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 (similar tomg) of low molecular weight membrane protein of photosystem 11, 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 mug protein/ml of bacterial culture. The procedure as we optimized is applicable for isolation of small membrane proteins for structural studies. (C) 2003 Elsevier Inc. All rights reserved.