Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin.

Autor: M Schopfer L; Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska, United States of America., David E; CHEMFORASE, Mont-Saint-Aignan, France., Hinrichs SH; Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America., Lockridge O; Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2023 Jan 13; Vol. 18 (1), pp. e0280380. Date of Electronic Publication: 2023 Jan 13 (Print Publication: 2023).
DOI: 10.1371/journal.pone.0280380
Abstrakt: Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2023 M. Schopfer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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