BACTERIAL EXPRESSION OF HUMAN CHORIONIC-GONADOTROPIN ALPHA-SUBUNIT - STUDIES ON REFOLDING, DIMER ASSEMBLY AND INTERACTION WITH 2 DIFFERENT BETA-SUBUNITS

Human chorionic gonadotropin (hCG) is a member of a family of heterodi meric glycoprotein hormones that have a common ct subunit but differ i n their hormone-specific beta subunit. The common a subunit contains t wo asparagine (N)-linked oligosaccharides. To study the function of ca rbohydrates on in vitro refolding of alpha subunit and dimer assembly, we generated recombinant non-glycosylated hCG alpha subunit (rNG-hCG alpha) from E. coli. The expression vector was constructed by insertin g hCG alpha cDNA coding for the mature form in-frame into a pQE-30 vec tor, which contains a 6 x His sequence immediately before the 5'-end o f hCG alpha cDNA for subsequent purification of rNG-hCG alpha. The rNG -hCG alpha expressed in inclusion bodies was efficiently purified by i mmobilized metal chelate affinity chromatography on Ni-NTA resin. SDS- PAGE, solid-phase binding assay and immunoblotting demonstrated the ex pression of rNG-hCG. Its alpha molecular weight on SDS-PAGE was 14.7 k Da under reducing conditions and 15 kDa for a monomer accompanied with some higher molecular weight oligomer under non-reducing conditions. Reconstitution of rNG-hCG alpha with native hCG beta and oFSH beta occ urred in very low yield under standard conditions. However, the oxidat ion-reduction system cystamine (1.34 mM) and cysteamine (7.3 mM) facil itated both the refolding of rNG-hCG alpha and reconstitution of rNG-h CG alpha with native hCG beta to regain partially correct conformation . These were revealed by conformationally sensitive antibody and recep tor binding assays. Cystamine and cysteamine were more effective in th e recombination of rNG-hCG alpha with oFSH beta as indicated by a 22-3 6-fold decrease in the amount required to cause a 50% competitive inhi bition in radioreceptor assay. They have no effect on assembly of rNG- hCG alpha with oLH beta. 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 s ubunit in adequate amounts for further study on their biological and t opographical features in complete absence of carbohydrate.