STRUCTURE-BASED DESIGN AND PROTEIN ENGINEERING OF INTERSUBUNIT DISULFIDE BONDS IN GONADOTROPINS

Pairs of cystine residues were introduced in the alpha- and beta-subun its of human choriogonadotropin at positions with optimal geometries f or the formation of disulfide bonds. Using the homology with luteinizi ng hormone and follicle stimulating hormone, similar mutations were ca rried out in these glycoprotein hormones. In nearly all mutants the co rresponding disulfide bonds were formed leading to a non-natural, cova lent linkage between the alpha- and beta-subunits. The mutants typical ly display wild-type receptor binding and bioactivity. The mutants wit h non-natural intersubunit disulfide bonds display enhanced thermostab ilities relative to the corresponding heterodimeric glycoprotein hormo nes, rendering them candidates for long acting gonadotropins with enha nced shelf lives.