Production of biologically active tethered tilapia LH by the methylotrophic yeast Pichia pastoris

In Wsh, luteinizing hormone (LH) stimulates processes leading to Wnal 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 tLH and mature protein-coding sequences were joined to form a fusion gene that encodes a “tethered” polypeptide in which the tLH -chain forms the N-terminal part and the -chain forms the C-terminal part. A “linker” sequence of six amino acids (three Gly-Ser pairs) was placed between the and -chains to assist in the chimerization of the subunits, and a six-His tail was placed at the end of the -subunit, to enable puriWcation of the recombinant protein. Western blot analysis of the pituitary LH resolved by SDS–PAGE yielded a band of 35 kDa, while the recombinant tLH had a molecular mass of 45 kDa, and was found to possess only N-linked carbohydrates. Recombinant tLH stimulated the release of 11-ketotestosterone from mature testes, whereas its release from immature testes was less pronounced.  2004 Elsevier Inc. All rights reserved.