Recombinant Mouse Fatty acyl-CoA reductase 1 (Far1)

What is mouse Fatty acyl-CoA reductase 1 (Far1) and what is its functional role?

Mouse Far1 (gene ID: 67420) is an enzyme that catalyzes the reduction of fatty acyl-CoA substrates to fatty alcohols . It plays a crucial role in the biosynthetic pathway of various lipid molecules, including waxes and ether lipids. Far1 functions by converting long-chain fatty acyl-CoA esters into corresponding fatty alcohols through an NADPH-dependent reductive mechanism. This conversion represents a key step in several lipid biosynthetic pathways .

The enzyme is widely expressed across multiple tissues and is particularly important in lipid metabolism pathways. Far1 contributes to the biosynthesis of fatty alcohols that serve diverse biological roles, including components of cellular membranes, signaling molecules, and specialized lipids found in various tissues .

How conserved is Far1 across different species?

Far1 shows remarkable evolutionary conservation across numerous species, suggesting its fundamental biological importance. Analysis of sequence homology reveals:

Notably, human and mouse SDF-1α (a signaling protein that can interact with lipid pathways) share 99% sequence identity, highlighting the extreme conservation of proteins involved in these pathways across mammals . The Far1 protein sequence is particularly well-conserved within mammalian species, with bumble bee species FARs from the same ortholog group showing 97.2-99.7% protein sequence identity .

What is the molecular structure and theoretical properties of mouse Far1?

Mouse Far1 is characterized by the following molecular properties:

• Molecular Weight: Approximately 8 kDa for the monomeric form (based on related proteins)

• Amino Acid Composition: Contains critical domains for substrate binding and catalysis

• Protein Structure: Features an NAD(P)H binding domain characteristic of the short-chain dehydrogenase/reductase (SDR) family

• Functional Domains: Contains substrate recognition sites that determine fatty acyl chain length specificity

The protein contains key structural motifs that are essential for its catalytic activity, including a Rossmann fold for cofactor binding and substrate recognition domains that determine its specificity for various fatty acyl chain lengths.

How can I determine the expression pattern of Far1 in different mouse tissues?

To determine the expression pattern of Far1 across mouse tissues:

• RT-PCR Analysis: Use tissue-specific RNA extraction followed by reverse transcription and PCR with Far1-specific primers to detect transcript levels across tissues.

• Western Blot Analysis: Utilize specific anti-Far1 antibodies (such as rabbit-produced antibodies) to detect protein expression in tissue lysates .

• Immunohistochemistry: Apply commercially available antibodies like HPA017322 for tissue localization studies. This approach allows visualization of Far1 expression at the cellular and subcellular levels .

• RNA-Seq Analysis: Analyze publicly available transcriptome datasets to compare Far1 expression across different tissues and developmental stages.

Proper normalization against housekeeping genes or proteins is essential for accurate quantitative comparison between tissues.

What are the optimal conditions for expressing recombinant mouse Far1?

Optimal expression of recombinant mouse Far1 can be achieved through careful consideration of the following parameters:

• Expression System Selection: E. coli has been successfully used for Far1 expression , though yeast expression systems (S. cerevisiae) have shown good results for related FARs .

• Expression Vector Design:

  • Include a His-tag for purification and detection purposes

  • Consider codon optimization for the expression host (critical for reducing ribosome stalling)

  • Use a strong but inducible promoter to control expression timing

• Culture Conditions:

  • Temperature: Lower temperatures (16-20°C) after induction often improve proper folding

  • Induction timing: Induce at mid-log phase (OD600 ~0.6-0.8)

  • Media supplements: Consider adding fatty acids that may stabilize the enzyme

• Protein Solubility Enhancement:

  • Co-expression with chaperones may improve folding

  • Fusion tags (MBP, SUMO) can enhance solubility

  • Addition of mild detergents during lysis may improve recovery

Research has shown that codon optimization can significantly improve the expression of full-length Far1, as demonstrated in research where synthetic FARs with codon usage optimized for S. cerevisiae showed predominantly full-length protein expression compared to non-optimized versions .

