Recombinant Pongo abelii Long-chain fatty acid transport protein 4 (SLC27A4)

What is SLC27A4 and what is its role in fatty acid metabolism?

SLC27A4, also known as Fatty Acid Transport Protein 4 (FATP4), is a critical membrane protein involved in the cellular uptake and activation of long-chain fatty acids. In Pongo abelii (Sumatran orangutan), this protein functions similarly to its human counterpart, facilitating the transport of fatty acids across cellular membranes and contributing to their subsequent metabolism. The protein possesses an acyl-CoA synthetase activity (EC 6.2.1.-) that catalyzes the ATP-dependent esterification of long-chain fatty acids to CoA, preparing them for further metabolic processes . This dual transport-activation function distinguishes SLC27A4 from other membrane transporters and positions it as a key regulator of cellular lipid homeostasis. Understanding the structure and function of Pongo abelii SLC27A4 provides valuable insights into evolutionary conservation of fatty acid metabolism across primate species.

How does Pongo abelii SLC27A4 compare structurally to human SLC27A4?

Pongo abelii SLC27A4 shares significant sequence homology with human SLC27A4, reflecting their close evolutionary relationship. The full amino acid sequence of Pongo abelii SLC27A4 consists of 643 amino acids, containing conserved functional domains important for fatty acid transport and activation . Comparative analysis reveals that both proteins contain a highly conserved AMP-binding domain crucial for the acyl-CoA synthetase activity. The transmembrane domains responsible for membrane insertion and fatty acid transport functionality show particularly high conservation between the species. Notable sequence differences appear primarily in non-catalytic regions, suggesting evolutionary adaptation while maintaining core functionality. When expressed as recombinant proteins, both human and Pongo abelii SLC27A4 demonstrate similar substrate preferences, with high affinity for long-chain fatty acids, particularly arachidonic acid. These structural similarities make Pongo abelii SLC27A4 a valuable model for understanding human fatty acid transport mechanisms.

What are the typical molecular characteristics of recombinant Pongo abelii SLC27A4?

Recombinant Pongo abelii SLC27A4 is typically produced with specific molecular characteristics optimized for research applications. The full-length protein has a calculated molecular mass around 70-75 kDa, though truncated versions focusing on specific functional domains are also commonly used in research . Commercial recombinant preparations often include fusion tags, with N-terminal His-tags being particularly common to facilitate purification via metal affinity chromatography. The purity of commercially available recombinant Pongo abelii SLC27A4 generally exceeds 85% as determined by SDS-PAGE analysis . The protein is typically supplied in either lyophilized form or as a frozen liquid in a Tris-based buffer containing 50% glycerol to maintain stability. Full-length constructs contain the complete functional domains including the AMP-binding region and transmembrane segments, while truncated versions (such as Met1-Phe237) may focus on specific domains of interest for specialized research applications . Researchers should verify the exact molecular specifications of their recombinant protein to ensure compatibility with planned experiments.

What expression systems are most effective for producing recombinant Pongo abelii SLC27A4?

The optimal expression system for recombinant Pongo abelii SLC27A4 largely depends on the specific research requirements, with bacterial systems being most commonly employed for basic structural studies. E. coli represents the predominant expression host due to its cost-effectiveness, rapid growth, and high protein yields . When expressing in E. coli, codon optimization for bacterial expression may significantly improve yields, as primate codon usage differs considerably from bacterial preferences. For applications requiring post-translational modifications, yeast expression systems provide advantages, balancing reasonable yields with eukaryotic processing capabilities . Insect cell systems (Sf9, Sf21) represent intermediate options that can accommodate larger proteins with some post-translational modifications. Mammalian expression systems, while less commonly used due to cost and complexity, may be necessary for studies requiring native protein conformation and full glycosylation patterns. The choice of vector system significantly impacts expression efficiency, with pGEX vectors being commonly used for GST-tagged proteins and pET vectors for His-tagged variants .

What are the recommended purification strategies for recombinant Pongo abelii SLC27A4?

Purification of recombinant Pongo abelii SLC27A4 typically employs affinity chromatography as the primary isolation step, taking advantage of fusion tags engineered into the protein. For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides efficient initial purification, with elution achieved using imidazole gradients . GST-tagged variants can be purified using glutathione agarose columns, with elution accomplished through reduced glutathione buffer systems . Following initial affinity purification, size exclusion chromatography (SEC) serves as an effective secondary purification step to remove aggregates and achieve >90% purity. For membrane-associated constructs containing the full transmembrane domains, detergent solubilization using mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin during purification is essential to maintain protein stability and prevent aggregation. Ion exchange chromatography may serve as an additional polishing step, particularly for removing contaminant proteins with similar molecular weights but different isoelectric points. Researchers should verify final purity through analytical techniques like SDS-PAGE with Coomassie Brilliant Blue staining .

