The Ovis aries lhr gene was the aim of the present work. In sheep, this gene is composed of eleven exons and is located on chromosome 3. LH, along with FSH and TSH receptors, forms a subfamily characterized by a large ectodomain bearing a leucine-rich region and glycosylated N-terminus, which actually is the binding site of their respective hormones. For this reason, through a heterologous system, the recombinant protein of the LHR external domain was expressed and termed lhr-bed. The starting point was the Ovis aries Oar_v4.0 genome assembly with the accession ID: GCF_000298735.2 and locus: NC_040254.1, belonging to the lhr gene, which has a size of 1,965 bp (Fig. 1a1). Related to the lhr gene, a downstream genome sequence of 1,308 bp is located at the GTF2A1L gene, while the upstream genome sequence regarding the lhr gene has 1,484 bp and is found at the LOC114113602 gene (Fig. 1a1). The expressed LHR transcription has a mRNA of 1,965 bp comprising 11 exons. The extracellular region is encoded by exons 1–10 while the seven-transmembrane domain is encoded by exon eleven (Fig. 1a2).
The computational analysis of the large N-terminal extracellular domain, including the LRR (Fig. 1a2) related to LHR-LH binding and the transmembrane region, shows a complete protein composed of 654 a.a. However, in the present work, the amplified lhr-bed mRNA from O. aries testis included only the region between exons 2 and 10, that is, 762 bp encoding only 273 a.a (Fig. 1a2). Interestingly, the LHR gene has already been reported to be expressed in rodent Leydig cells .
One important goal was to reach a reliable production of recombinant lhr-bed. Therefore, cold shock expression technology was applied; that is, cloning and subcloning the lhr-bed gene ligated to the pCOLD II vector expression. After that, the positive selection cloning vector pJET1-2-blunt-lhr-bed was subcloned for expression and efficient recovery. Consequently, the DNA insert of 762 bp cloned from pJET1.2/BLUNT (Fig. 1b) was ligated into the pCOLD II vector. The subcloning process included both NdeI and XhoI digestion enzymes.
Special care was taken for the correct size and alignment of the pCOLDII vector as well as the insert lhr-bed gene in gel agarose (Fig. 1c). Regarding this gel, lane 2 shows the corresponding insert to the lhr-bed with 762 bp size, and the linearized pCOLD II shows a size of 4392 bp, in lane 3 (Fig. 1c). The results of cloning and subcloning are depicted in Fig. 1b and c. It is important to remark that the pCOLDII vector is suitable for application in proteins intrinsically difficult to express, which require low temperature (15°C) and show a slow translation process. Additionally, it ensures improved folding and stability of the recombinant protein during its expression, and, very importantly, produces high amounts of protein in E. coli .
The induction experiment yielded several clones resulting from the pCOLDII-lhr-bed construction, which actually were transformed into BL21 strain bacteria. Thus, the experiment to induce the recombinant protein of the extracellular domain of LHR Ovis aries (rLHR-Bed) was designed as follows: BL21 cells transformed with pCOLDII-empty were labeled negative induction, while clones 2 and 4 were positive, resulting in the transformed pCOLDII-lhr-bed construction.
The extent of protein expression was evaluated through SDS-PAGE profiles. Likewise, recombinant protein with a histidine tag of 50 kDa was added as a WB positive control (Fig. 2a, lane 10). In the case of BL21 clone 2, no expression of the recombinant protein was found (Fig. 2a, lanes 3 and 5). In contrast, a band of ~ 28 kDa corresponding to the induction elicited by clone 4 was observed (Fig. 2a, lane 7). To corroborate that the band located between 25 and 37 kDa was actually the rLHR-Bed, a WB analysis was applied using an anti-His tag antibody and mediated with a twin gel corresponding to the protein profile of the induction experiment (Fig. 2b).
Unexpectedly, the expression study carried out under standard conditions using BL21 transformed with constructed pCOLDII-lhr-bed growing at 180 rpm/15°C/18 h failed, somewhat yielding low expression of rLHR-Bed (data not shown). Therefore, a modified procedure was implemented according to Spadiut et al. , raising the temperature from 15°C to 25°C and performing a preinduction course, resulting in optimum induction of 24 U/L recombinant protein. Accordingly, applying those modifications successfully induced rLHR-Bed, showing that the pCOLDII system is stable and functional at 25°C .
