Recombinant Rat Fatty acyl-CoA reductase 1 (Far1)

What is rat FAR1 and what is its biological function?

Fatty acyl-CoA reductase 1 (FAR1) is an enzyme that catalyzes the reduction of fatty acyl-CoA to fatty alcohols, playing a crucial role in lipid metabolism. In rats, as in humans, FAR1 is primarily involved in the biosynthesis of ether lipids and waxes. The enzyme demonstrates both fatty-acyl-CoA reductase (alcohol-forming) activity and long-chain-fatty-acyl-CoA reductase activity, as confirmed by functional studies. FAR1 participates in the peroxisome pathway, where it contributes to the metabolism of very long-chain fatty acids and interacts with multiple peroxisomal proteins including PEX14 . This enzyme represents an important component in cellular lipid homeostasis, with implications for membrane structure and function across mammalian species.

What expression systems are recommended for producing functional recombinant rat FAR1?

Several expression systems can be employed for producing recombinant rat FAR1, each with distinct advantages:

• Bacterial expression (E. coli): The pGEX vector system, which creates GST-fusion proteins, offers a robust platform for initial expression attempts. This approach allows for inducible expression using IPTG (typically 0.1 mM) with optimal induction periods of approximately 6 hours . The primary advantage is high yield, though proper folding may be compromised.

• Yeast expression systems: These provide a eukaryotic environment with some post-translational processing capabilities while maintaining relatively high yields.

• Insect cell expression: Baculovirus-infected insect cells offer improved folding and post-translational modifications compared to prokaryotic systems.

• Mammalian cell expression: For the most physiologically relevant form of rat FAR1, mammalian expression systems such as HEK293 cells provide appropriate folding and post-translational modifications . This approach is particularly relevant when investigating protein-protein interactions or when enzymatic activity requires specific modifications.

Selection of an appropriate expression system should be guided by the specific experimental requirements, balancing protein yield with functional considerations.

How can I optimize purification protocols to maintain rat FAR1 enzymatic activity?

Optimizing purification of recombinant rat FAR1 requires careful attention to several critical factors:

• Lysis conditions: Use gentle lysis methods such as sonication (3-5 cycles of 30 seconds on/off) in buffers containing protease inhibitors and reducing agents (DTT or β-mercaptoethanol) . This prevents protein degradation and oxidation of cysteine residues.

• Affinity purification: For GST-tagged FAR1, glutathione-Sepharose affinity chromatography represents an effective first purification step. After binding, wash extensively with PBS to remove non-specifically bound proteins before eluting with 10 mM reduced glutathione in buffer .

• Tag removal: If a tag-free protein is desired, include a protease cleavage site between the tag and FAR1. For GST-tagged proteins, PreScission Protease can efficiently cleave the tag under mild conditions while maintaining enzyme activity .

• Buffer optimization: Test different buffers (pH 7.0-8.0) containing stabilizing agents such as glycerol (10-20%) and reducing agents. Include cofactor (NADPH) at low concentrations to stabilize the active site.

• Storage conditions: Aliquot purified FAR1 into small volumes to avoid freeze-thaw cycles and store at -80°C in buffer containing 10-20% glycerol and reducing agent .

The purification process should be monitored at each step using activity assays to ensure the final product retains catalytic function.

What analytical methods can verify the purity and identity of recombinant rat FAR1?

Multiple complementary techniques should be employed to verify the purity and identity of recombinant rat FAR1:

• SDS-PAGE: Assess protein purity and approximate molecular weight. Expect a band at approximately 60 kDa for rat FAR1 (or higher if fusion tags are present) .

• Western blotting: Confirm identity using antibodies specific to FAR1 or to the fusion tag (e.g., anti-GST) . This provides specificity that SDS-PAGE alone cannot offer.

• Mass spectrometry: MALDI-QqTOF mass spectrometry can provide precise molecular weight determination and, following protease digestion, peptide mapping for sequence confirmation . This approach can identify post-translational modifications and verify sequence integrity.

• Size exclusion chromatography: Analyze the oligomeric state and detect potential aggregates or degradation products.

