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 α-and β-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 α-and β-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the β-chain joined to the N-terminus of the α-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 (oFSHβα) was expressed at approximately 0·1 µg/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.

Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

“…The b-a configuration was widely used to produce recombinant single-chain glycoprotein hormones of many species (Narayan et al 1995, Boime & BenMenahem 1999, Dirnberger et al 2001, Fidler et al 2003, Min et al 2004, Jablonka-Shariff et al 2007, sometimes with heterologous CTP from hCG inserted between fused a-and b-subunits to enhance the secretion of these single-chain protein variants (Sugahara et al 1996, Garcia-Campayo et al 1997, Grossmann et al 1997, Fares et al 1998. These earlier studies showed that the CTP played the role of a flexible hydrophilic spacer allowing genetically fused a-and b-subunits to adopt a functional conformation and that secretion in the presence of CTP was enhanced due to its specific O-glycans.…”

Aptamer-Based Regionally Protected PCR for Protein Detection

“…For this study, we prepared recombinant oFSH␣ (roFSH␣) as described previously to generate the aptamer (9 ). roFSH containing both ␣ and ␤ subunits (roFSH␣␤) was also produced with transfected insect cells (High Five™; Invitrogen NZ) with the expressed protein secreted into serum-free culture medium.…”

“…roFSH containing both ␣ and ␤ subunits (roFSH␣␤) was also produced with transfected insect cells (High Five™; Invitrogen NZ) with the expressed protein secreted into serum-free culture medium. The roFSH␣␤ was demonstrated to be biologically active by in vitro bioassay (9 ). A highly purified sheep pituitary gland extract of native oFSH (Ova2) was a gift of Dr. Lloyd Moore (AgResearch Invermay, Mosgiel, New Zealand).…”

“…A highly purified sheep pituitary gland extract of native oFSH (Ova2) was a gift of Dr. Lloyd Moore (AgResearch Invermay, Mosgiel, New Zealand). This material was comparable in biological activity to the highly purified oFSH RP2 standard produced by the National Hormone and Pituitary Program (USA), as assessed by both RIA and in vitro bioassay (9 ). Both the roFSH␣␤ and Ova2 were used for characterization of the aptamer that had been generated against the roFSH␣.…”

Involvement of equine chorionic gonadotropin (eCG) carbohydrate side chains in its bioactivity; lessons from recombinant hormone expressed in insect cells

“…However, the great advantage of non-mammalian systems like yeasts, insect cell lines or plants would be to produce recombinant proteins in large amounts, at a lower cost, and in the absence of fetal calf serum. Some gonadotropins of zootechnical interest have already been expressed in baculovirus systems [11][12][13][14], but also in the methylotropic yeast Pichia pastoris [15][16][17], and in plants [18]. However, only in vitro biological activities have been reported in these studies with no information concerning in vivo potencies.…”