Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/128050
- author
- Halbhuber, Z ; Petrmichlov, Z ; Alexciev, K ; Thulin, Eva LU and Stys, D
- organization
- publishing date
- 2003
- 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}}, }