How can I optimize protein yield when expressing recombinant Pongo abelii SLC27A4?

Optimizing recombinant Pongo abelii SLC27A4 expression requires systematic adjustment of multiple parameters to balance protein yield with proper folding. Induction conditions represent a critical optimization point, with lower temperatures (16-25°C) often favoring proper folding of mammalian proteins in bacterial systems, while extended induction times (16-24 hours) can compensate for slower expression rates. The concentration of induction agents (typically IPTG for bacterial systems) should be titrated, as excessive concentrations may lead to inclusion body formation. Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ) can significantly enhance folding efficiency, particularly for complex multi-domain proteins like SLC27A4. For challenging constructs, expression of soluble domains rather than the full-length protein may dramatically improve yield, though at the cost of losing transmembrane functionality. Optimizing media composition through supplementation with trace elements and specific carbon sources can further boost expression levels. Sequential purification steps should be optimized to minimize protein loss, particularly by adjusting buffer compositions to maintain stability during each chromatography stage. Documentation of systematic optimization experiments will facilitate reproducible production of high-quality protein for downstream applications.

What are the optimal storage conditions for maintaining activity of recombinant Pongo abelii SLC27A4?

Optimal storage of recombinant Pongo abelii SLC27A4 requires careful consideration of buffer composition and temperature to maintain structural integrity and functional activity. For short-term storage (1-2 weeks), the protein can be maintained at 2-8°C in an appropriate buffer system, typically containing 50-100 mM phosphate or Tris buffer (pH 7.4-8.0) with physiological salt concentrations . For long-term storage, proteins should be stored at -20°C to -80°C, with the lower temperature preferred for extended periods exceeding 6 months. Addition of 50% glycerol acts as a cryoprotectant to prevent freeze-damage during storage at subzero temperatures . For lyophilized preparations, storage at -20°C in sealed containers with desiccant is recommended, with reconstitution performed immediately before use. Multiple freeze-thaw cycles significantly reduce protein activity, necessitating aliquoting of purified protein into single-use volumes before freezing. Addition of reducing agents (1-5 mM DTT or 2-mercaptoethanol) may be beneficial for maintaining the native state of cysteine-containing proteins like SLC27A4. For detergent-solubilized full-length preparations, maintaining detergent concentrations above the critical micelle concentration during storage is essential to prevent protein aggregation.

What is the recommended protocol for reconstituting lyophilized Pongo abelii SLC27A4?

Reconstitution of lyophilized Pongo abelii SLC27A4 requires careful handling to ensure complete solubilization while maintaining protein structure and function. The optimal procedure begins with equilibrating the lyophilized protein vial to room temperature before opening to prevent condensation that could affect protein stability. Reconstitution should be performed using sterile water or an appropriate buffer (typically phosphate-buffered saline pH 7.4 or a Tris-based buffer system) to a concentration of 0.25 μg/μl for optimal results . The solution should be gently mixed by rotating or inverting the vial rather than vortexing, which can cause protein denaturation through excessive shearing forces. Centrifugation of the vial before opening is recommended to collect any protein powder that may adhere to the cap or vial walls during transportation . Complete solubilization typically requires 10-15 minutes of gentle mixing, with visual inspection to ensure no particulate matter remains. For long-term storage of reconstituted protein, addition of an equal volume of glycerol is recommended, bringing the final glycerol concentration to 50% . Reconstituted protein should be aliquoted into single-use volumes before storage at -20°C to -80°C to avoid repeated freeze-thaw cycles that significantly reduce activity.

How can researchers assess the stability and activity of stored recombinant Pongo abelii SLC27A4?

