A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide
(2009) In The FEBS Journal 276(5). p.1266-1281- Abstract
- We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (A beta), together with a scaleable and inexpensive purification procedure. A beta(1-40) and A beta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-A beta(1-40) and Met-A beta(1-42), referred to as A beta(M1-40) and A beta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method... (More)
- We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (A beta), together with a scaleable and inexpensive purification procedure. A beta(1-40) and A beta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-A beta(1-40) and Met-A beta(1-42), referred to as A beta(M1-40) and A beta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure A beta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized A beta 1-40 and A beta 1-42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1372222
- author
- Walsh, Dominic M. ; Thulin, Eva LU ; Minogue, Aedin M. ; Gustavsson, Niklas LU ; Pang, Eric ; Teplow, David B. and Linse, Sara LU
- organization
- publishing date
- 2009
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- amyloid, aggregation, A beta, fibrillogenesis, Alzheimer's disease
- in
- The FEBS Journal
- volume
- 276
- issue
- 5
- pages
- 1266 - 1281
- publisher
- Wiley-Blackwell
- external identifiers
-
- wos:000263451600011
- scopus:60349100306
- pmid:19175671
- ISSN
- 1742-464X
- DOI
- 10.1111/j.1742-4658.2008.06862.x
- language
- English
- LU publication?
- yes
- id
- 1c7e328e-f80e-4d83-907c-066f73d6fbf8 (old id 1372222)
- date added to LUP
- 2016-04-01 12:59:56
- date last changed
- 2022-04-06 01:55:25
@article{1c7e328e-f80e-4d83-907c-066f73d6fbf8,
abstract = {{We report the development of a high-level bacterial expression system for the Alzheimer's disease-associated amyloid beta-peptide (A beta), together with a scaleable and inexpensive purification procedure. A beta(1-40) and A beta(1-42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-A beta(1-40) and Met-A beta(1-42), referred to as A beta(M1-40) and A beta(M1-42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure A beta peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized A beta 1-40 and A beta 1-42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.}},
author = {{Walsh, Dominic M. and Thulin, Eva and Minogue, Aedin M. and Gustavsson, Niklas and Pang, Eric and Teplow, David B. and Linse, Sara}},
issn = {{1742-464X}},
keywords = {{amyloid; aggregation; A beta; fibrillogenesis; Alzheimer's disease}},
language = {{eng}},
number = {{5}},
pages = {{1266--1281}},
publisher = {{Wiley-Blackwell}},
series = {{The FEBS Journal}},
title = {{A facile method for expression and purification of the Alzheimer's disease-associated amyloid beta-peptide}},
url = {{http://dx.doi.org/10.1111/j.1742-4658.2008.06862.x}},
doi = {{10.1111/j.1742-4658.2008.06862.x}},
volume = {{276}},
year = {{2009}},
}