title: Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.
abstract: The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.
authors.affiliation: AgResearch Wallaceville, PO Box 40063, Upper Hutt, New Zealand.
authors.forename: A E
authors.initials: AE
authors.lastname: Fidler
authors.forename: J S
authors.initials: JS
authors.lastname: Lin
authors.forename: S
authors.initials: S
authors.lastname: Lun
authors.forename: W
authors.initials: W
authors.lastname: Ng Chie
authors.forename: A
authors.initials: A
authors.lastname: Western
authors.forename: V
authors.initials: V
authors.lastname: Stent
authors.forename: K P
authors.initials: KP
authors.lastname: McNatty
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D002417:Cattle; D003062:Codon; D005260:Female; D037201:Follicle Stimulating Hormone, beta Subunit / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D015292:Glycoprotein Hormones, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011962:Receptors, FSH / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012756:Sheep
chemical_list: D003062:Codon; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D011962:Receptors, FSH; D011993:Recombinant Fusion Proteins