• Circular dichroism: Assess secondary structure to confirm proper folding, particularly when comparing different expression systems or purification methods.

• Enzymatic activity assays: Measure the conversion of fatty acyl-CoA substrates to fatty alcohols using spectrophotometric methods tracking NADPH consumption as the definitive test of functional integrity.

These methods collectively provide a comprehensive characterization profile for recombinant rat FAR1 preparations.

What approaches can resolve inconsistent activity data between different recombinant rat FAR1 batches?

Inconsistent activity between recombinant rat FAR1 batches represents a common challenge requiring systematic troubleshooting:

• Standardization of expression conditions: Maintain consistent cell density before induction (OD600 of 0.6-0.8), IPTG concentration (0.1 mM has been demonstrated effective for recombinant proteins), and induction time (6 hours optimum for many GST-fusion proteins) . Document growth curves for each production batch.

• Protein characterization matrix:

  Parameter	Method	Acceptance Criteria
  Purity	SDS-PAGE	>95% single band
  Identity	Western blot	Single band at expected MW
  Oligomeric state	Size exclusion	Predominantly monomeric
  Activity	NADPH consumption	>80% of reference standard
  Stability	Thermal shift assay	Tm within ±2°C of reference

• Reference standard implementation: Maintain a well-characterized reference standard preparation and include it in each activity assay as an internal control.

• Cofactor and substrate quality: Use fresh NADPH preparations and verify the quality of fatty acyl-CoA substrates, as these can degrade and significantly impact measured activity.

• Environmental variables: Control temperature precisely during activity assays, as enzymatic rates are highly temperature-dependent. Standardize all buffer components, including salt concentration and pH.

• Statistical validation: Perform at least triplicate measurements for each activity determination and apply appropriate statistical analyses to determine if observed differences are significant.

Implementing these approaches creates a robust framework for identifying sources of variability and establishing reliable quality control parameters.

How can I design experiments to investigate rat FAR1 substrate specificity?

Investigating substrate specificity of rat FAR1 requires a systematic experimental design:

• Substrate panel preparation: Assemble a diverse panel of fatty acyl-CoA substrates including:

  • Varying chain lengths (C8-C24)

  • Saturated and unsaturated fatty acids

  • Branched-chain fatty acids (particularly relevant based on peroxisomal catabolism studies)

  • Hydroxylated or otherwise modified fatty acids

• Kinetic characterization protocol:

  • Measure initial reaction rates across a range of substrate concentrations (typically 1-100 μM)

  • Maintain constant enzyme concentration and reaction conditions

  • Determine Km, Vmax, and catalytic efficiency (kcat/Km) for each substrate

  • Plot structure-activity relationships based on chain length, degree of unsaturation, etc.

• Competition experiments:

  • Perform assays with multiple substrates simultaneously

  • Analyze product formation using GC-MS or LC-MS to determine preference ratios

  • Use varying ratios of competing substrates to establish preference profiles

• Structure-function analysis:

  • Design and express FAR1 variants with mutations in the predicted substrate binding pocket

  • Compare substrate profiles of wild-type and mutant proteins

  • Correlate changes in specificity with structural features

• Computational modeling:

  • Develop homology models of rat FAR1 based on related enzymes

  • Perform molecular docking with various substrates

  • Identify key residues involved in substrate recognition

This comprehensive approach provides both qualitative and quantitative insights into the substrate preference landscape of rat FAR1.

What methodologies effectively investigate rat FAR1's role in peroxisomal lipid metabolism?

