High-level expression of the Arabidopsis thaliana ethylene receptor protein ETR1 in Escherichia coli and purification of the recombinant protein.
Ethylene responses in plants are mediated by a small family of membrane integral receptors including the ETR1 gene product which are similar to the two-component bacterial histidine kinase regulators. Detailed biochemical and structural analysis of the ethylene-receptor family was hampered by the scarce amount of pure protein. Here, we report the construction, expression, and single-step purification of the ETR1 receptor protein from Arabidopsis thaliana in a bacterial expression system. The DNA fragment encoding the mature ETR1 receptor protein was subcloned into the pET15b expression vector and highly expressed in derivatives C41(DE3) and C43(DE3) of the Escherichia coli strain BL21(DE3). The recombinant protein was solubilised from the bacterial cells using mild non-denaturing detergents and purified to homogeneity by Ni-NTA affinity chromatography, yielding approximately 2-3 mg pure protein per litre of cells. The molecular mass of the purified protein was estimated to be 78 kDa by SDS-PAGE. The expression and purification of recombinant ETR1 reported here provide a basis for detailed functional and structural studies of the receptor protein, which might help to understand signal perception and signal transduction of the phytohormone ethylene on the molecular level.
Voet-van-Vormizeele
Jan
J
Heinrich-Heine-Universität, Biochemie der Pflanzen, Universitätsstr. 1, Düsseldorf D-40225, Germany.
Groth
Georg
G
D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000145:classification / Q000235:genetics*; D015870:Gene Expression; D009532:Nickel / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature
D029681:Arabidopsis Proteins; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C083750:ethylene receptors, plant; D009532:Nickel