What methods are most effective for purifying recombinant Far1?

Effective purification of recombinant Far1 typically follows this workflow:

• Affinity Chromatography: Using His-tagged Far1 constructs, Ni-NTA affinity chromatography serves as an effective first purification step. Elution can be performed with imidazole gradients (typically 250-300 mM for final elution) .

• Size Exclusion Chromatography: To separate monomeric Far1 from aggregates and incomplete translation products that are commonly observed in heterologous expression systems .

• Ion Exchange Chromatography: As a polishing step to remove remaining contaminants and achieve higher purity.

• Buffer Optimization:

  • Final storage buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol

  • Addition of 0.1% BSA for prolonged storage has been recommended for related proteins

  • Consider addition of reducing agents like DTT (1 mM) to prevent oxidation

• Storage Recommendations:

  • For prolonged storage, dilute to working aliquots in a 0.1% BSA solution

  • Store at -80°C and avoid repeated freeze-thaw cycles

Western blot analysis using anti-His antibodies should be performed to confirm the identity and integrity of the purified protein .

How can I assess the activity of recombinant Far1 in vitro?

In vitro assessment of Far1 activity can be conducted using the following approaches:

• GC (Gas Chromatography) Analysis:

  • Incubate purified Far1 with fatty acyl-CoA substrates and NADPH

  • Extract fatty alcohols using organic solvents

  • Analyze products by GC to quantify fatty alcohol production

• Spectrophotometric Assays:

  • Monitor NADPH consumption at 340 nm as an indirect measure of enzymatic activity

  • Calculate reaction rates under different substrate concentrations

• Activity Calculation:

  • Fatty alcohol ratios can be calculated using the equation:
    Fatty alcohol ratio = Quantity of specific fatty alcohol / Quantity of fatty acyl precursors × 100%

  • Ratios >50% suggest high conversion efficiency, with some monounsaturated >C20 fatty alcohols approaching 100% conversion rates

• Substrate Specificity Analysis:

  • Test activity against various chain length fatty acyl-CoAs (C12-C24)

  • Examine preference for saturated versus unsaturated substrates

  • Analyze kinetic parameters (Km, Vmax) for different substrates

For example, in research with related FARs, fatty alcohol ratios greater than 50% were observed for most fatty alcohols in labial glands (LGs), with some monounsaturated >C20 fatty alcohols approaching complete conversion (nearly 100%) .

How should I design experiments to study Far1 substrate specificity?

Designing robust experiments to determine Far1 substrate specificity requires:

• Substrate Panel Preparation:

  • Assemble a diverse panel of fatty acyl-CoA substrates varying in:

    • Chain length (C12-C24)

    • Degree of unsaturation (saturated, mono-, poly-unsaturated)

    • Position of unsaturation (Δ9, Δ11, etc.)

• Reaction Conditions Optimization:

  • Buffer composition: Typically 100 mM phosphate buffer pH 7.4

  • Cofactor concentration: 1-2 mM NADPH (freshly prepared)

  • Enzyme concentration: 1-5 μg purified Far1 per reaction

  • Temperature and time: 30°C for 30-60 minutes

• Kinetic Parameter Determination:

  • Conduct time-course experiments to ensure linearity

  • Perform substrate concentration gradients (typically 1-100 μM)

  • Calculate Km and Vmax for each substrate

  • Create Lineweaver-Burk plots to visualize enzyme kinetics

• Competition Assays:

  • Offer multiple substrates simultaneously to determine preference

  • Analyze product formation ratios when substrates compete

• Analytical Methods:

  • GC analysis of extracted products following derivatization

  • LC-MS for more sensitive detection of products

  • Relative quantification against standard curves

Research has shown that heterologous expression in yeast followed by GC analysis is an effective approach for determining substrate specificity of FARs .

What controls are essential when working with recombinant Far1?

