Overexpression and purification of recombinant membrane PsbH protein in Escherichia coli.
In this work, we featured an expression system that enables the production of sufficient quantities ( approximately 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 microg protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies.
Halbhuber
Zbynek
Z
Photosynthesis Research Center, Institute of Physical Biology, University of South Bohemia, Zamek 136, 373 33 Nove Hrady, Czech Republic.
Petrmichlová
Zdenka
Z
Alexciev
Kassimir
K
Thulin
Eva
E
Stys
Dalibor
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D000458:Cyanobacteria; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D045332:Photosystem II Protein Complex / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D008565:Membrane Proteins; D010750:Phosphoproteins; D045332:Photosystem II Protein Complex; D011993:Recombinant Fusion Proteins; C063224:photosystem II, psbH subunit; D015951:Factor Xa