Recombinant Mouse Long-chain fatty acid transport protein 3 (Slc27a3)

What is the primary biochemical function of mouse Slc27a3?

Mouse Slc27a3 primarily functions as an acyl-CoA ligase for long-chain and very-long-chain fatty acids. Despite its classification within the fatty acid transport protein family, research demonstrates that Slc27a3 does not exhibit fatty acid transport activity . Its main role involves activating fatty acids through the addition of Coenzyme A, preparing these molecules for subsequent metabolic processes such as β-oxidation or lipid synthesis. This enzymatic activity is conserved between mouse and human orthologs, with studies showing 88% amino acid sequence identity between species .

How does Slc27a3 differ structurally and functionally from other FATP family members?

Slc27a3 (FATP3) contains the characteristic domains of the FATP family, including the ATP/AMP binding motif and the FATP signature sequence, but differs functionally from other family members:

Unlike FATP1, which forms an oligomeric complex with ACSL1 to facilitate fatty acid transport through vectorial acylation , Slc27a3 functions primarily as an acyl-CoA synthetase without directly participating in membrane transport processes.

What expression patterns does mouse Slc27a3 exhibit across tissues and developmental stages?

Mouse Slc27a3 shows a distinct tissue distribution pattern, with significant expression in brain and lung tissues. This pattern is important when considering appropriate experimental models and interpreting research findings. In melanocytic cells, Slc27a3 gene expression may be regulated by the microphthalmia-associated transcription factor (MITF) , suggesting tissue-specific regulatory mechanisms. Developmental expression patterns indicate temporal regulation during embryogenesis and postnatal development, which may be relevant for studies examining its role in developmental processes.

What are the optimal conditions for assessing recombinant mouse Slc27a3 enzymatic activity?

For accurate assessment of mouse Slc27a3 acyl-CoA ligase activity:

• Buffer composition: Use physiological pH (7.4) with appropriate concentrations of ATP (2-5 mM), CoA (0.5-1 mM), and Mg²⁺ (5-10 mM)

• Substrate selection: Test a range of long-chain (C12-C20) and very-long-chain (>C20) fatty acids to determine substrate preference

• Detection methods:

  • Spectrophotometric assays coupling AMP production to NAD⁺/NADH conversion

  • HPLC-based methods for direct quantification of acyl-CoA products

  • Radiometric assays using ¹⁴C-labeled fatty acids as substrates

Controls should include reactions without ATP or CoA to verify dependence on these cofactors and confirm specific enzymatic activity versus non-enzymatic processes.

How should researchers validate antibodies for mouse Slc27a3 in experimental applications?

A comprehensive validation approach for mouse Slc27a3 antibodies should include:

• Western blotting with recombinant mouse Slc27a3 protein fragments (such as aa 94-228 or aa 407-518) as positive controls

• Pre-incubation controls using recombinant protein fragments at 100x molar excess relative to antibody concentration

• Tissue specificity verification using lysates from tissues known to express Slc27a3

• Knockout validation using Slc27a3-deficient cell lines or tissues

• Cross-reactivity assessment against other FATP family members

When using commercial antibodies, researchers should note that human and mouse orthologs share 88% sequence identity, which may affect antibody specificity . Pre-incubation of antibody with recombinant protein fragments for 30 minutes at room temperature is recommended for blocking experiments in immunohistochemistry, immunocytochemistry, and Western blotting applications .

What genotyping strategies are effective for identifying Slc27a3 polymorphisms in mouse models?

Based on methodologies applied to SLC27A3 in other species, researchers can employ:

• PCR-RFLP analysis targeting specific exons (particularly exons 2, 3, and 4) with appropriate restriction enzymes

• Primer design strategies that target conserved regions while allowing for polymorphism detection

• DNA extraction protocols using established methods such as phenol-chloroform extraction from whole blood samples

• Statistical analysis to determine allele frequencies and genotype distributions in experimental populations

For PCR conditions, researchers have successfully amplified SLC27A3 exonic regions using primers designed to flank potential polymorphic sites, with subsequent restriction enzyme digestion to identify specific variants . Hardy-Weinberg equilibrium testing should be performed to validate the distribution of identified polymorphisms within study populations.