In addition, the rLHR-Bed induction elicited by clone 4 was submitted to solubility evaluation. The resulting SDS-PAGE showed a marked band in the insoluble fraction, especially in the range between 25 and 37 kDa, while the soluble fraction in that range showed lower intensity (Fig. 2c). The rLHR-Bed was clearly confirmed in the insoluble fraction by the WB experiment (Fig. 2c, lane 5). However, the solubility analyses of rLHR-BED showed that the expressed protein yielded inclusion bodies, compelling us to implement a column protocol. That is, a denaturation-refolding scheme to obtain the recombinant protein soluble to finish the rLHR-Bed purification. In this way, the denaturing condition was achieved by modifying the solubility buffer to 0.1% CHAPS, which is considered the minimal micellar concentration to improve the solubility of recombinant proteins . The solubility results, including the complete profile of purification, are shown by SDS-PAGE (Fig. 2d). Interestingly, in the case of insoluble protein aggregates forming inclusion bodies, the protein conserves its native-state secondary structure . Thus, as anticipated, the expressed and purified protein was actually rLHR-Bed and was fully validated by two reliable techniques: WB mediated with anti-histidine monoclonal antibody and mass spectrometric (MS) analysis (Supplementary file). These final results corroborated that the induced protein indeed corresponds to the rLHR-Bed of O. aries .
On the other hand, two vials containing the purified recombinant protein with a performance of 0.25 mg/L were submitted to acquire an electrophoretic profile, and the band was cut and sent for MS identification to the proteomic unit of the Instituto de Biotecnología-UNAM, México. Seven peptides were identified corresponding to 100% probability to the Lutropin-choriogonadotropin-hormone receptor OS = Ovis aries, according to the UniProt source database (Table 1). Clearly, the spectrum mass/charge (M/Z) vs. relative intensity corresponding to each peptide showed the sequenced peptide, protein identification probability, percent of the best peptide and spectrum M/Z (Table 1).
LHR has been expressed in different models not exempt from drawbacks; for instance, in human embryonic kidney cells, the expressed extracellular domain shows high affinity to hCG but remains trapped within the cells . Similarly, LHR expressed in baculovirus-infected insect cells is inactive and remains trapped in aggregate pools . The rat extracellular domain of LHR has been expressed in E. coli, with high binding affinity for hCG; however, proper folding is achieved only after laborious refolding in vitro . Additionally, in E. coli, the extracellular domain of the human receptor expressed in a chimeric fusion model, including thioredoxin reductase and glutathione reductase genes in the cloning plasmid vector, maintained the disulfide bonds of the expressed LHR extracellular domain and confirmed that regardless of the lack of glycans, the truncated receptor may show high affinity to hCG, similar to the native receptor . Nevertheless, this elegant design is still quite elaborated. More complex chimeric expression of the extracellular domain of the LH receptor has been elaborated but rather more expensive, as has been reported in CD8 lymphocyte membranes . In the present work, certain advantages of the improved technique were achieved; for instance, disulfide bonds were retained since β–mercaptoethanol was not used. Likewise, dialysis was avoided, allowing easy refolding of the protein in the repurification procedure, leaving the receptor in a friendly refolding buffer ready for future crystallization.
In addition to crystallization as well as molecular properties and physicochemical studies, many possibilities arise mostly in the field of biology of reproduction and the livestock industry. In our group, we are willing to test the binding of LH isoforms in the expressed extracellular LHR motive, since working with LH isoforms previously, some differences have been observed in cAMP and vascular endothelial growth factor production .
Briefly, in the present work, the extracellular domain of LHR was expressed successfully based on a reliable and minimal effort molecular biology strategy. The protein rLHR-bed from O aries, expressed from the lhr-bed gene, was obtained from testicular Leydig cells. BL21 competent cells and the pCOLDII vector were selected to amplify the gene. Thus, based on the current literature, we proposed the strategy of induction-solubilization-refolding and purification in a raw sample. The procedure was β-mercaptoethanol-free, and dialysis was omitted. In this way, the recombinant protein was expressed in significant amounts and ready for subsequent structural analyses. The results suggest that this strategic pathway could be applied to express almost any membrane protein effortlessly.