Investigating rat FAR1's role in peroxisomal lipid metabolism requires a multi-faceted approach:

• Subcellular localization studies:

  • Immunofluorescence microscopy with co-staining for peroxisomal markers (e.g., PEX14)

  • Subcellular fractionation followed by Western blot analysis

  • Live-cell imaging with fluorescently tagged FAR1 to monitor dynamic localization

• Protein-protein interaction network:

  • Co-immunoprecipitation with known peroxisomal proteins

  • Proximity labeling using BioID-FAR1 fusion proteins

  • Analyze interactions with peroxisome biogenesis factors and metabolic enzymes

  • Cross-reference with known FAR1 interacting proteins such as PEX14 and GSTK1

• Metabolic impact assessment:

  • Lipidomic analysis comparing wild-type and FAR1-deficient cells

  • Focus on ether lipid precursors and derivatives

  • Stable isotope labeling to track metabolic flux through FAR1-dependent pathways

  • Analysis of peroxisomal β-oxidation efficiency

• Genetic manipulation strategies:

  • siRNA knockdown of FAR1 in rat hepatocytes or other relevant cell types

  • CRISPR-Cas9 genome editing to create FAR1-null cells

  • Rescue experiments with wild-type and mutant FAR1 constructs

  • Conditional knockout models to study tissue-specific effects

• Disease model correlations:

  • Examine FAR1 expression and function in rodent models of peroxisomal disorders

  • Investigate potential compensatory mechanisms involving FAR2

These approaches collectively provide a comprehensive understanding of FAR1's contribution to peroxisomal metabolism and potential implications for metabolic disorders.

How can structure-function relationships in rat FAR1 be systematically investigated?

Systematic investigation of structure-function relationships in rat FAR1 requires a coordinated experimental approach:

• Structural analysis foundations:

  • Generate homology models based on related enzymes in the SDR (short-chain dehydrogenase/reductase) family

  • Identify conserved domains through sequence alignment with FAR2 and other homologs

  • Predict catalytic residues and substrate-binding regions

• Domain mapping strategy:

  • Create truncation mutants to identify minimal functional units

  • Design chimeric proteins with domains from related enzymes (e.g., FAR2)

  • Systematically replace regions with the corresponding sequences from other species

• Site-directed mutagenesis design:

  • Target predicted catalytic residues (typically involving S, Y, K catalytic triad in SDR enzymes)

  • Modify residues in the NAD(P)H-binding region (typically a Rossmann fold)

  • Alter potential membrane-association domains

  • Create charge-reversal mutations at potential protein-protein interaction interfaces

• Functional characterization matrix:

  Construct	Enzymatic Activity	Substrate Specificity	Protein Interactions	Cellular Localization
  Wild-type	Baseline	Baseline	Baseline	Peroxisomal
  Catalytic mutants	Reduced/eliminated	Altered	Maintained	Maintained
  Binding pocket mutants	Maintained	Altered	Maintained	Maintained
  Localization mutants	Maintained	Maintained	Altered	Cytosolic/mislocalized

• Biophysical analysis:

  • Circular dichroism to assess secondary structure changes

  • Thermal stability measurements to identify destabilizing mutations

  • Intrinsic fluorescence to monitor conformational changes upon substrate/cofactor binding

This systematic approach correlates structural elements with specific functional properties, providing mechanistic insights into rat FAR1's catalytic activity and biological role.

What are the optimal assay conditions for measuring rat FAR1 enzymatic activity in vitro?

Establishing optimal assay conditions for rat FAR1 requires careful optimization of multiple parameters:

• Basic assay components:

  • Buffer: 100 mM phosphate buffer or 50 mM Tris-HCl, pH 7.4-7.8

  • Reducing environment: 1-5 mM DTT or β-mercaptoethanol

  • Cofactor: 0.1-0.5 mM NADPH (freshly prepared)

  • Substrate: 10-100 μM fatty acyl-CoA (chain length appropriate for experiment)

  • Enzyme: 0.1-1.0 μg purified recombinant FAR1

• Spectrophotometric measurement protocol:

  • Monitor NADPH consumption at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Maintain temperature at 25°C or 37°C (consistent between experiments)

  • Measure initial rates (first 1-5 minutes) to ensure linearity

  • Include appropriate controls (no enzyme, no substrate, heat-inactivated enzyme)

• Alternative product detection methods:

  • Gas chromatography analysis of fatty alcohols following extraction

  • HPLC separation with UV or fluorescence detection (may require derivatization)

  • LC-MS/MS for highest sensitivity and specificity

• Optimization considerations:

