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

1998 ◽  
Vol 21 (3) ◽  
pp. 327-336 ◽  
Author(s):  
AE Fidler ◽  
S Lun ◽  
W Young ◽  
KP McNatty
Keyword(s):  
Pichia Pastoris ◽  
Follicle Stimulating Hormone ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Beta Subunit ◽  
Fusion Genes ◽  
Glycoprotein Hormone ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Stimulating Hormone

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.

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

2003 ◽  
Vol 30 (2) ◽  
pp. 213-225 ◽  
Author(s):  
AE Fidler ◽  
JS Lin ◽  
S Lun ◽  
W Ng Chie ◽  
A Western ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

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.

Production of a biologically active recombinant marsupial growth factor using the methylotrophic yeast Pichia pastoris

2002 ◽  
Vol 14 (6) ◽  
pp. 327 ◽  
Author(s):  
Andrew E. Fidler ◽  
Andrea H. Western ◽  
Nicole Griffith ◽  
Lynne Selwood ◽  
Vicki Stent ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Germ Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Brushtail Possum ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

The cytokine stem cell factor (SCF) and its interaction with its receptor c-kit plays an important role in the development of germ cells in eutherians. To investigate the putative roles of the SCF/c-kit system in marsupials, recombinant Australian brushtail possum (Trichosurus vulpecula) SCF was purified after secretion by the methylotrophic yeast Pichia pastoris. The purification procedure utilized Ni2+ affinity chromatography with a poly-histidine tag engineered onto the C-terminus of the recombinant SCF. The recombinant possum SCF had a molecular weight of 48 kDa, as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and was biologically active with respect to its ability to maintain and induce proliferation of marsupial primordial germ cells in vitro. Furthermore, the recombinant possum SCF stimulated proliferation of the cell line TF1 and this bioactivity could be inhibited using an antibody directed against recombinant mouse SCF. This source of biologically active marsupial SCF may prove useful in future studies of marsupial development.

scholarly journals Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

1999 ◽  
Vol 22 (3) ◽  
pp. 273-283 ◽  
Author(s):  
C Sen Gupta ◽  
RR Dighe
Keyword(s):  
Pichia Pastoris ◽  
Structure Function ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Recombinant Expression ◽  
Expression System ◽  
Biologically Active ◽  
Maximal Response ◽  
Glycoprotein Hormone

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.

Expression of Biologically Active Recombinant Pokeweed Antiviral Protein in Methylotrophic Yeast Pichia pastoris

2000 ◽  
Vol 18 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Francis Rajamohan ◽  
Sekou O. Doumbia ◽  
Cherri R. Engstrom ◽  
Sharon L. Pendergras ◽  
Daniell L. Maher ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Biologically Active ◽  
Pokeweed Antiviral Protein ◽  
Antiviral Protein ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals Expression of Biologically Active Fusion Genes Encoding the Common Subunit and the Follicle-stimulating Hormone Subunit

1996 ◽  
Vol 271 (18) ◽  
pp. 10445-10448 ◽  
Author(s):  
Tadashi Sugahara ◽  
Asomi Sato ◽  
Masataka Kudo ◽  
David Ben-Menahem ◽  
Mary R. Pixley ◽  
...  
Keyword(s):  
Follicle Stimulating Hormone ◽  
Biologically Active ◽  
Fusion Genes ◽  
Genes Encoding ◽  
The Common ◽  
Stimulating Hormone

scholarly journals Actions and Roles of FSH in Germinative Cells

2021 ◽  
Vol 22 (18) ◽  
pp. 10110
Author(s):  
Kaiana Recchia ◽  
Amanda Soares Jorge ◽  
Laís Vicari de Figueiredo Pessôa ◽  
Ramon Cesar Botigelli ◽  
Vanessa Cristiane Zugaib ◽  
...  
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Follicle Stimulating Hormone ◽  
Adult Life ◽  
Target Cells ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Stimulating Hormone

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic–pituitary–gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

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