Recombinant Mouse Long-chain fatty acid transport protein 4 (Slc27a4)

What is Slc27a4 and what are its primary functions?

Slc27a4, also known as FATP4 or ACSVL4, is a member of the solute carrier family 27 of fatty acid transporters. It primarily functions in the uptake and activation of long-chain fatty acids (LCFAs) and demonstrates significant activity towards both palmitic acid (C16:0) and lignoceric acid (C24:0), with notably higher affinity for the latter . Additionally, it activates arachidonic acid (C20:4n-6) and participates in the production of triglycerides, cholesterol esters, and ceramide . In the central nervous system, Slc27a4 works collaboratively with SLC27A1 to facilitate fatty acid transport across the blood-brain barrier .

What expression systems are commonly used for producing recombinant mouse Slc27a4?

Recombinant mouse Slc27a4 can be produced using several expression systems, each with distinct advantages depending on research objectives:

• E. coli expression system: Commonly used for high protein yield, as seen in commercial preparations of His-tagged Slc27a4

• HEK-293 cells: Mammalian system that provides proper folding and post-translational modifications

• Cell-free protein synthesis (CFPS): Used for producing Strep-tagged versions with reasonable purity (>70-80%)

The choice of expression system impacts protein folding, post-translational modifications, and ultimately functional characteristics for downstream applications.

How should recombinant Slc27a4 protein be stored and handled to maintain activity?

For optimal storage and handling of recombinant Slc27a4:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage, add 5-50% glycerol (final concentration recommendation: 50%) and store in aliquots at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

• Centrifuge vials briefly before opening to ensure all content is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

Adherence to these storage protocols is essential for maintaining enzymatic activity and structural integrity in functional assays.

What functional assays can verify the activity of recombinant Slc27a4?

To verify the enzymatic activity and transport function of recombinant Slc27a4, researchers can employ several approaches:

When selecting assays, consider that Slc27a4 demonstrates variable activity toward different fatty acids, with particularly strong activity toward lignoceric acid (C24:0) compared to palmitic acid (C16:0) .

How does the substrate specificity of mouse Slc27a4 compare to other FATP family members?

The FATP family (SLC27) consists of six members with varying substrate preferences and tissue distributions:

Slc27a4 is distinguished by its particularly high activity toward very-long-chain fatty acids (VLCFAs), especially lignoceric acid (C24:0), while maintaining substantial activity toward LCFAs including palmitic acid (C16:0) and arachidonic acid (C20:4n-6) . This broader substrate range compared to other family members makes Slc27a4 particularly important in tissues requiring diverse fatty acid metabolism.

What is known about tissue-specific expression patterns of Slc27a4 and their physiological significance?

Slc27a4 demonstrates a distinct tissue expression pattern with significant physiological implications:

• Brain: Highly expressed in brain tissue where it cooperates with SLC27A1 to transport fatty acids across the blood-brain barrier

• Intestine: Important for intestinal fatty acid absorption

• Skin: Critical for maintaining epidermal barrier function

• Placenta: Involved in maternal-fetal fatty acid transport

In pathological contexts, reduced expression of Slc27a4 has been observed in glioblastoma tumors compared to peritumoral areas, suggesting altered fatty acid metabolism in these cancers . This altered expression may contribute to the metabolic reprogramming observed in glioblastoma, potentially offering therapeutic targeting opportunities.

What are the optimal purification methods for recombinant His-tagged Slc27a4?

Purification of His-tagged Slc27a4 requires careful optimization to maintain protein integrity and activity:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins under native conditions

• Buffer Components:

  • Inclusion of 5-10% glycerol helps stabilize membrane-associated domains

  • Low concentrations of non-ionic detergents (0.01-0.05% Triton X-100 or n-dodecyl-β-D-maltoside) aid solubilization

  • Addition of reducing agents (1-2 mM DTT or β-mercaptoethanol) prevents oxidation

• Polishing Step: Size exclusion chromatography (SEC) or ion exchange chromatography

• Quality Control: Assessment via SDS-PAGE, Western blot, and analytical SEC (HPLC) to confirm >90% purity

For functional studies, maintaining the native conformation is critical, which may necessitate milder purification conditions than those used for structural characterization.

