Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli.
Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly alpha-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent K(D) of about 0.6 microM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.
Mennes
Nadine
N
Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Klare
Johann P
JP
Chizhov
Igor
I
Seidel
Ralf
R
Schlesinger
Ramona
R
Engelhard
Martin
M
D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D007834:Lasers; D010782:Photolysis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D019843:Archaeal Proteins; C434451:HtrII protein, Natronobacterium pharaonis; D011994:Recombinant Proteins