Overproduction of rat 1,25-dihydroxyvitamin D3 receptor in insect cells using the baculovirus expression system.
The rat 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor has been expressed at elevated levels in Spodoptera frugiperda cells using the baculovirus expression vector system. The recombinant 1,25-(OH)2D3 receptor is full-length, binds 1,25-(OH)2D3, and is recognized by a monoclonal antibody specific for 1,25-(OH)2D3 receptor. Densitometric scanning of Coomassie brilliant blue-stained SDS/polyacrylamide gels indicated a recombinant receptor protein level comprising 5% of the total soluble protein from the insect cells. The hydroxylapatite binding assay revealed average levels of 2 nmol of unoccupied 1,25-(OH)2D3 receptor per mg of protein in insect cells at 72 hr after infection with recombinant baculovirus. A measure of total 1,25-(OH)2D3 receptor using a ligand-independent, immunoradiometric assay disclosed average levels of 2.3 nmol of receptor per mg of protein produced by these same cells. A monoclonal antibody directed against the 1,25-(OH)2D3 receptor, and reported to cross-react with this receptor derived from several species, recognized the recombinant rat 1,25-(OH)2D3 receptor upon Western analysis. A monoclonal antibody directed specifically against the porcine receptor failed to recognize the recombinant rat 1,25-(OH)2D3 receptor protein. The cytosolic preparation of insect cells infected with recombinant baculovirus exhibited an equilibrium dissociation constant of 1 x 10(-11) M as determined by a 1,25-(OH)2D3 saturation analysis plotted by the method of Scatchard. This expression system provides an adequate source from which abundant quantities of 1,25-(OH)2D3 receptor can be purified for subsequent x-ray crystallographic analyses.
Ross
T K
TK
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Prahl
J M
JM
DeLuca
H F
HF
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D010957:Plasmids; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D002117:Calcitriol