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Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli
Aggregation of amyloid-β protein (Aβ) is hypothesized to be a seminal neuropathological event in Alzheimer's disease (AD). Recombinant expression and purification of Aβ represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a no...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9087987/ https://www.ncbi.nlm.nih.gov/pubmed/35546794 http://dx.doi.org/10.1039/c8ra00042e |
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author | Jia, Longgang Wang, Wenjuan Shang, Jinzhao Zhao, Wenping Wei, Wei Wang, Ying Li, Li Lu, Fuping Liu, Fufeng |
author_facet | Jia, Longgang Wang, Wenjuan Shang, Jinzhao Zhao, Wenping Wei, Wei Wang, Ying Li, Li Lu, Fuping Liu, Fufeng |
author_sort | Jia, Longgang |
collection | PubMed |
description | Aggregation of amyloid-β protein (Aβ) is hypothesized to be a seminal neuropathological event in Alzheimer's disease (AD). Recombinant expression and purification of Aβ represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a novel high-yield expression and purification method for Aβ42 based on fusion with maltose binding protein (MBP) followed by the soluble polypeptide linker (NANP)(3) and a modified tobacco etch virus (TEV) cleavage site before the Aβ42. We obtained a final yield of ∼18 mg L(−1) of recombinant Aβ42 that was confirmed by SDS-PAGE, protein immunoblotting and MALDI-TOF. Finally, thioflavin T fluorescence and atomic force microscopy revealed that the recombinant Aβ42 aggregated into long, branched fibrils. Furthermore, the aggregates of the recombinant peptide had a strong cytotoxic effect on PC12 cells. The method described here can therefore be used to efficiently express the soluble fusion protein MBP-Aβ42 and obtain high-purity Aβ42 peptide, which can be used to understand the molecular mechanism of Aβ42 fibrillization and screen new candidate drugs for AD. |
format | Online Article Text |
id | pubmed-9087987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
spelling | pubmed-90879872022-05-10 Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli Jia, Longgang Wang, Wenjuan Shang, Jinzhao Zhao, Wenping Wei, Wei Wang, Ying Li, Li Lu, Fuping Liu, Fufeng RSC Adv Chemistry Aggregation of amyloid-β protein (Aβ) is hypothesized to be a seminal neuropathological event in Alzheimer's disease (AD). Recombinant expression and purification of Aβ represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. Herein, we report a novel high-yield expression and purification method for Aβ42 based on fusion with maltose binding protein (MBP) followed by the soluble polypeptide linker (NANP)(3) and a modified tobacco etch virus (TEV) cleavage site before the Aβ42. We obtained a final yield of ∼18 mg L(−1) of recombinant Aβ42 that was confirmed by SDS-PAGE, protein immunoblotting and MALDI-TOF. Finally, thioflavin T fluorescence and atomic force microscopy revealed that the recombinant Aβ42 aggregated into long, branched fibrils. Furthermore, the aggregates of the recombinant peptide had a strong cytotoxic effect on PC12 cells. The method described here can therefore be used to efficiently express the soluble fusion protein MBP-Aβ42 and obtain high-purity Aβ42 peptide, which can be used to understand the molecular mechanism of Aβ42 fibrillization and screen new candidate drugs for AD. The Royal Society of Chemistry 2018-05-21 /pmc/articles/PMC9087987/ /pubmed/35546794 http://dx.doi.org/10.1039/c8ra00042e Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by/3.0/ |
spellingShingle | Chemistry Jia, Longgang Wang, Wenjuan Shang, Jinzhao Zhao, Wenping Wei, Wei Wang, Ying Li, Li Lu, Fuping Liu, Fufeng Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title | Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title_full | Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title_fullStr | Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title_full_unstemmed | Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title_short | Highly efficient soluble expression, purification and characterization of recombinant Aβ42 from Escherichia coli |
title_sort | highly efficient soluble expression, purification and characterization of recombinant aβ42 from escherichia coli |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9087987/ https://www.ncbi.nlm.nih.gov/pubmed/35546794 http://dx.doi.org/10.1039/c8ra00042e |
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