Assessment of recombinant Pongo abelii SLC27A4 stability and activity following storage requires implementation of appropriate analytical techniques targeting both structural integrity and functional capacity. Structural integrity can be evaluated through size exclusion chromatography to detect aggregation or degradation, with shifts in elution profiles indicating structural changes during storage. SDS-PAGE analysis under reducing and non-reducing conditions provides information about potential degradation products and disulfide bond integrity. Thermal shift assays using differential scanning fluorimetry with dyes like SYPRO Orange can quantitatively assess conformational stability, with changes in melting temperature (Tm) serving as sensitive indicators of protein stability alterations. Functional activity assessment typically employs fatty acid uptake assays using fluorescently labeled fatty acids (e.g., BODIPY-labeled fatty acids) or radioisotope-labeled substrates in reconstituted liposome systems. The acyl-CoA synthetase activity can be quantified through ATP consumption assays or direct measurement of CoA-esterified product formation. Circular dichroism spectroscopy provides valuable information about secondary structure maintenance, with changes in alpha-helical or beta-sheet content indicating structural perturbations during storage. Implementation of multiple complementary techniques provides comprehensive assessment of protein quality before experimental use.

How can recombinant Pongo abelii SLC27A4 be utilized in biochemical and structural studies?

Recombinant Pongo abelii SLC27A4 serves as a valuable tool for diverse biochemical and structural investigations of fatty acid transport mechanisms. For kinetic analyses, purified protein can be reconstituted into liposomes or nanodiscs to study transport rates and substrate specificities using fluorescently labeled fatty acids, enabling determination of Michaelis-Menten parameters (Km, Vmax) for various fatty acid substrates. Structural studies may employ X-ray crystallography of soluble domains or cryo-electron microscopy for full-length protein to elucidate the three-dimensional architecture, though membrane protein crystallization presents significant technical challenges requiring detergent screening and lipid cubic phase methodologies. Hydrogen-deuterium exchange mass spectrometry offers insights into protein dynamics and substrate-induced conformational changes without requiring crystallization. Site-directed mutagenesis of conserved residues, followed by functional assays, enables mapping of the catalytic site and transport channel, with particular focus on the AMP-binding domain critical for acyl-CoA synthetase activity. Protein-protein interaction studies using techniques such as pull-down assays, surface plasmon resonance, or crosslinking experiments can identify potential regulatory partners or multiprotein complexes involved in fatty acid metabolism. Comparative studies between Pongo abelii and human SLC27A4 provide evolutionary insights into conserved mechanisms of fatty acid transport across primate species.

What are the recommended protocols for using recombinant Pongo abelii SLC27A4 in immunological assays?

Implementation of recombinant Pongo abelii SLC27A4 in immunological assays requires optimization of several parameters to ensure specificity and sensitivity. For enzyme-linked immunosorbent assays (ELISA), the recombinant protein can serve as a coating antigen at concentrations of 1-5 μg/ml in carbonate-bicarbonate buffer (pH 9.6) . Blocking with 3-5% bovine serum albumin effectively minimizes non-specific binding, while detection may employ species-specific secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Western blotting applications typically require 50-100 ng of recombinant protein per lane, with transfer to PVDF membranes preferred over nitrocellulose due to improved retention of hydrophobic membrane proteins. For immunoprecipitation studies, approximately 1-5 μg of protein-specific antibody per 100-500 μg of total protein sample yields optimal results, with pre-clearing using protein A/G beads recommended to reduce background . Flow cytometry applications may employ the recombinant protein as a standard or as a competitive inhibitor when analyzing cellular expression. When developing new antibodies, the recombinant protein serves as an ideal immunogen, with protein purity exceeding 90% critical for producing highly specific antibodies. Cross-reactivity testing against human SLC27A4 is advisable to determine species specificity of resulting antibodies.

How can recombinant Pongo abelii SLC27A4 be employed in functional transport assays?

Functional characterization of recombinant Pongo abelii SLC27A4 requires establishment of transport assays that accurately recapitulate its dual roles in fatty acid uptake and activation. Cellular uptake assays represent the most accessible approach, wherein recombinant protein is expressed in heterologous systems (HEK293, CHO cells) followed by incubation with fluorescently labeled fatty acids (BODIPY-FA) or radiolabeled substrates ([³H]-palmitate, [¹⁴C]-oleate). Quantification via fluorescence microscopy, flow cytometry, or scintillation counting enables determination of transport kinetics. For mechanistic studies, reconstitution of purified protein into proteoliposomes or planar lipid bilayers allows precise control of membrane composition and transmembrane gradients. Enzyme-coupled spectrophotometric assays targeting the acyl-CoA synthetase activity measure either ATP consumption (coupled to pyruvate kinase/lactate dehydrogenase) or CoA consumption (using dithionitrobenzoic acid for colorimetric detection). Multi-parametric analysis combining transport measurements with membrane potential monitoring (using potential-sensitive dyes) can elucidate the role of electrochemical gradients in transport mechanisms. Competition assays with non-labeled fatty acids of varying chain lengths and saturation states reveal substrate preferences and structure-activity relationships. For comprehensive functional characterization, parallel assessment of both transport and activation functions is essential, as these activities may be differentially affected by experimental conditions or mutations.

