title: 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.
authors.affiliation: Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universität Berlin, Albrecht-Thaer-Weg 6, D-14195, Berlin, Germany.
authors.forename: Klaas
authors.initials: K
authors.lastname: Wulfetange
authors.forename: Wolfram
authors.initials: W
authors.lastname: Saenger
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmülling
authors.forename: Alexander
authors.initials: A
authors.lastname: Heyl
mesh_terms: D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002474:Cell-Free System; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029681:Arabidopsis Proteins; D011956:Receptors, Cell Surface; D011494:Protein Kinases; C421783:WOL protein, Arabidopsis