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Expression of biologically active beta subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris. 1997

M Samaddar, and J F Catterall, and R R Dighe
Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore, India.

Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an alpha subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific beta subunit. The objective of the present study is to develop a recombinant DNA expression system for the beta subunit of FSH that can be applied to study structure-function relationships while producing large quantities of the hormone subunit for immuno-contraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSH beta (bFSH beta) in the methylotrophic yeast Pichia pastoris. The Pichia-expressed FSH beta (pFSH beta) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSH beta. Replacement of cognate signal peptide with the yeast alpha mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 micrograms/ml (alpha mating factor signal peptide) of the culture supernatant. pFSH beta His.tag (pFSH beta with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSH beta His.tag was approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSH beta. pFSH beta His.tag could assemble with the alpha subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSH beta His.tag is properly folded and biologically active.

UI MeSH Term Description Entries
D007986 Luteinizing Hormone A major gonadotropin secreted by the adenohypophysis (PITUITARY GLAND, ANTERIOR). Luteinizing hormone regulates steroid production by the interstitial cells of the TESTIS and the OVARY. The preovulatory LUTEINIZING HORMONE surge in females induces OVULATION, and subsequent LUTEINIZATION of the follicle. LUTEINIZING HORMONE consists of two noncovalently linked subunits, alpha and beta. Within a species, the alpha subunit is common in the three pituitary glycoprotein hormones (TSH, LH and FSH), but the beta subunit is unique and confers its biological specificity. ICSH (Interstitial Cell Stimulating Hormone),Interstitial Cell-Stimulating Hormone,LH (Luteinizing Hormone),Lutropin,Luteoziman,Luteozyman,Hormone, Interstitial Cell-Stimulating,Hormone, Luteinizing,Interstitial Cell Stimulating Hormone
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D010843 Pichia Yeast-like ascomycetous fungi of the family Saccharomycetaceae, order SACCHAROMYCETALES isolated from exuded tree sap. Hansenula,Hansenulas,Pichias
D011863 Radioimmunoassay Classic quantitative assay for detection of antigen-antibody reactions using a radioactively labeled substance (radioligand) either directly or indirectly to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Non-immunogenic substances (e.g., haptens) can be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. Radioimmunoassays
D011962 Receptors, FSH Cell surface proteins that bind FOLLICLE STIMULATING HORMONE with high affinity and trigger intracellular changes influencing the behavior of cells. FSH Receptors,Follicle-Stimulating Hormone Receptors,Receptors, Follicle-Stimulating Hormone,FSH Receptor,Follicle-Stimulating Hormone Receptor,Follicle Stimulating Hormone Receptor,Follicle Stimulating Hormone Receptors,Hormone Receptor, Follicle-Stimulating,Hormone Receptors, Follicle-Stimulating,Receptor, FSH,Receptor, Follicle-Stimulating Hormone,Receptors, Follicle Stimulating Hormone
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002417 Cattle Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor. Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography

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