan, Kalimuthu ; Rodriguez, Raymond L. ; Lebrilla, Carlito B ; McDonald, Karen A. ; Nandi, Somen. / Purification and site-specific N-glycosylation analysis of human recombinant butyrylcholinesterase from Nicotiana benthamiana. In: Biochemical Engineering Journal. 2019 ; Vol. 142. pp. 58-67.
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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{\circledR}) 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{\circledR} 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.",
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