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

Harel Kasuto, Berta Levavi-Sivan

Research output: Contribution to journalArticlepeer-review

70 Scopus citations

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 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.

Original languageAmerican English
Pages (from-to)222-232
Number of pages11
JournalGeneral and Comparative Endocrinology
Volume140
Issue number3
DOIs
StatePublished - Feb 2005

Bibliographical note

Funding Information:
This study is supported by a research grant from the Israeli Ministry of Science, Culture and Sport No. 1.18.372. We thank Dr. David Kime, Sheffield, for providing the detailed ELISA protocol for 11-KT, as well as the antiserum.

Keywords

  • 11-Ketotestosterone
  • Glycosylation
  • Gonadotropin
  • Recombinant
  • Tilapia

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