Overexpression and purification of recombinant membrane protein PsbH in Escherichia coli
Zbyněk Halbhuber1, Zdeňka Petrmichlová 1,2, Kassimir Alexciev3, Eva Thulin4 and Dalibor Štys1,2,5
1Photosynthesis Research Center, Institute of Physical Biology, University of South Bohemia, Zamek 136, 373 33 Nové Hrady, Czech Republic
2 Department of Autotrophic Microorganisms, Institute of Microbiology CAS, 37901 Třeboň - Opatovický mlýn, Czech Republic
3 CanAg Diagnostics, 414 55 Gothenburg , Sweden
4 Biophysical Chemistry, Lund University, Box 124, 22100 Lund, Sweden5
5Institute of Landscape Ecology, Academy of Science of the Czech Republic, Zamek 136, 373 33 Nové Hrady, Czech Republic
In this work we featured an expression system that enables the production of sufficient quantities of membrane PsbH protein (~mg´s quantities) for solid-state NMR as well as other biophysical studies. PsbH is a small membrane protein associated with the photosystem II complex in higher plants, algae and cyanobacteria. Although the exact role of PsbH is not clear, it seems to be important for the structure and function of photosystem II.
In this approach a synthetic psbH gene from cyanobacterium Synechocystis sp. PCC 6803 was cloned into a plasmid expression vector, which allowed a direct synthesis of the PsbH protein as a glutathione‑S transferase (GST) fusion protein in E. coli BL21(DE3) cells. A relatively large GST anchor overcome 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 immobilised glutathione under non-denaturating 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 µg protein/ml of bacterial culture. Details of sample optimization for small membrane proteins as well as the impact constitutive cell protection mechanism against host membrane proteins are discussed.