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

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 proce...

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Autores principales: M. Schopfer, Lawrence, David, Emilie, Hinrichs, Steven H., Lockridge, Oksana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9838835/
https://www.ncbi.nlm.nih.gov/pubmed/36638134
http://dx.doi.org/10.1371/journal.pone.0280380
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author M. Schopfer, Lawrence
David, Emilie
Hinrichs, Steven H.
Lockridge, Oksana
author_facet M. Schopfer, Lawrence
David, Emilie
Hinrichs, Steven H.
Lockridge, Oksana
author_sort M. Schopfer, Lawrence
collection PubMed
description 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.
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spelling pubmed-98388352023-01-14 Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin M. Schopfer, Lawrence David, Emilie Hinrichs, Steven H. Lockridge, Oksana PLoS One Research Article 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. Public Library of Science 2023-01-13 /pmc/articles/PMC9838835/ /pubmed/36638134 http://dx.doi.org/10.1371/journal.pone.0280380 Text en © 2023 M. Schopfer et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
M. Schopfer, Lawrence
David, Emilie
Hinrichs, Steven H.
Lockridge, Oksana
Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title_full Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title_fullStr Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title_full_unstemmed Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title_short Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin
title_sort human butyrylcholinesterase in cohn fraction iv-4 purified in a single chromatography step on hupresin
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9838835/
https://www.ncbi.nlm.nih.gov/pubmed/36638134
http://dx.doi.org/10.1371/journal.pone.0280380
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