Essential controls for Far1 research include:

• Negative Controls:

  • Enzyme-free reactions to account for non-enzymatic reduction

  • Heat-inactivated Far1 to confirm activity is enzyme-dependent

  • Empty vector transformations in expression systems

  • Reactions without NADPH to confirm cofactor requirement

• Positive Controls:

  • Known FAR enzyme with well-characterized activity

  • Purified commercial enzymes from related pathways

  • Synthetic standards of expected fatty alcohol products

• Expression Controls:

  • Western blot analysis to confirm expression of full-length Far1

  • Activity controls with well-characterized substrates

• Specificity Controls:

  • Include related enzymes (e.g., Far2) to distinguish isoform-specific activities

  • Test structurally related non-substrate molecules

• Technical Replicates:

  • Minimum of three independent biological replicates

  • Multiple technical replicates within each experiment

When expressing FARs in heterologous systems, western blot analysis is critical to confirm proper expression. Research has shown that without codon optimization, incomplete translation products are commonly observed alongside the expected full-length protein .

How can I investigate Far1's interaction with other enzymes in fatty alcohol synthesis pathways?

Investigating Far1's interactions requires multi-faceted approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use anti-Far1 antibodies to precipitate protein complexes

  • Analyze co-precipitated proteins by mass spectrometry

  • Confirm specific interactions with targeted western blots

• Yeast Two-Hybrid Screening:

  • Use Far1 as bait to screen for interacting proteins

  • Focus on candidate enzymes from related metabolic pathways

• Proximity Labeling Approaches:

  • BioID or APEX2 fusions to identify proteins in close proximity to Far1

  • Temporal analysis to detect transient interactions

• Functional Enzyme Coupling Assays:

  • Design assays where Far1 activity is coupled to upstream or downstream enzymes

  • Measure product formation in coupled versus uncoupled reactions

  • Analyze kinetic coupling effects

• Metabolic Flux Analysis:

  • Trace isotope-labeled substrates through the pathway

  • Compare flux with and without Far1 inhibition or overexpression

  • Identify rate-limiting steps and regulatory points

Research indicates that fatty alcohol biosynthesis involves complex enzyme interactions, with in vivo specificity differing from isolated enzyme activity , highlighting the importance of studying Far1 in its pathway context.

How do I calculate and interpret fatty alcohol ratios in Far1 studies?

Fatty alcohol ratios provide insight into the in vivo specificity and efficiency of Far1:

• Calculation Method:
  The fatty alcohol ratio is calculated using the equation:

  Fatty alcohol ratio (%) = [Quantity of specific fatty alcohol / Quantity of hypothetical fatty acyl precursors] × 100%

• Interpretation Guidelines:

  • Ratios >50%: Indicate high conversion efficiency

  • Ratios approaching 100%: Suggest near-complete conversion of precursors

  • Varying ratios across chain lengths: Reflect substrate preferences

• Comparative Analysis:

  • Compare ratios between different fatty alcohol species to determine substrate preferences

  • Analyze differences between in vitro and in vivo ratios to identify potential regulatory mechanisms

• Visualization Methods:

  • Plot ratios against carbon chain length to visualize preference patterns

  • Use heat maps to compare ratios across different conditions or enzyme variants

Research has shown that in labial glands (LGs), fatty alcohol ratios exceed 50% for most fatty alcohols and approach 100% for some monounsaturated >C20 fatty alcohols, suggesting highly efficient conversion in these tissues .

How can I resolve contradictory results in Far1 substrate specificity studies?

Resolving contradictions in substrate specificity findings requires systematic investigation:

• Source of Contradiction Analysis:

  • Examine differences in experimental conditions:

    • Expression systems (bacterial vs. yeast vs. mammalian cells)

    • Enzyme preparation methods (purification tags, buffers)

    • Assay conditions (pH, temperature, cofactor concentration)

  • Consider post-translational modifications present in different systems

• Methodological Approaches:

  • Perform side-by-side comparisons using standardized methods

  • Use multiple analytical techniques (GC, LC-MS) to confirm findings

  • Validate with both in vitro and cellular assays

• Protein Structure Considerations:

  • Investigate if protein truncation or tagging affects activity

  • Examine if variants or isoforms were used in different studies

  • Consider the effect of oligomerization state on activity

• Data Integration Strategies:

  • Meta-analysis of multiple studies with weighted confidence scores

  • Bayesian approaches to reconcile contradictory findings

  • Collaborative validation across different laboratories

Research demonstrates that heterologous expression can lead to variations in protein functionality, with factors like codon optimization significantly affecting enzyme production and activity .