How can recombinant mouse Slc27a3 be utilized to investigate its role in pulmonary disease models?

Recent research has identified SLC27A3 as a valuable diagnostic biomarker for Chronic Obstructive Pulmonary Disease (COPD), with high diagnostic value demonstrated through area under the curve (AUC) analyses . To investigate this function in mouse models:

• Expression profiling: Quantify Slc27a3 expression in lung tissue from control versus disease models using the recombinant protein as standards

• Functional assessment: Examine how altered Slc27a3 activity affects pulmonary inflammation and immune cell infiltration patterns

• Biomarker validation: Evaluate whether Slc27a3 expression correlates with disease severity in mouse COPD models

• Diagnostic development: Assess the potential of Slc27a3 as a diagnostic marker using receiver operating characteristic (ROC) curve analysis

Machine learning approaches combining Slc27a3 with other markers have shown promise, as SLC27A3 demonstrated an AUC of 0.900 in human studies using the GSE106986 dataset . Similar methodologies could be applied to mouse models to validate translational relevance.

What role does Slc27a3 play in cancer metabolism and how can this be studied using recombinant protein?

Evidence indicates that SLC27A3 is upregulated in pazopanib-resistant clear cell renal cell carcinoma (ccRCC) and predicts poor prognosis in lipid-rich tumors . To investigate Slc27a3's role in cancer metabolism:

• Metabolic profiling: Use recombinant Slc27a3 to establish standard curves for quantitative analysis of tumor samples

• Lipid droplet assessment: Employ techniques such as Oil Red O and BODIPY staining to visualize lipid accumulation in relation to Slc27a3 expression

• Acyl-CoA profiling: Implement micro-targeted lipidomics to identify specific metabolites associated with Slc27a3 activity

• Mitochondrial function assessment: Examine the relationship between Slc27a3 expression and mitochondrial membrane potential, reactive oxygen species levels, and mitophagy

Research suggests that Slc27a3-mediated lipid metabolism reprogramming may contribute to drug resistance mechanisms through effects on mitochondrial function and cellular stress responses in cancer models.

How does Slc27a3 interact with mitophagy pathways, and what experimental approaches can investigate this relationship?

Recent studies have identified connections between SLC27A3, mitophagy, and lipid metabolism remodeling, particularly in disease contexts . To study these interactions:

• Microscopy techniques: Employ electron microscopy and fluorescent Mtphagy probes to visualize mitophagy events in relation to Slc27a3 activity

• Protein interaction studies: Investigate potential interactions between Slc27a3 and mitophagy regulators (such as PINK1) through co-immunoprecipitation and Western blotting

• Functional assays: Measure mitochondrial membrane potential using JC-1 probes and reactive oxygen species levels using DCF probes in systems with modulated Slc27a3 expression

• Transcriptional regulation: Explore the role of transcription factors (such as STAT2) in coordinating Slc27a3 expression and mitophagy regulation through dual-luciferase reporter assays

These approaches can help elucidate how Slc27a3-mediated fatty acid metabolism affects mitochondrial quality control and cellular adaptation to stress conditions.

How does mouse Slc27a3 compare with human SLC27A3 in structure, function, and expression patterns?

A comparative analysis reveals important similarities and differences with translational implications:

When using mouse models to study Slc27a3 functions relevant to human disease, researchers should consider these similarities and differences for appropriate experimental design and interpretation.

What can mouse models reveal about Slc27a3 polymorphisms and their phenotypic consequences?

