Recombinant Human Putative olfactory receptor ENSP00000348552

What is Recombinant Human Putative olfactory receptor ENSP00000348552?

Recombinant Human Putative olfactory receptor ENSP00000348552 (UniProt ID: Q8NH95) is a full-length human protein belonging to the G protein-coupled receptor family, specifically to the olfactory receptor subfamily. The protein is encoded by the gene OR13C6P, which is classified as a pseudogene in the olfactory receptor family 13, subfamily C, member 6. It is also known by several synonyms including Putative olfactory receptor 13C6, Olfactory receptor family 13 subfamily C member 6 pseudogene, and Putative olfactory receptor 13C7 .

This recombinant protein represents a member of a large subfamily of genes sharing extensive sequence similarities with putative rat olfactory receptors. Research suggests that olfactory receptors may function beyond their traditional role in odorant sensing, potentially participating in cellular recognition and chemotaxis processes .

How is Recombinant Human Putative olfactory receptor ENSP00000348552 expressed and purified?

The commercially available Recombinant Human Putative olfactory receptor ENSP00000348552 is typically expressed in E. coli expression systems. The full-length protein (amino acids 1-320) is produced with an N-terminal His-tag fusion to facilitate purification .

Methodology for expression and purification:

• Expression system: The protein is expressed in E. coli, which provides several advantages for recombinant protein production including rapid growth, high protein yields, and well-established genetic manipulation techniques.

• Purification process: The His-tagged protein is likely purified using immobilized metal affinity chromatography (IMAC), leveraging the affinity of histidine residues for metal ions such as nickel or cobalt.

• Quality control: Purity is typically assessed using SDS-PAGE, with commercial preparations achieving greater than 90% purity .

What are the optimal storage conditions for ENSP00000348552?

For optimal stability and activity retention of Recombinant Human Putative olfactory receptor ENSP00000348552, the following storage conditions are recommended:

• Temperature: Store at -20°C or preferably -80°C upon receipt .

• Aliquoting: Dividing the protein into smaller working aliquots is necessary for multiple use to avoid repeated freeze-thaw cycles .

• Short-term storage: Working aliquots can be stored at 4°C for up to one week .

• Form: The protein is typically provided as a lyophilized powder in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

• Freeze-thaw stability: Repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of activity .

How should Recombinant Human Putative olfactory receptor ENSP00000348552 be reconstituted for experimental use?

For optimal reconstitution of lyophilized Recombinant Human Putative olfactory receptor ENSP00000348552, the following protocol is recommended:

• Pre-reconstitution preparation: Briefly centrifuge the vial prior to opening to bring the contents to the bottom .

• Reconstitution solution: Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Glycerol addition: For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) .

• Aliquoting: Divide the reconstituted protein into small working aliquots to minimize freeze-thaw cycles .

• Storage after reconstitution: Store reconstituted aliquots at -20°C or -80°C for long-term storage .

This reconstitution procedure helps maintain protein integrity and activity for subsequent experimental applications.

What are the potential research applications of Recombinant Human Putative olfactory receptor ENSP00000348552?

Recombinant Human Putative olfactory receptor ENSP00000348552 offers multiple research opportunities across various scientific domains:

• Olfactory system studies: As a member of the olfactory receptor family, this protein can be used to investigate odorant recognition mechanisms and signal transduction pathways in the olfactory system .

• Axonal guidance research: Olfactory receptors are implicated in guiding axons of olfactory neurons to appropriate glomeruli in the olfactory bulb. The recombinant protein can be valuable for studying these guidance mechanisms .

• Reproductive biology research: Given that olfactory receptors have been detected in testis and may be involved in sperm chemotaxis during fertilization, ENSP00000348552 could be useful in reproductive biology studies .

• Pseudogene functionality exploration: Though classified as a pseudogene, OR13C6P may have regulatory functions or encode functional fragments, making this recombinant protein valuable for investigating pseudogene biology .

• GPCR structural and functional studies: As a G protein-coupled receptor, this protein serves as a model for studying GPCR structure, ligand binding, and activation mechanisms.

How can researchers verify the functionality of Recombinant Human Putative olfactory receptor ENSP00000348552?

Verifying the functionality of Recombinant Human Putative olfactory receptor ENSP00000348552 requires several complementary approaches:

• Ligand binding assays: Employ radioligand binding or fluorescence-based assays to assess binding of potential odorants or other ligands.

• Calcium mobilization assays: Since G protein-coupled receptors often trigger calcium release upon activation, calcium flux assays using fluorescent calcium indicators can detect receptor functionality.

• GTPγS binding assays: Measure G protein activation using non-hydrolyzable GTP analogs to assess receptor coupling to G proteins.

• Receptor trafficking studies: Use fluorescently labeled antibodies or tagged receptors to monitor cellular localization and trafficking in response to potential activators.

• Electrophysiological recordings: In more advanced systems, patch-clamp techniques can measure electrical responses in cells expressing the receptor.

• Bioluminescence Resonance Energy Transfer (BRET): This technique can assess protein-protein interactions between the receptor and downstream signaling components.