What techniques are available for studying structure-function relationships in Pongo abelii SLC27A4?

Investigation of structure-function relationships in Pongo abelii SLC27A4 requires integration of structural data with functional analyses through complementary methodologies. Computational approaches represent initial steps, with homology modeling based on crystallized related proteins generating structural predictions that can guide experimental design. Advanced protein visualization techniques, including hydrogen-deuterium exchange mass spectrometry (HDX-MS), reveal conformational dynamics and substrate-induced structural changes with peptide-level resolution. Site-directed mutagenesis of conserved residues identified through multiple sequence alignments, followed by functional assays, enables experimental validation of computational predictions regarding catalytic sites and transport channels. Deletion constructs focusing on specific domains help delineate the minimal functional units required for transport versus acyl-CoA synthetase activities. Cross-linking studies using bifunctional reagents with varying spacer lengths can map proximity relationships between domains during different functional states. Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling provides distance measurements between specific residues in the native membrane environment. Integration of structural data from X-ray crystallography of soluble domains with cryo-electron microscopy of full-length protein can generate comprehensive structural models, though membrane protein crystallization presents significant technical challenges requiring specialized approaches like lipid cubic phase crystallization or detergent screening.

How can researchers address challenges in comparing Pongo abelii SLC27A4 to human orthologs?

Comparative analysis between Pongo abelii SLC27A4 and human orthologs presents several methodological challenges requiring systematic approaches for meaningful interpretation. Sequence alignment using specialized algorithms optimized for membrane proteins (e.g., PRALINE-TM) provides the foundation for identifying conserved functional domains versus divergent regions that may reflect species-specific adaptations. Expression of both proteins under identical conditions in heterologous systems enables direct functional comparisons, though differences in codon usage may necessitate optimization for the expression system. Substrate specificity profiles should be generated using identical fatty acid panels, with special attention to polyunsaturated fatty acids that often show species-specific handling. Kinetic parameters (Km, Vmax) determined under standardized conditions provide quantitative comparison metrics, though temperature optimization might differ between species. Pharmacological profiling using inhibitor panels can reveal subtle differences in binding site architecture not apparent from sequence comparisons alone. When comparing tissue expression patterns, adjustment for evolutionary differences in tissue-specific promoter elements is essential for proper interpretation. Chimeric constructs containing domains from both species can pinpoint regions responsible for functional differences. For physiological relevance, comparative studies should consider species differences in plasma fatty acid composition and metabolism. Integration of structural, functional, and expression data provides comprehensive understanding of evolutionary conservation versus divergence in fatty acid transport mechanisms.

What are the critical considerations when designing experiments to investigate SLC27A4 regulation?

Experimental investigation of SLC27A4 regulation requires careful consideration of multiple regulatory mechanisms spanning transcriptional, post-transcriptional, and post-translational levels. Transcriptional regulation studies should examine promoter activity using reporter constructs (luciferase, GFP) in response to metabolic stimuli, with particular attention to PPAR response elements and sterol regulatory elements known to regulate lipid metabolism genes. Chromatin immunoprecipitation (ChIP) assays enable identification of transcription factors directly binding the promoter region. Post-transcriptional regulation investigation requires analysis of mRNA stability through actinomycin D chase experiments and examination of potential microRNA binding sites through reporter constructs containing the 3'UTR. Alternative splicing analysis using RT-PCR with isoform-specific primers can reveal tissue-specific expression patterns of variant transcripts. Post-translational regulation studies should assess phosphorylation status using phospho-specific antibodies or mass spectrometry-based phosphoproteomics, with particular focus on potential protein kinase A and protein kinase C sites. Protein stability and turnover can be examined through cycloheximide chase experiments combined with proteasome and lysosome inhibitors. For comprehensive understanding, integration of multiple regulatory layers is essential, as is consideration of species-specific regulatory mechanisms when extrapolating from Pongo abelii to human systems. Time-course experiments following metabolic stimuli can reveal sequential regulatory events, providing insights into the dynamic regulation of fatty acid transport in response to changing metabolic demands.

