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Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease

Frankel, Rebecca LU ; Bernfur, Katja LU ; Sparr, Emma LU and Linse, Sara LU (2021) In Protein Expression and Purification 187.
Abstract
Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus... (More)
Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol. (Less)
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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Apolipoprotein A-I, Autoprotease, Expression, Purification, High-density lipoprotein
in
Protein Expression and Purification
volume
187
article number
105946
pages
10 pages
publisher
Academic Press
external identifiers
  • scopus:85111208778
  • pmid:34298139
ISSN
1046-5928
DOI
10.1016/j.pep.2021.105946
language
English
LU publication?
yes
id
7e4d70c4-8075-49b6-be75-be35cfbaf808
date added to LUP
2021-10-11 09:43:06
date last changed
2023-11-08 20:49:08
@article{7e4d70c4-8075-49b6-be75-be35cfbaf808,
  abstract     = {{Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in <i>E. coli</i>. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.}},
  author       = {{Frankel, Rebecca and Bernfur, Katja and Sparr, Emma and Linse, Sara}},
  issn         = {{1046-5928}},
  keywords     = {{Apolipoprotein A-I; Autoprotease; Expression; Purification; High-density lipoprotein}},
  language     = {{eng}},
  publisher    = {{Academic Press}},
  series       = {{Protein Expression and Purification}},
  title        = {{Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease}},
  url          = {{http://dx.doi.org/10.1016/j.pep.2021.105946}},
  doi          = {{10.1016/j.pep.2021.105946}},
  volume       = {{187}},
  year         = {{2021}},
}