Researchers should note that as ENSP00000348552 is derived from a putative olfactory receptor pseudogene, functional verification may be challenging and requires appropriate controls.

What experimental challenges are associated with studying olfactory receptors like ENSP00000348552?

Researchers face several significant challenges when working with Recombinant Human Putative olfactory receptor ENSP00000348552 and similar olfactory receptors:

• Membrane protein expression difficulties: As a seven-transmembrane protein, achieving proper folding and membrane insertion in heterologous expression systems is challenging.

• Hydrophobic nature: The hydrophobic domains of olfactory receptors create solubility issues during purification and functional studies.

• Ligand identification: The natural ligands for many olfactory receptors, including ENSP00000348552, remain unknown, complicating functional characterization.

• Pseudogene status: As OR13C6P is classified as a pseudogene, determining whether it produces a functional protein requires careful validation .

• Low expression levels: Olfactory receptors often express poorly in heterologous systems, requiring optimization of expression conditions.

• Receptor trafficking issues: Proper localization to the cell membrane in non-native cell types often requires co-expression with chaperone proteins.

• Functional assay sensitivity: Detecting potentially low-affinity interactions with odorants requires highly sensitive assay systems.

How does OR13C6P compare to other members of the olfactory receptor family?

The OR13C6P gene, encoding ENSP00000348552, belongs to the olfactory receptor family 13, subfamily C, and shares several characteristics with other olfactory receptors while maintaining distinct features:

The olfactory receptor gene family represents one of the largest gene families in the human genome, with members sharing sequence similarities while exhibiting distinct odorant specificities . OR13C6P's classification as a pseudogene suggests potential evolutionary divergence from functional olfactory receptors, though it retains significant sequence homology with other family members .

What techniques can be used to study the binding properties of ENSP00000348552?

To characterize the binding properties of Recombinant Human Putative olfactory receptor ENSP00000348552, researchers can employ several advanced techniques:

• Surface Plasmon Resonance (SPR): This label-free technique enables real-time monitoring of binding interactions between the immobilized receptor and potential ligands, providing association and dissociation rate constants.

• Isothermal Titration Calorimetry (ITC): ITC measures heat changes during binding interactions, providing thermodynamic parameters including binding affinity, enthalpy, and stoichiometry.

• Microscale Thermophoresis (MST): This technique detects changes in the movement of molecules along microscopic temperature gradients upon binding, requiring minimal protein amounts.

• Fluorescence-based assays: Including:

  • Förster Resonance Energy Transfer (FRET)

  • Fluorescence Polarization (FP)

  • Fluorescent ligand binding assays

• Computational methods: Molecular docking and virtual screening can predict potential ligands and binding modes before experimental validation.

• Covalent labeling approaches: Photoaffinity labeling with modified ligands can identify binding sites within the receptor structure.

• NMR spectroscopy: For fragment screening and identifying binding epitopes, though challenging with membrane proteins.

These techniques can be complementary, with multiple approaches providing more comprehensive binding characterization.

Are there known ligands for the Recombinant Human Putative olfactory receptor ENSP00000348552?

Currently, no specific ligands have been definitively identified for Recombinant Human Putative olfactory receptor ENSP00000348552 based on the available research data. This knowledge gap is common for many olfactory receptors, particularly those derived from pseudogenes like OR13C6P .

Several factors contribute to this lack of identified ligands:

• Pseudogene classification: As OR13C6P is annotated as a pseudogene, less research has focused on identifying its potential ligands compared to functional olfactory receptors .

• Deorphanization challenges: The process of identifying ligands for orphan receptors (deorphanization) is technically challenging, requiring high-throughput screening of diverse chemical libraries.

• Expression system limitations: Functional expression in heterologous systems, necessary for ligand screening, is particularly difficult for olfactory receptors.

• Promiscuous binding: Olfactory receptors often respond to multiple structurally related compounds with varying affinities, complicating definitive ligand identification.

Researchers interested in identifying potential ligands would typically employ systematic screening approaches using chemical libraries of odorants, coupled with functional assays measuring receptor activation.

How can researchers investigate the role of ENSP00000348552 in non-olfactory tissues?

Investigating the role of Recombinant Human Putative olfactory receptor ENSP00000348552 in non-olfactory tissues requires a multifaceted approach:

• Expression profiling:

  • RT-PCR and qPCR to quantify gene expression across tissues

  • RNAseq analysis for comprehensive transcriptome profiling

  • In situ hybridization to localize expression within specific cell types

  • Northern blotting to detect transcript presence in tissues such as testis

• Protein detection:

  • Immunohistochemistry using specific antibodies against ENSP00000348552

  • Western blotting for protein expression quantification

  • Immunofluorescence microscopy for subcellular localization

• Functional studies:

  • RNA interference or CRISPR-mediated gene silencing to assess loss-of-function effects

  • Overexpression studies to identify gain-of-function phenotypes

  • Cell migration assays to investigate potential chemotactic functions

• Signaling pathway analysis:

  • Second messenger assays (cAMP, calcium) to identify downstream signaling

  • Protein-protein interaction studies using co-immunoprecipitation or proximity ligation assays

• Physiological relevance:

  • Ex vivo tissue studies focusing on tissues where expression is detected

  • Correlation analyses between receptor expression and tissue-specific functions

The detection of olfactory receptor transcripts in testis suggests potential roles in sperm chemotaxis during fertilization, making reproductive tissues particularly relevant for investigation .

