Efficient eukaryotic expression system for authentic human sex hormone-binding globulin

Abstract

Sex hormone-binding globulin (SHBG) is the main carrier for androgens and oestrogens in humans. It mediates the transport of steroid hormones in the circulation and testicular fluid, and regulates their bioavailability to steroid-responsive tissues. In addition, the protein interacts with membrane receptors expressed in target tissues. Binding to the receptors is suspected to facilitate the uptake of steroid hormones and/or elicit cellular signal transduction. The identity of the SHBG receptor has not yet been resolved, in part due to a lack of sufficient quantities of authentic SHBG for receptor purification and molecular characterization. We have successfully addressed this problem by establishing an episomal expression system in human embryonic kidney cells that produces 5 mg of fully active human SHBG per litre. The recombinant protein resembles native SHBG in terms of structure, glycosylation pattern and steroid-binding activity. Moreover, the protein interacts with plasma membranes in steroid target tissues, an activity not observed with SHBG from other recombinant expression systems. Thus our studies have removed an important obstacle to the further elucidation of the role SHBG plays in steroid hormone action.

Differential Binding of Ligands to the Apolipoprotein E Receptor 2

“…For production of the various receptor domains, the encoding PCR fragments were recovered by NheI and NotI digest from pGemTeasy and cloned into plasmid pCEP-Pu-sp-his-mys-fXa that allows expression of the proteins as carboxyl-terminal fusions with a hexahistidine tag, a myc epitope, and a factor Xa cleavage site (26). All constructs were introduced into 293 EBNA cells (Invitrogen, www.invitrogen.com).…”

Estrogen and the inner ear: megalin knockout mice suffer progressive hearing loss

“…GST-tagged rat receptor-associated protein (RAP) was purified from HEK293 EBNA (Epstein-Barr virus nuclear antigen) cells as described previously (10) and bathed in a solution of fluorescein isothiocyanate (FITC)-estradiol BSA (22.7 g/l; final concentration of 4 M). FITC-labeled 17␤-estradiol was synthesized from 1,3,5(10)-estradien-3-17␤-diol-17-hemisuccinate: BSA (Steraloids, London, UK) with FITC.…”

Effect of mutations of the human serpin protein corticosteroid-binding globulin on cortisol-binding, thermal and protease sensitivity

“…For functional analyses we employed two different expression vectors. Wild-type CBG cDNA including the signal sequence was subcloned into pCEP-Pu [28,29] to obtain CBG without a tag. Alternatively, wild-type CBG and the different CBG mutants without CBG signal sequence were subcloned into the pCEP-Pu-SP-his-myc-fX vector [28,29], which provides an N-terminal human BM-40 signal sequence and a His 6 -tag.…”