Production of biologically active tethered tilapia LHβα 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 tLHβ and α mature protein-coding sequences were joined to form a fusion gene that encodes a “tethered” polypeptide in which the tLHβ-chain forms the N-terminal part and the α-chain forms the C-terminal part. A “linker” sequence of six amino acids (three Gly-Ser pairs) was placed between the β- and α-chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the β-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 tLHβα had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLHβα stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.