{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:46:55Z","timestamp":1760244415113,"version":"build-2065373602"},"reference-count":29,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2022,11,17]],"date-time":"2022-11-17T00:00:00Z","timestamp":1668643200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>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 \u00b0C, 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\u2032UTR on pCOLD II vector is stable at 15 \u00b0C. We predicted and obtained RNAfold stability at 25 \u00b0C. 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.<\/jats:p>","DOI":"10.3390\/cimb44110387","type":"journal-article","created":{"date-parts":[[2022,11,18]],"date-time":"2022-11-18T06:22:28Z","timestamp":1668752548000},"page":"5718-5727","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the Ovis aries Testis"],"prefix":"10.3390","volume":"44","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2563-9313","authenticated-orcid":false,"given":"Jos\u00e9 Luis","family":"Villalpando-Aguilar","sequence":"first","affiliation":[{"name":"Instituto de Investigaciones en Matem\u00e1ticas Aplicadas y en Sistemas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, M\u00e9rida 97302, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3864-260X","authenticated-orcid":false,"given":"Itzel","family":"L\u00f3pez-Rosas","sequence":"additional","affiliation":[{"name":"Consejo Nacional de Ciencia y Tecnolog\u00eda, Ciudad de M\u00e9xico 03940, Mexico"},{"name":"Colegio de Postgraduados Campus Campeche, Campeche 24050, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8836-4591","authenticated-orcid":false,"given":"Arnulfo","family":"Montero-Pardo","sequence":"additional","affiliation":[{"name":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Aut\u00f3noma de Sinaloa, San Benito 80260, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6392-170X","authenticated-orcid":false,"given":"Elisa","family":"Azuara-Liceaga","sequence":"additional","affiliation":[{"name":"Posgrado en Ciencias Gen\u00f3micas, Campus del Valle, Universidad Aut\u00f3noma de la Ciudad de M\u00e9xico, Ciudad de M\u00e9xico 03100, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Javier de Jes\u00fas","family":"Valencia-M\u00e9ndez","sequence":"additional","affiliation":[{"name":"Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cynthia R.","family":"Trejo-Mu\u00f1oz","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Biom\u00e9dicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carlos","family":"Kubli-Garfias","sequence":"additional","affiliation":[{"name":"Instituto de Investigaciones Biom\u00e9dicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Ciudad de M\u00e9xico 04510, Mexico"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1126\/science.2502842","article-title":"Lutropin-Choriogonadotropin Receptor: An Unusual Member of the G Protein-Coupled Receptor Family","volume":"245","author":"McFarland","year":"1989","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1477-7827-1-1","article-title":"Multiple Luteinizing Hormone Receptor (LHR) Protein Variants, Interspecies Reactivity of Anti-LHR MAb Clone 3B5, Subcellular Localization of LHR in Human Placenta, Pelvic Floor and Brain, and Possible Role for LHR in the Development of Abnormal Pregnancy, Pelvic Floor Disorders and Alzheimer\u2019s Disease","volume":"1","author":"Bukovsky","year":"2003","journal-title":"Reprod. 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