E. coli-Based Cell-Free Expression, Purification and Characterization of the Membrane-Bound Ligand-Binding CHASE-TM Domain of the Cytokinin Receptor CRE1/AHK4 of Arabidopsis thaliana

Abstract

The plant hormone cytokinin is implicated in a large number of developmental and physiological processes. In the model plant Arabidopsis thaliana cytokinin is perceived by a class of membrane-bound receptor histidine kinases with three members, namely AHK2, AHK3, and CRE1/AHK4. These receptors possess an N-terminally located putative extracellular cyclases/histidine kinases associated sensor extracellular (CHASE) domain, which is responsible for hormone recognition. This hydrophilic domain and the two flanking transmembrane regions (CHASE-TM) were expressed using a cell-free protein expression system based on a bacterial ribosomal extract. To obtain soluble CHASE-TM protein, different detergents were directly added to the cell-free reaction and their effect on the yield of soluble protein was studied. After optimising the experimental set-up and employing Brij 58 as a detergent more than 3 mg/ml soluble protein of the CHASE-TM domain were obtained. Affinity purification via a C-terminally fused His-tag resulted in greater than 90% purity. The identity of the purified domain was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Circular dichroism spectroscopy was used and a predominantly α-helical folding pattern was shown, which is in good accordance with secondary structure prediction. A newly developed cytokinin binding assay confirmed the functionality of the thus expressed and purified CHASE-TM domain. The work presented clearly demonstrates the feasibility of producing high amounts of a plant membrane protein using a cell-free protein expression system. This opens the possibility of further biochemical and pharmacological analysis, as well as structural studies on this type of receptor protein.