Purification and aqueous phase atomic force microscopic observation of recombinant P2X2 receptor.
Recombinant P2X2 receptor was observed by atomic force microscope in the aqueous phase. The P2X2 receptor was expressed in an insect cell line, and recombinant proteins were prepared under native conditions. The membrane fractions were extracted, and histidine-tagged receptor protein was purified from the fractions by column chromatography. When the purified protein fraction was diluted with water and served for atomic force microscopy, dispersed particles of about 3 nm in height were observed. In the presence of 1 mM ATP, the assembly-like images of the particles were obtained. More densely assembled images of the particles were achieved when the protein was dissolved in a Tris buffer containing 1 mM ATP. Under this condition, imaging of the surface of the particles exhibited a circular structure with a diameter of about 10 nm having a pore-like structure. These results suggest that atomic force microscopy provides structural information about P2X2 receptor in aqueous phase.
Nakazawa
Ken
K
Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan. nakazawa@nihs.go.jp
Yamakoshi
Yoko
Y
Tsuchiya
Toshie
T
Ohno
Yasuo
Y
D000818:Animals; D018625:Microscopy, Atomic Force / Q000379:methods*; D010316:Particle Size; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014867:Water / Q000737:chemistry
D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D011994:Recombinant Proteins; D014867:Water