Studies examining SLC27A3 polymorphisms in other species provide a framework for investigating mouse Slc27a3 variants:

• Genotype-phenotype correlations: Analysis of SNPs in exons 2, 3, and 4 has revealed associations with traits such as milk composition in sheep

• Allele frequency distribution: Polymorphisms at specific loci (analogous to those identified in sheep with frequencies of 0.59(G)/0.41(T) for SNP1, 0.57(G)/0.43(C) for SNP2, and 0.58(A)/0.42(C) for SNP3) may also exist in mouse populations

• Functional consequences: Different genotypes may correlate with altered expression levels or enzymatic activities that affect fatty acid metabolism

Researchers investigating mouse Slc27a3 polymorphisms should employ PCR-RFLP techniques similar to those used in other species, with subsequent analysis of how identified variants affect protein function and associated phenotypes.

How can findings from mouse Slc27a3 studies be translated to understanding human disease mechanisms?

Translational approaches connecting mouse Slc27a3 research to human health applications include:

• Biomarker validation: Evaluate whether Slc27a3 expression patterns in mouse disease models reflect observations in human conditions, such as the diagnostic value of SLC27A3 in COPD

• Drug resistance mechanisms: Investigate whether mouse models recapitulate the association between SLC27A3 upregulation and resistance to tyrosine kinase inhibitors observed in human cancers

• Therapeutic target evaluation: Assess whether modulation of Slc27a3 activity affects disease progression in models of conditions where human SLC27A3 plays a role

• Comparative pathway analysis: Determine whether Slc27a3-associated signaling pathways, such as STAT2/SLC27A3/PINK1-mediated mitophagy , are conserved between mouse models and human disease

The high sequence homology (88%) between mouse and human orthologs supports translational relevance, though researchers should remain attentive to species-specific differences in regulatory mechanisms and tissue expression patterns.

What expression systems are optimal for producing functional recombinant mouse Slc27a3?

For successful production of functional recombinant mouse Slc27a3:

• Mammalian expression systems (HEK293, CHO cells) typically yield properly folded and functionally active protein with appropriate post-translational modifications

• Insect cell systems (Sf9, Hi5) using baculovirus expression vectors can provide higher yields for structural studies

• Bacterial expression systems may be suitable for producing protein fragments for antibody validation , but may not yield fully functional full-length protein

When designing expression constructs, researchers should consider:

• Affinity tag placement (preferably C-terminal to avoid interference with enzymatic activity)

• Codon optimization for the selected expression system

• Inclusion of appropriate secretion signals if extracellular production is desired

What purification strategies ensure high-quality recombinant mouse Slc27a3 for research applications?

To obtain pure, active recombinant mouse Slc27a3:

• Affinity chromatography using tag-specific resins (His-tag, FLAG-tag) as the initial capture step

• Size exclusion chromatography to separate properly folded protein from aggregates

• Ion exchange chromatography to remove contaminating proteins

• Activity-based verification to confirm that purified protein retains acyl-CoA ligase function

Quality control assessments should include:

• SDS-PAGE to verify size and purity

• Western blotting to confirm identity

• Enzymatic activity assays to ensure functionality

• Endotoxin testing for applications involving cell culture or in vivo studies

How should recombinant mouse Slc27a3 be stored and handled to maintain activity?

For optimal stability and activity maintenance:

• Storage conditions:

  • Short-term (1-2 weeks): 4°C in buffer containing glycerol (10-20%)

  • Long-term: -80°C in small aliquots to avoid repeated freeze-thaw cycles

  • Lyophilized for extended shelf life when appropriate

• Buffer composition:

  • Physiological pH (7.2-7.4)

  • Stabilizing agents such as glycerol or low concentrations of reducing agents

  • Protease inhibitors to prevent degradation

• Handling precautions:

  • Minimize exposure to room temperature

  • Avoid repeated freeze-thaw cycles

  • Use low-binding tubes to prevent protein adsorption to container surfaces

Activity should be verified periodically using established enzymatic assays to ensure the recombinant protein remains functional throughout the experimental timeframe.