Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris.
The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MFalpha) leader and ovine follicle stimulating hormone (oFSH) alpha and beta 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 alpha and one copy of the beta 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 alpha and beta subunit concentrations indicated less than 10% of alpha and 25-33% of beta 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.
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AgResearch Wallaceville, PO Box 40063, Ward Street, Upper Hutt, New Zealand.
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Young
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McNatty
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D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D037201:Follicle Stimulating Hormone, beta Subunit; D015870:Gene Expression; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D014170:Transformation, Genetic
D017931:DNA Primers; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone