A glutathione-S-transferase-FSHalpha subunit hybrid associates with FSHbeta, retains biological activity, and facilitates purification.
The glycoprotein hormones are heterodimeric proteins that share a common alpha subunit and have unique beta subunits that confer receptor selectivity. One member of this family, follicle-stimulating hormone (FSH), is secreted by the pituitary and is involved in the control of male and female reproduction. Herein, we describe the construction of baculoviruses for glutathione-S-transferase (GST) fusions of the human FSH (hFSH) subunits and their expression in insect cells, either alone or with the complementary non-fused FSH subunits (FSHalpha or FSHbeta). Only the GST-BV-hFSHalpha monomer and the GST-BV-hFSHalpha/BV-hFSHbeta (GST-BV-hFSH) heterodimer were efficiently secreted into the culture supernatant. The hybrid molecule, GST-BV-hFSH, was affinity purified in one step, and demonstrated activity in receptor-radioligand binding assays and in a cAMP accumulation assay. The use of GST-BV-hFSHalpha provides a novel and efficient method for purifying and studying members of the glycoprotein hormone family derived from the culture supernatant or subcellular fractions of the cell.
Stevis
Panayiotis E
PE
Women's Health and Bone, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA. stevisp@wyeth.com
Arey
Brian J
BJ
Deecher
Darlene C
DC
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019281:Dimerization; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D008297:Male; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011962:Receptors, FSH / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D021122:Protein Subunits; D011962:Receptors, FSH; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D005982:Glutathione Transferase