Production of biologically active tethered tilapia LHbetaalpha by the methylotrophic yeast Pichia pastoris.
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.
Kasuto
Harel
H
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, P.O. Box 12, Rehovot 76100, Israel.
Levavi-Sivan
Berta
B
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
D012333:RNA, Messenger; D011994:Recombinant Proteins; D013739:Testosterone; D007986:Luteinizing Hormone; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C003600:11-ketotestosterone