  • Temperature dependence: Test activity at 25°C, 30°C, and 37°C

  • pH profile: Test activity across pH range 6.5-8.5

  • Salt sensitivity: Test NaCl concentrations from 0-500 mM

  • Divalent cation effects: Test impact of Mg²⁺, Mn²⁺, Ca²⁺ (0-5 mM)

• Data analysis:

  • Calculate specific activity as nmol NADPH consumed/min/mg protein

  • For kinetic studies, fit data to Michaelis-Menten equation to determine Km and Vmax

  • For inhibition studies, determine IC₅₀ values and inhibition mechanisms

These optimized conditions ensure reproducible and physiologically relevant measurement of rat FAR1 activity.

What techniques can effectively analyze post-translational modifications of recombinant rat FAR1?

Post-translational modifications (PTMs) of recombinant rat FAR1 can be analyzed using multiple complementary techniques:

• Mass spectrometry-based identification:

  • Digest purified FAR1 with trypsin or endoproteinase Glu-C

  • Analyze peptides by MALDI-QqTOF MS in positive ionization mode

  • Compare observed masses with theoretical peptide masses

  • Identify mass shifts corresponding to potential modifications

  • Confirm PTM identity and location using MS/MS fragmentation patterns

• Modification-specific enrichment strategies:

  • Phosphorylation: Immobilized metal affinity chromatography (IMAC) or phospho-specific antibodies

  • Glycosylation: Lectin affinity chromatography

  • Ubiquitination: Affinity purification with anti-ubiquitin antibodies

  • Acetylation: Anti-acetyllysine antibodies for enrichment

• Site-directed mutagenesis approach:

  • Identify potential modification sites by in silico prediction

  • Create site-specific mutants (e.g., S→A to prevent phosphorylation, K→R to prevent acetylation)

  • Compare properties of wild-type and mutant proteins

  • Generate phosphomimetic mutants (S→D or S→E) to simulate phosphorylation

• Expression system comparison:

  • Express rat FAR1 in different systems (E. coli, yeast, insect cells, mammalian cells)

  • Compare PTM patterns between expression systems

  • Correlate modifications with functional properties

• Enzymatic demodification:

  • Treat purified FAR1 with phosphatases, deglycosylases, or deacetylases

  • Monitor changes in electrophoretic mobility and activity

  • Perform peptide mass fingerprinting before and after treatment

These approaches provide comprehensive characterization of FAR1 PTMs and their functional significance.

What methods can detect and characterize protein-protein interactions involving rat FAR1?

Multiple complementary approaches can effectively detect and characterize protein-protein interactions involving rat FAR1:

• Affinity-based isolation techniques:

  • Co-immunoprecipitation using antibodies against FAR1 or known/suspected interacting partners

  • Pull-down assays with recombinant GST-tagged FAR1 as bait

  • Tandem affinity purification for higher stringency

  • Analysis of isolated complexes by mass spectrometry to identify novel interacting proteins

• Proximity-based detection methods:

  • Bimolecular fluorescence complementation (BiFC) for direct visualization in cells

  • Förster resonance energy transfer (FRET) to measure proximity between fluorescently tagged proteins

  • Proximity ligation assay (PLA) for detection of endogenous protein interactions

  • BioID or APEX2 proximity labeling to identify proteins in the vicinity of FAR1

• Genetic interaction screens:

  • Yeast two-hybrid screening using rat FAR1 as bait

  • Mammalian two-hybrid assays for verification in more relevant cell types

  • Synthetic lethality screens to identify functional interactions

• Quantitative interaction characterization:

  • Surface plasmon resonance (SPR) to measure binding kinetics and affinity

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for interaction studies in solution

• Interaction mapping:

  • Domain mapping through truncation mutants

  • Alanine scanning of predicted interaction surfaces

  • Crosslinking mass spectrometry to identify interaction interfaces

• Functional validation:

  • Co-localization studies in cells using immunofluorescence

  • Mutagenesis of interaction interfaces and functional testing

  • Competition assays with peptides derived from interaction regions

These approaches collectively provide robust identification and characterization of the FAR1 interactome, including interactions with peroxisomal proteins such as PEX14 and metabolic enzymes like GSTK1 .

