Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants.
Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus-Sf9 insect cell system either as a single-chain with the C-terminus of the beta-subunit fused to the N-terminus of the alpha-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of alpha-subunit and the other at the C-terminus of the beta-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 degrees C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T(1/2), 74-77 degrees C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.
Legardinier
Sébastien
S
INRA, CNRS, Université François Rabelais de Tours, Physiologie de la Reproduction et des Comportements, INRA Centre de Recherches de Tours-Nouzilly, 37 380 Nouzilly, France.
Poirier
Jean-Claude
JC
Klett
Danièle
D
Combarnous
Yves
Y
Cahoreau
Claire
C
D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D003001:Cloning, Molecular; D019281:Dimerization; D004355:Drug Stability; D018397:Equidae; D006064:Gonadotropins, Equine / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D006639:Histidine / Q000235:genetics; D007986:Luteinizing Hormone / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D011489:Protein Denaturation; D015202:Protein Engineering; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D018411:Spodoptera; D014162:Transfection
D006063:Chorionic Gonadotropin; D006064:Gonadotropins, Equine; D020033:Protein Isoforms; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D007986:Luteinizing Hormone