How can researchers address solubility issues when working with recombinant SLC27A4?

Addressing solubility challenges with recombinant Pongo abelii SLC27A4 requires systematic optimization of expression and purification conditions to accommodate its membrane-associated domains. Expression of truncated constructs focusing on soluble domains represents the most straightforward approach, though at the cost of losing transmembrane functionality. Co-expression with solubility-enhancing fusion partners (MBP, SUMO, thioredoxin) significantly improves soluble yields compared to conventional His or GST tags alone. For full-length protein expression, detergent screening is essential, with initial trials typically including mild detergents like DDM, digitonin, or LMNG that effectively solubilize membrane proteins while preserving native structure. Buffer optimization should explore various pH ranges (typically 6.5-8.5) and salt concentrations (100-500 mM) to identify conditions promoting solubility without compromising stability. Addition of glycerol (5-10%) or specific lipids (cholesterol, phosphatidylcholine) to purification buffers can maintain the native lipid environment crucial for membrane protein stability. Temperature reduction during expression (16-20°C) slows protein synthesis, allowing proper folding and insertion into membranes. For particularly challenging constructs, cell-free expression systems provide alternatives, allowing direct incorporation into nanodiscs or liposomes during synthesis. Implementation of high-throughput screening approaches using fluorescent fusion proteins enables rapid identification of optimal solubilization conditions before scaling to preparative levels.

What strategies can be employed to overcome issues with protein stability during experimental procedures?

Maintaining stability of recombinant Pongo abelii SLC27A4 throughout experimental procedures requires implementation of protective strategies addressing multiple degradation pathways. Buffer optimization represents the primary approach, with inclusion of protease inhibitor cocktails (PMSF, leupeptin, aprotinin) essential for preventing proteolytic degradation during initial extraction and purification. Stability-enhancing additives including glycerol (10-20%), specific lipids (cholesterol, phosphatidylcholine), and osmolytes (trehalose, sucrose) significantly improve protein stability by preferential hydration and protection against denaturation . Temperature management is crucial, with all procedures ideally performed at 4°C and sample exposure to room temperature minimized to prevent thermal denaturation. Oxidative damage can be mitigated through addition of reducing agents (DTT, 2-mercaptoethanol) and operation under nitrogen atmosphere for particularly sensitive preparations. For membrane proteins like SLC27A4, maintaining appropriate detergent concentrations above critical micelle concentration throughout all procedures prevents protein aggregation resulting from micelle disruption. Centrifugation steps should employ the minimum force required to achieve separation, as excessive g-forces can cause protein denaturation at phase interfaces. Storage of purified protein as multiple single-use aliquots prevents activity loss from repeated freeze-thaw cycles. For particularly unstable constructs, chemical crosslinking or complex formation with binding partners can significantly enhance stability, though potentially at the cost of altered functionality.

How can researchers validate the functional activity of their recombinant Pongo abelii SLC27A4 preparations?

Comprehensive validation of recombinant Pongo abelii SLC27A4 functional activity requires implementation of multiple complementary assays targeting its dual transport and enzymatic functions. Fatty acid uptake assays represent the primary functional validation, typically using fluorescently labeled fatty acids (BODIPY-C12) in cellular systems expressing the recombinant protein or in reconstituted proteoliposomes for purified protein. Quantification via fluorescence microscopy, flow cytometry, or plate reader measurements provides direct evidence of transport functionality. Acyl-CoA synthetase activity can be assessed through coupled enzyme assays measuring either ATP consumption (linked to pyruvate kinase/lactate dehydrogenase) or CoA consumption (using DTNB for colorimetric detection), with kinetic parameters compared to established values to confirm enzymatic competence. Binding assays using isothermal titration calorimetry or microscale thermophoresis with fatty acid ligands provide thermodynamic parameters (Kd, stoichiometry) indicative of properly folded binding sites. Competitive inhibition profiles using known inhibitors (e.g., triacsin C) should match established patterns for the native protein. Circular dichroism spectroscopy confirms proper secondary structure content, while thermal shift assays assess conformational stability, with properly folded protein showing cooperative unfolding transitions. For membrane-inserted constructs, reconstitution efficiency into liposomes or nanodiscs can be verified through sucrose gradient centrifugation followed by protein and lipid quantification to determine protein:lipid ratios. Integration of multiple validation techniques provides comprehensive confirmation of functional integrity before proceeding to experimental applications.