Why might my recombinant Far1 show low activity in heterologous expression systems?

Low activity of recombinant Far1 may stem from several factors:

• Expression-Related Issues:

  • Incomplete translation: Western blot analysis often reveals multiple protein bands of lower molecular weight than the full-length Far1, indicating truncated products

  • Protein aggregation: Higher molecular weight bands on western blots may represent aggregated Far1 proteins

  • Codon usage: Non-optimized codons can cause ribosome stalling and incomplete translation

• Folding and Structural Problems:

  • Improper folding in heterologous systems

  • Incorrect disulfide bond formation

  • Incomplete post-translational modifications

• Cofactor Availability:

  • Insufficient NADPH in the expression system

  • Suboptimal redox environment

• Substrate Accessibility:

  • Limited substrate uptake by cells

  • Compartmentalization issues preventing substrate-enzyme interaction

• Solution Approaches:

  • Codon optimization: Synthetic Far1 coding regions with optimized codon usage show improved expression of full-length protein

  • Lower expression temperature to improve folding

  • Co-expression with chaperone proteins

  • Addition of relevant cofactors to growth media

Research clearly demonstrates that codon-optimized synthetic versions of FARs show significantly improved expression patterns with predominant full-length protein production compared to non-optimized versions .

How can codon optimization enhance recombinant Far1 expression?

Codon optimization significantly improves Far1 expression through several mechanisms:

Research demonstrates that the shortened heterologously expressed proteins observed in western blots presumably represent incompletely transcribed versions of full-length FARs resulting from ribosome stalling, a problem effectively addressed through codon optimization .

What are promising applications of engineered Far1 variants?

Engineered Far1 variants offer several promising research and biotechnological applications:

• Biofuel Production Enhancement:

  • Engineering Far1 variants with improved activity toward specific fatty acid chain lengths could enhance production of fatty alcohols used in biofuel applications

  • Variants with increased thermostability and solvent tolerance would be valuable for industrial processes

• Metabolic Engineering Tools:

  • Modified Far1 variants can serve as key enzymes in synthetic pathways for production of specialty chemicals

  • Substrate specificity-altered variants could enable production of novel fatty alcohol derivatives

• Biomedical Applications:

  • Far1 variants as potential therapeutic targets for lipid metabolism disorders

  • Development of inhibitors or activators for specific Far1 variants could modulate lipid metabolism pathways

• Research Tools:

  • Reporter-fused Far1 variants for studying lipid metabolism in real-time

  • Tagged variants for identification of interaction partners and regulatory mechanisms

  • Temperature-sensitive variants for conditional studies

The FAR gene family has undergone significant expansion through evolution, suggesting adaptability to different biochemical contexts and substrates , which provides a foundation for engineering variants with novel properties.

What emerging technologies might advance Far1 research?

Emerging technologies poised to transform Far1 research include:

• Structural Biology Advancements:

  • Cryo-EM for high-resolution structure determination of Far1 complexes

  • AlphaFold2 and related AI approaches for structure prediction and rational engineering

  • Time-resolved crystallography to capture catalytic intermediates

• High-Throughput Screening Platforms:

  • Droplet microfluidics for rapid screening of Far1 variants

  • Cell-free expression systems coupled with activity assays

  • Multiplexed activity assays for simultaneous testing of multiple substrates

• Genome Editing Tools:

  • CRISPR-Cas9 for precise genomic editing to study Far1 function in vivo

  • Base editing for introducing specific amino acid changes without double-strand breaks

  • Prime editing for precise modification of Far1 sequences

• Systems Biology Approaches:

  • Multi-omics integration to place Far1 in broader metabolic networks

  • Machine learning models to predict Far1 activity based on substrate structures

  • Metabolic flux analysis to understand Far1's role in lipid homeostasis

These technologies will enable more comprehensive understanding of Far1 structure-function relationships and its integration in cellular metabolism.