Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris

Abstract

The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MF ) leader and ovine follicle stimulating hormone (oFSH) and subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51·0 ng/ml and 17·5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the and one copy of the subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay).It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH and subunit concentrations indicated less than 10% of and 25-33% of subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines

“…However, the great advantage of non-mammalian systems like yeasts, insect cell lines or plants would be to produce recombinant proteins in larger 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 (Huang et al 1991, Inaba et al 1998, van de Wiel et al 1998, in the methylotropic yeast Pichia pastoris (Fidler et al 1998, Richard et al 1998, and in plants (Dirnberger et al 2001). However, only in vitro biological activities have been reported in these studies, with no information concerning the in vivo potencies of the recombinant hormones produced.…”

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris

“…Supernatants from two strains expressing tethered oFSH protein (GS115/ pPIC9 FSH Mut S and GS115/pPIC9 EAFSH Mut S ) competitively displaced radiolabelled oFSH in a similar manner to the reference oFSH standard (RP2) as shown by the slopes of their displacement regression lines being similar ( 0·868 and 0·861 respectively) to that of the native oFSH standard RP2 ( 0·814). Furthermore the supernatant from a previously described strain which expresses the oFSH -and -chains separately (GS115/pAOM M Mut S , Fidler et al (1998)) produced a displacement curve parallel to that of the strains expressing the tethered oFSH protein.…”

“…In a previous paper we reported the production of biological active ovine FSH (oFSH) using transformed Pichia strains coexpressing oFSHand -subunit cDNA sequences fused to nucleotide sequences encoding the mating factor alpha (MF ) leader sequence (Fidler et al 1998). Such Pichia strains expressed biologically active oFSH, albeit at low levels.…”

“…Such Pichia strains expressed biologically active oFSH, albeit at low levels. However, most of the subunits that were secreted had not dimerised and furthermore it appeared that only one-third of the dimers produced were biologically active (Fidler et al 1998). Therefore the primary aim of this study was to test whether the issue of the low level of interchain dimerisation could be addressed by using P. pastoris to express recombinant tethered proteins formed by joining the C-terminus of the oFSH -chain to the N-terminus of the -chain, with a two amino acid linker sequence.…”

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.…”