What methods can be used to analyze contradictory findings in ENSP00000348552 research?

When confronted with contradictory findings in research concerning Recombinant Human Putative olfactory receptor ENSP00000348552, investigators should implement a systematic analytical approach:

• Methodological comparison and standardization:

  • Analyze differences in experimental protocols, expression systems, and assay conditions

  • Implement standardized positive and negative controls across studies

  • Consider interlaboratory validation studies using identical protocols

• Statistical re-evaluation:

  • Perform meta-analysis of available data when sufficient studies exist

  • Assess statistical power and sample sizes in contradictory studies

  • Consider Bayesian approaches to integrate prior knowledge with new findings

• Technical validation approaches:

  • Cross-validate findings using orthogonal techniques

  • Implement antibody validation procedures to confirm specificity

  • Use multiple cell lines or experimental models to assess consistency

• Sequence and structural analysis:

  • Verify the exact sequence used in different studies (full-length vs. fragments)

  • Consider post-translational modifications that might affect function

  • Analyze potential splice variants and their functional implications

• Biological context consideration:

  • Assess different cellular contexts that might explain contradictory findings

  • Consider receptor homo/hetero-dimerization that might affect function

  • Evaluate potential species-specific differences in receptor function

• Dialogue contradiction detection framework:

  • Implement formal contradiction detection methodologies to systematically identify incompatible findings

  • Classify contradictions as direct, indirect, or contextual to guide resolution strategies

How can ENSP00000348552 be incorporated into functional assays?

Incorporating Recombinant Human Putative olfactory receptor ENSP00000348552 into functional assays requires careful experimental design to accommodate the challenges of working with membrane proteins:

• Cell-based reporter systems:

  • Stable or transient transfection in HEK293, CHO, or Sf9 cells

  • Co-expression with G protein chimeras to enhance coupling efficiency

  • Integration with luciferase, GFP, or β-lactamase reporters downstream of receptor activation

• Membrane preparation approaches:

  • Purification of receptor-containing membrane fractions for binding studies

  • Reconstitution into liposomes or nanodiscs for enhanced stability

  • Development of soluble receptor constructs using fusion partners

• Biosensor development:

  • Surface immobilization on SPR chips via His-tag

  • Integration into field-effect transistor (FET) based biosensors

  • Development of whole-cell biosensors expressing the receptor

• High-throughput screening platforms:

  • Fluorescence-based calcium mobilization assays in 384-well format

  • Automated patch-clamp systems for electrophysiological measurements

  • Label-free cellular impedance measurements

• Axonal guidance assays:

  • In vitro growth cone turning assays to assess chemotactic properties

  • Co-culture systems with receptor-expressing cells

  • Microfluidic devices for controlled gradient formation

The experimental design should account for the receptor's N-terminal His-tag, which may affect function in some assays but can facilitate purification and detection . Additionally, researchers should consider the pseudogene status of OR13C6P when interpreting functional results .

What are the implications of ENSP00000348552 being classified as a pseudogene?

The classification of OR13C6P (encoding ENSP00000348552) as a pseudogene has several important implications for research:

• Evolutionary significance: Pseudogenes often represent evolutionary relics that have lost their protein-coding function but may retain regulatory roles. The persistence of OR13C6P in the human genome suggests possible selective pressure for retention .

• Regulatory potential: Pseudogenes can function as competing endogenous RNAs (ceRNAs), regulating the expression of their functional counterparts through microRNA sequestration. OR13C6P transcripts may play such regulatory roles in olfactory receptor gene expression.

• Truncated protein products: Some pseudogenes produce truncated or altered proteins that retain partial functionality or acquire novel functions. The successful expression of full-length ENSP00000348552 in recombinant systems suggests the potential for protein production despite pseudogene classification .

• Tissue-specific expression: The detection of olfactory receptor transcripts in non-olfactory tissues like testis suggests functions beyond olfaction . Pseudogene expression is often more tissue-restricted than functional genes, potentially indicating specialized roles.

• Experimental design considerations: Researchers should exercise caution when interpreting functional data, as the pseudogene status suggests potential differences from canonical olfactory receptors. Control experiments comparing ENSP00000348552 with functional olfactory receptors are advisable.

• Therapeutic relevance: Understanding pseudogene functionality has implications for therapeutic target validation, as pseudogenes have been implicated in various pathologies and could represent novel drug targets.

Research indicates that olfactory receptor genes may function in both olfaction and sperm chemotaxis , raising the possibility that even pseudogenized members like OR13C6P might retain functional roles in specific biological contexts.