Current Issues in Molecular Biology (Nov 2022)

Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the <i>Ovis aries</i> Testis

  • José Luis Villalpando-Aguilar,
  • Itzel López-Rosas,
  • Arnulfo Montero-Pardo,
  • Elisa Azuara-Liceaga,
  • Javier de Jesús Valencia-Méndez,
  • Cynthia R. Trejo-Muñoz,
  • Carlos Kubli-Garfias

DOI
https://doi.org/10.3390/cimb44110387
Journal volume & issue
Vol. 44, no. 11
pp. 5718 – 5727

Abstract

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The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5′UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future.

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