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The Chloroplast Small Heat Shock Protein-Purification and Characterization of Pea Recombinant Protein

Härndahl, Ulrika; Tufvesson, Ellen LU and Sundby, Cecilia (1998) In Protein Expression and Purification 14(1). p.87-96
Abstract
We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in... (More)
We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens @a-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-@b proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% @a-helical secondary structure. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
14
issue
1
pages
87 - 96
publisher
Academic Press
external identifiers
  • scopus:0344222184
ISSN
1046-5928
DOI
10.1006/prep.1998.0921
language
English
LU publication?
yes
id
6a63dd93-cf62-49ed-a2a9-a4168d3086cb (old id 125547)
date added to LUP
2007-07-05 14:55:50
date last changed
2017-06-04 03:46:13
@article{6a63dd93-cf62-49ed-a2a9-a4168d3086cb,
  abstract     = {We report here on a procedure to obtain large amounts of a chloroplast-localized heat shock protein (HSP21) with unknown structure and function, by using anEscherichia coliexpression system for the pea (Pisum sativum) protein and a purification procedure based on perfusion ion-exchange chromatography. After initial precipitation steps, the sample was applied to cation- and anion-exchange on two columns connected in sequence, which allowed rapid purification of HSP21 in one equilibration and one elution step. The purified recombinant protein had an isoelectric point of 5.0 and appeared in assembled, oligomeric form (approximately 200 kDa) composed of 21-kDa monomers, similar to the native HSP21 protein as detected by immunoblotting in plants after heat-stress treatment. This chloroplast-localized heat shock protein belongs to a special group of small heat shock proteins (sHSPs), which share an evolutionary conserved C-terminal domain with the vertebrate eye lens @a-crystallin. The crystallins are known from both crystallographic and spectroscopic data to be all-@b proteins. In contrast, this paper presents circular dichroism spectroscopy data which shows that the purified recombinant HSP21 oligomer has a content of more than 30% @a-helical secondary structure.},
  author       = {Härndahl, Ulrika and Tufvesson, Ellen and Sundby, Cecilia},
  issn         = {1046-5928},
  language     = {eng},
  number       = {1},
  pages        = {87--96},
  publisher    = {Academic Press},
  series       = {Protein Expression and Purification},
  title        = {The Chloroplast Small Heat Shock Protein-Purification and Characterization of Pea Recombinant Protein},
  url          = {http://dx.doi.org/10.1006/prep.1998.0921},
  volume       = {14},
  year         = {1998},
}