Bacterial expression of human chorionic gonadotropin α subunit: studies on refolding, dimer assembly and interaction with two different β subunits

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common α subunit but differ in their hormone-specific β subunit. The common α subunit contains two asparagine (N)-linked oligosaccharides. To study the function of carbohydrates on in vitro refolding of α subunit and dimer assembly, we generated recombinant non-glycosylated hCG α subunit (rNG-hCGα) from E. coli. The expression vector was constructed by inserting hCGα cDNA coding for the mature form in-frame into a pQE-30 vector, which contains a 6 × His sequence immediately before the 5′-end of hCGα cDNA for subsequent purification of rNG-hCGα. The rNG-hCGα expressed in inclusion bodies was efficiently purified by immobilized metal chelate affinity chromatography on Ni-NTA resin. SDS-PAGE, solid-phase binding assay and immunoblotting demonstrated the expression of rNG-hCG. Its α molecular weight on SDS-PAGE was 14.7 kDa under reducing conditions and 15 kDa for a monomer accompanied with some higher molecular weight oligomer under non-reducing conditions. Reconstitution of rNG-hCGα with native hCGβ and oFSHβ occurred in very low yield under standard conditions. However, the oxidation-reduction system cystamine (1.34 mM) and cysteamine (7.3 mM) facilitated both the refolding of rNG-hCGα and reconstitution of rNG-hCGα with native hCGβ to regain partially correct conformation. These were revealed by conformationally sensitive antibody and receptor binding assays. Cystamine and cysteamine were more effective in the recombination of rNG-hCGα with oFSHβ as indicated by a 22–36-fold decrease in the amount required to cause a 50% competitive inhibition in radioreceptor assay. They have no effect on assembly of rNG-hCGα with oLHβ. Our results suggest the carbohydrate moieties confer greater conformational flexibility to the backbone of the β subunit and the relative rigidity of the β subunit may serve as a conformational template of the α subunit. The present approach has made it possible to prepare the non-glycosylated gonadotropin α subunit in adequate amounts for further study on their biological and topographical features in complete absence of carbohydrate.