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Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli.

Halbhuber, Z ; Petrmichlov, Z ; Alexciev, K ; Thulin, Eva LU and Stys, D (2003) In Protein Expression and Purification 32(1). p.18-27
Abstract
In this work, we featured an expression system that enables the production of sufficient quantities (~mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies. PsbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione-S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells. A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism. As a result, the majority of fusion protein was obtained in a soluble state and... (More)
In this work, we featured an expression system that enables the production of sufficient quantities (~mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies. PsbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione-S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells. A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism. As a result, the majority of fusion protein was obtained in a soluble state and could be purified from crude bacterial lysate by affinity chromatography on immobilized glutathione under non-denaturing conditions. The PsbH protein was cleaved from the carrier protein with Factor Xa protease and purified on DEAE–cellulose column with yields of up to 2.1 g protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies. (Less)
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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Expression and Purification
volume
32
issue
1
pages
18 - 27
publisher
Academic Press
external identifiers
  • wos:000186502100003
  • pmid:14680935
  • scopus:0344961712
ISSN
1046-5928
DOI
10.1016/S1046-5928(03)00188-8
language
English
LU publication?
yes
id
53c01075-adc6-47c1-9767-bfbf1e559b0c (old id 128050)
date added to LUP
2016-04-01 12:13:25
date last changed
2022-01-27 00:38:36
@article{53c01075-adc6-47c1-9767-bfbf1e559b0c,
  abstract     = {{In this work, we featured an expression system that enables the production of sufficient quantities (~mg) of low molecular weight membrane protein of photosystem II, PsbH protein, for solid-state NMR as well as other biophysical studies. PsbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed expression of the PsbH protein as a glutathione-S transferase (GST) fusion protein in Escherichia coli BL21(DE3) cells. A relatively large GST anchor overcomes foreseeable problems with the low solubility of membrane proteins and the toxicity caused by protein incorporation into the membrane of the host organism. As a result, the majority of fusion protein was obtained in a soluble state and could be purified from crude bacterial lysate by affinity chromatography on immobilized glutathione under non-denaturing conditions. The PsbH protein was cleaved from the carrier protein with Factor Xa protease and purified on DEAE–cellulose column with yields of up to 2.1 g protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies.}},
  author       = {{Halbhuber, Z and Petrmichlov, Z and Alexciev, K and Thulin, Eva and Stys, D}},
  issn         = {{1046-5928}},
  language     = {{eng}},
  number       = {{1}},
  pages        = {{18--27}},
  publisher    = {{Academic Press}},
  series       = {{Protein Expression and Purification}},
  title        = {{Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli.}},
  url          = {{http://dx.doi.org/10.1016/S1046-5928(03)00188-8}},
  doi          = {{10.1016/S1046-5928(03)00188-8}},
  volume       = {{32}},
  year         = {{2003}},
}