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 journalArticlepeer-review

3 Scopus citations

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

Keywords

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

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Environmental Engineering
  • Biomedical Engineering

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