How can I design rigorous controls for rat FAR1 knockdown or overexpression experiments?

Designing rigorous controls for rat FAR1 knockdown or overexpression experiments requires addressing several potential sources of experimental variability:

• Knockdown experiment controls:

  • Non-targeting siRNA/shRNA with similar GC content to FAR1-targeting sequences

  • Multiple independent siRNA/shRNA sequences targeting different regions of FAR1 mRNA

  • Rescue experiments with siRNA-resistant FAR1 cDNA (containing silent mutations)

  • Quantification of knockdown efficiency by qRT-PCR and Western blot

  • Assessment of potential off-target effects using transcriptome analysis

  • Evaluation of compensatory upregulation of FAR2 or other related enzymes

• Overexpression experiment controls:

  • Empty vector transfection maintaining identical promoter and regulatory elements

  • Expression of unrelated protein of similar size (e.g., GFP) from the same vector

  • Catalytically inactive mutant FAR1 (for separating enzymatic from structural functions)

  • Titration of expression levels to avoid non-physiological effects

  • Subcellular localization verification to ensure proper targeting

  • Time-course experiments to distinguish acute from adaptive responses

• Experimental design considerations:

  • Cell-type specific controls (primary cells vs. cell lines)

  • Appropriate timepoints based on protein half-life and cellular processes

  • Paired biological replicates to minimize batch effects

  • Randomization of sample processing order

  • Blinded analysis of experimental outcomes where possible

• Phenotypic validation:

  • Multiple independent methods to assess the same endpoint

  • Complementary approaches (e.g., genetic and pharmacological)

  • Dose-dependency of observed effects

  • Correlation between degree of knockdown/overexpression and phenotype magnitude

These comprehensive controls ensure that observed phenotypes can be confidently attributed to specific alterations in FAR1 expression or function.

What strategies can overcome expression and solubility challenges with recombinant rat FAR1?

Overcoming expression and solubility challenges with recombinant rat FAR1 requires systematic optimization at multiple levels:

• Vector design optimization:

  • Test multiple fusion tags (GST, MBP, SUMO, TRX) to improve solubility

  • Optimize codon usage for the expression host

  • Include solubility-enhancing partners upstream of FAR1

  • Design constructs with removable tags using specific proteases

  • Consider domain boundaries carefully for truncated constructs

• Expression host selection:

  • E. coli strains specialized for membrane or difficult proteins (C41/C43, Rosetta, SHuffle)

  • Yeast systems (Pichia pastoris) for improved folding

  • Insect cell systems for complex eukaryotic proteins

  • Mammalian expression for fully native folding and modifications

• Expression condition optimization matrix:

  Parameter	Variables to Test	Expected Impact
  Temperature	16°C, 25°C, 30°C, 37°C	Lower temperatures reduce inclusion body formation
  Induction	0.01-1.0 mM IPTG	Lower inducer concentrations can improve solubility
  Media	LB, TB, 2xYT, minimal	Rich media can support proper folding
  Additives	Glycerol, sucrose, sorbitol	Osmolytes can stabilize folding intermediates
  Duration	3-24 hours	Optimal time balances yield vs. degradation

• Protein extraction and purification strategies:

  • Gentle lysis methods (enzymatic lysis, freeze-thaw cycles)

  • Addition of detergents for membrane-associated proteins

  • Inclusion of cofactors (NADPH) during purification

  • On-column refolding for inclusion body protein

  • Size-exclusion chromatography to remove aggregates

• Buffer optimization:

  • Screen buffer compositions (HEPES, Tris, phosphate)

  • Vary pH conditions (pH 6.0-9.0)

  • Test salt concentrations (0-500 mM NaCl)

  • Include stabilizing additives (glycerol, arginine, trehalose)

  • Add reducing agents (DTT, β-mercaptoethanol) to prevent oxidation

These strategies have successfully addressed similar challenges in recombinant protein expression as documented for other challenging enzymes .