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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2023-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0280380 |
| _version_ | 1797943983834398720 |
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| author | Lawrence M Schopfer Emilie David Steven H Hinrichs Oksana Lockridge |
| author_facet | Lawrence M Schopfer Emilie David Steven H Hinrichs Oksana Lockridge |
| author_sort | Lawrence M Schopfer |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-10T20:31:29Z |
| format | Article |
| id | doaj.art-276bc035f5dc4e2db13f98512aa3fbd3 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-10T20:31:29Z |
| publishDate | 2023-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-276bc035f5dc4e2db13f98512aa3fbd32023-01-25T05:33:01ZengPublic Library of Science (PLoS)PLoS ONE1932-62032023-01-01181e028038010.1371/journal.pone.0280380Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin.Lawrence M SchopferEmilie DavidSteven H HinrichsOksana LockridgeProtection 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.https://doi.org/10.1371/journal.pone.0280380 |
| spellingShingle | Lawrence M Schopfer Emilie David Steven H Hinrichs Oksana Lockridge Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin. PLoS ONE |
| 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 |
| url | https://doi.org/10.1371/journal.pone.0280380 |
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