How can researchers effectively design loss-of-function and gain-of-function models to study Slc27a4?

Strategic approaches for manipulating Slc27a4 function include:

Loss-of-Function Models:

• CRISPR/Cas9 Gene Editing: For complete knockout or targeted mutations in specific domains

• siRNA/shRNA: For transient or stable knockdown in cell culture models

• Dominant-Negative Mutants: Creation of catalytically inactive mutants that compete with endogenous protein

Gain-of-Function Models:

• Overexpression Systems: Using mammalian expression vectors with strong promoters

• Inducible Expression: Tet-On/Off systems for temporal control

• Tissue-Specific Transgenic Models: For studying effects in specific tissues

When designing functional studies, researchers should consider:

• Compensatory upregulation of other FATP family members (particularly SLC27A1) may occur in Slc27a4 knockout models

• Metabolic phenotypes may vary based on nutritional status and dietary fat content

• Both transport and enzymatic activation functions should be assessed independently

How should researchers address contradictory findings between in vitro and in vivo Slc27a4 functional studies?

When confronting discrepancies between in vitro and in vivo findings:

• Protein Context Considerations:

  • Recombinant proteins may lack critical interacting partners present in vivo

  • Membrane environment significantly impacts transport function

  • Post-translational modifications may differ between systems

• Methodological Reconciliation:

  • Compare substrate concentrations between systems (physiological vs experimental)

  • Assess differences in measurement techniques (direct vs indirect readouts)

  • Consider effects of compensatory mechanisms in vivo but absent in vitro

• Integrated Approaches:

  • Validate findings using multiple experimental systems

  • Use reconstituted proteoliposomes as intermediate models

  • Employ tissue-specific conditional knockout models rather than global knockouts

When analyzing contradictory results, researchers should systematically evaluate differences in experimental conditions, protein context, and the specific functions being measured (transport vs. activation) .

What are appropriate controls when using recombinant Slc27a4 in experimental systems?

Rigorous experimental design requires appropriate controls:

Additionally, researchers should include inter-assay calibrators when comparing results across different experimental batches or when using proteins from different expression systems .

How is Slc27a4 involved in pathological conditions, and what are the research implications?

Emerging research highlights Slc27a4's involvement in several pathological conditions:

• Neurological Disorders:

  • Reduced expression in glioblastoma tumors compared to peritumoral areas

  • Potential role in fatty acid transport across the blood-brain barrier, relevant to neurodegenerative diseases

• Metabolic Diseases:

  • Involvement in obesity-related conditions through regulation of fatty acid uptake

  • Potential contributor to insulin resistance in tissues with aberrant fatty acid accumulation

• Cancer Metabolism:

  • Altered expression in tumors may contribute to metabolic reprogramming

  • Sex-specific differences in expression observed in glioblastoma patients suggest hormonal regulation

These pathological connections present opportunities for:

• Development of Slc27a4 modulators as potential therapeutic agents

• Utilization as biomarkers for disease progression

• Targeting in metabolic reprogramming strategies for cancer treatment

What technological advances are improving our understanding of Slc27a4 structure-function relationships?

Cutting-edge technologies advancing Slc27a4 research include:

• Structural Biology Approaches:

  • Cryo-electron microscopy for membrane protein structure determination

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Molecular dynamics simulations for transport mechanism elucidation

• Advanced Functional Characterization:

  • Single-molecule tracking to monitor protein dynamics in membranes

  • Metabolic flux analysis using stable isotope-labeled fatty acids

  • Optogenetic control of Slc27a4 activity for temporal studies

• Systems Biology Integration:

  • Multi-omics approaches correlating Slc27a4 function with lipidome changes

  • Network analysis of Slc27a4 interactome in different physiological states

  • Machine learning models predicting functional impacts of Slc27a4 variants

These technological advances provide unprecedented opportunities to understand how the structure of Slc27a4 relates to its dual functions of fatty acid transport and activation at a molecular level.