Expression and characterization of the extracellular domain of guanylyl cyclase C from a baculovirus and Sf21 insect cells.
Guanylyl cyclase (GC)-C, a single-transmembrane receptor protein for heat-stable enterotoxin, guanylin, and uroguanylin, and its N-terminal extracellular domain were prepared at a high level of expression from a system constructed of Sf21 insect cells and recombinant baculovirus. The recombinant GC-C, containing the complete sequence, retained its binding affinity to heat-stable enterotoxin with a KD value (6.2 x 10(-10) M) and cyclase catalytic activity at a level similar to those of GC-C expressed in mammalian cell lines, such as COS-7. The N-terminal extracellular domain was prepared in a form which contained the hexahistidine tail at its C-terminus and was purified as a homogenous protein by Con A and Ni-chelating affinity chromatography from the culture medium of the insect cells. The purified N-terminal extracellular domain of GC-C exhibited the high (KD = 4 x 10(-10) M) and low (KD = 7 x 10(-8) M) affinity sites in binding to heat-stable enterotoxin. These results clearly indicate that the N-terminal extracellular domain of GC-C possesses the same biochemical characteristics as the complete GC-C protein even in the membrane-free form. Moreover, the extracellular domain is able to form an oligomer in a ligand-dependent manner, suggesting that the N-terminal extracellular domains interact with one another in binding to ligands.
Hasegawa
M
M
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565, Japan.
Kawano
Y
Y
Matsumoto
Y
Y
Hidaka
Y
Y
Fujii
J
J
Taniguchi
N
N
Wada
A
A
Hirayama
T
T
Shimonishi
Y
Y
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005768:Gastrointestinal Hormones; D005822:Genetic Vectors; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D045265:Natriuretic Peptides; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013552:Swine; D014162:Transfection
D005768:Gastrointestinal Hormones; D045265:Natriuretic Peptides; D010446:Peptide Fragments; D010455:Peptides; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C072685:guanylin; C084362:uroguanylin; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled