Expression and Purification of the Extracellular Domain of Luteinizing Hormone Receptor From Ovis Aries Testicle

Abstract

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptor superfamily. Physiologically, this receptor participates in corpus luteum formation and ovulation in females. In males, it acts in testosterone synthesis and spermatogenesis and is involved in some fertility disorders. RNA was extracted from Ovis aries testicles, and the corresponding cDNA was synthesized to amplify the lhr gene, termed lhr-bed here, consisting of 762 bp that encodes 273 amino acids of the extracellular domain of LHR. Thus, the lhr-bed was cloned into pJET1.2/blunt, subcloned into the pCOLD II expression vector and finally transformed into E. coli BL21 cells. Since the induced rLHR-Bed protein was found in the insoluble fraction, the purification protocol was modified as follows: induction at 25°C, denaturing conditions (8 M UREA and 0.1% CHAPS) and refolding in the column to increase solubility. The rLHR-Bed expression was corroborated by western blotting and mass spectrometry (MS) analysis. This successful method to obtain the recombinant LHR extracellular domain yields 0.2 mg/L of the protein with approximately 90% purity from a single chromatographic purification step. The present approach demonstrates the feasibility of obtaining large quantities of rLHR-Bed. This might be useful to accomplish future studies regarding the structure and functional analysis of the binding interplay with its ligand luteinizing hormone and its isoforms. Additionally, this biotechnological strategy might be used to improve and to develop new drugs for the treatment of reproductive disorders and might also be applied to species reproduction in the livestock industry.