Expression of biologically active beta subunit of bovine follicle-stimulating hormone in the methylotrophic yeast Pichia pastoris.
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.
Samaddar
M
M
Center for Reproductive Biology and Molecular Endocrinology, Indian Institute of Science, Bangalore, India.
Catterall
J F
JF
Dighe
R R
RR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D037201:Follicle Stimulating Hormone, beta Subunit; D015870:Gene Expression; D007986:Luteinizing Hormone / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011863:Radioimmunoassay; D011962:Receptors, FSH / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012756:Sheep
D037201:Follicle Stimulating Hormone, beta Subunit; D010455:Peptides; D021382:Protein Sorting Signals; D011962:Receptors, FSH; D011994:Recombinant Proteins; D000072235:Mating Factor; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone