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

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. The common alpha subunit contains two asparagine (N)-linked oligosaccharides. To study the function of carbohydrates on in vitro refolding of alpha subunit and dimer assembly, we generated recombinant non-glycosylated hCG alpha subunit (rNGl-hCGalpha) from E. coli. The expression vector was constructed by inserting hCGalpha cDNA coding for the mature form in-frame into a pQE-30 vector, which contains a 6 x His sequence immediately before the 5'-end of hCGalpha cDNA for subsequent purification of rNG-hCGalpha. The rNG-hCGalpha 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 alpha 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-hCGalpha with native hCGbeta and oFSHbeta 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-hCGalpha and reconstitution of rNG-hCGalpha with native hCGbeta 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-hCGalpha with oFSHbeta 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-hCGalpha with oLHbeta. Our results suggest the carbohydrate moieties confer greater conformational flexibility to the backbone of the beta subunit and the relative rigidity of the beta subunit may serve as a conformational template of the alpha subunit. The present approach has made it possible to prepare the non-glycosylated gonadotropin alpha subunit in adequate amounts for further study on their biological and topographical features in complete absence of carbohydrate.