title: Production of biologically active tethered tilapia LHbetaalpha by the methylotrophic yeast Pichia pastoris.
abstract: In fish, luteinizing hormone (LH) stimulates processes leading to final oocyte maturation and ovulation in females, and spermiation in males. The hormone is a heterodimeric glycoprotein composed of two non-covalently associated subunits. In this study, we describe the expression of tilapia LH (tLH) as a biologically active, single-chain polypeptide using the methylotrophic yeast Pichia pastoris. The tLHbeta and alpha mature protein-coding sequences were joined to form a fusion gene that encodes a "tethered" polypeptide in which the tLHbeta-chain forms the N-terminal part and the alpha-chain forms the C-terminal part. A "linker" sequence of six amino acids (three Gly-Ser pairs) was placed between the beta- and alpha-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the beta-subunit, to enable purification of the recombinant protein. Western blot analysis of the pituitary LH resolved by SDS-PAGE yielded a band of 35 kDa, while the recombinant tLHbetaalpha had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLHbetaalpha stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.
authors.affiliation: Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel.
authors.forename: Harel
authors.initials: H
authors.lastname: Kasuto
authors.forename: Berta
authors.initials: B
authors.lastname: Levavi-Sivan
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D023681:Cichlids / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D007124:Immunoenzyme Techniques / Q000662:veterinary; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008297:Male; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000031:analogs & derivatives* / Q000032:analysis
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D013739:Testosterone; D007986:Luteinizing Hormone; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C003600:11-ketotestosterone