Recombinant Galleria mellonella Apolipophorin-3
Description
Definition and Production of Recombinant Gm-apoLp-III
rGm-apoLp-III is synthesized by cloning the cDNA of Galleria mellonella apoLp-III into a pET vector, followed by overexpression in Escherichia coli . Key steps include:
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Gene Identification: Full cDNA sequencing via MOPAC and RACE-PCR.
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Expression: High-yield production in E. coli with subsequent purification.
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Functional Validation: Confirmation of lipid-binding capacity using human LDL turbidity assays and electron microscopy .
Table 1: Comparison of Native and Recombinant Gm-apoLp-III
| Property | Native apoLp-III | Recombinant apoLp-III |
|---|---|---|
| Source | G. mellonella hemolymph | E. coli expression system |
| Lipid-binding ability | Stabilizes LDLp particles | Retains LDL stabilization |
| Immune activation | Enhances antibacterial activity | Induces similar responses |
| Structural conformation | Lipid-free and lipid-bound | Mimics lipid-bound state |
Pathogen Recognition and Binding
rGm-apoLp-III acts as a pattern recognition receptor (PRR) with broad specificity:
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Fungal β-1,3-glucan: Direct binding to fungal conidia, facilitating encapsulation .
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Bacterial Surfaces: Targets Gram-negative (LPS) and Gram-positive (lipoteichoic acid) pathogens .
Antimicrobial Activity
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Injection Studies: Administering rGm-apoLp-III into G. mellonella larvae elevates lysozyme-like activity and suppresses E. coli growth by 60–80% within 24 hours .
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Synergy: Augments lysozyme perforation of bacterial membranes, increasing hydrophobicity and negative charge on microbial surfaces .
Cellular Immune Modulation
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Hemocyte Activation: Lipid-bound rGm-apoLp-III stimulates hemocyte adhesion and spreading, critical for nodule formation .
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Degradation by Pathogens: Pseudomonas aeruginosa protease IV cleaves apoLp-III at Lys70–Lys71, suggesting immune evasion tactics .
Table 2: Immune Functions of rGm-apoLp-III
| Function | Mechanism | Outcome |
|---|---|---|
| Pathogen binding | Binds β-1,3-glucan, LPS, LTA | Enhances encapsulation/phagocytosis |
| Antimicrobial induction | Upregulates lysozyme and AMPs | Reduces bacterial viability |
| Hemocyte modulation | Activates adhesion and spreading | Promotes multicellular encapsulation |
Applications in Research
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Infection Models: Used to study host-pathogen interactions, including fungal (Beauveria bassiana) and bacterial (P. aeruginosa) infections .
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Comparative Immunology: Insights into functional parallels with mammalian apolipoprotein E (apoE), particularly in LPS detoxification .
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Parasitology: Silencing apoLp-III homologs in mosquitoes reduces Plasmodium survival, highlighting cross-species relevance .
Challenges and Future Directions
Product Specs
Target Background
Q&A
What is Apolipophorin-3 and what are its functions in Galleria mellonella?
Apolipophorin-3 (apoLp-III) is an exchangeable insect apolipoprotein with dual primary functions. Traditionally, it has been understood to stabilize low-density lipophorin particles (LDLp) that cross the hemocoel during high energy consumption phases, delivering lipids from the fat body to flight muscle cells . More recently, research has revealed that apoLp-III plays a significant role in insect immune activation, functioning as an opsonin characterized by lipid and carbohydrate-binding properties . When injected into G. mellonella larvae, apoLp-III stimulates increased antibacterial activity against bacteria like Escherichia coli and enhances lysozyme-like activities . This establishes an important correlation between lipid physiology and immune defense mechanisms in insects .
How is recombinant G. mellonella Apolipophorin-3 produced?
Recombinant G. mellonella apoLp-III production typically follows this methodological approach:
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RNA isolation from G. mellonella larvae using protocols like TRIzol extraction
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cDNA synthesis via reverse transcription
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PCR amplification of the apoLp-III open reading frame (approximately 510 bp) using specific primers containing restriction enzyme sites (typically EcoRI and HindIII)
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Cloning the amplicon into a suitable expression vector (e.g., pCold I)
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Transformation of expression-optimized E. coli strains like BL21(DE3) Star
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Induction of protein expression using IPTG (typically 1.0 mM) at lower temperatures (15°C) for extended periods (20 hours)
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Cell harvesting by centrifugation and protein purification through metal affinity chromatography
Importantly, the native protein contains a signal peptide at the N-terminus, which is typically excluded from recombinant constructs. The expression vector often adds elements like TEE motifs, His-tags, and Factor Xa sites, resulting in a recombinant protein of approximately 21 kDa versus the 18 kDa native form .
What verification methods confirm recombinant apoLp-III functionality?
Verification of recombinant apoLp-III functionality requires multiple complementary approaches:
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Lipid-binding assays: Photometric turbidity assays with human low-density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs confirm the protein's lipid association capability .
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Immune stimulation tests: Injection of purified recombinant protein into G. mellonella larvae followed by measurement of antibacterial activity in cell-free hemolymph 24 hours later should show increased antimicrobial activity .
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Molecular characterization: SDS-PAGE analysis with both Coomassie blue and silver staining confirms purity and expected molecular weight .
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Comparative studies: Side-by-side testing with native apoLp-III in functional assays validates that the recombinant form behaves similarly to the native protein.
What are the optimal conditions for expressing recombinant G. mellonella apoLp-III in E. coli?
Based on published research, optimal expression conditions include:
| Parameter | Optimal Condition | Notes |
|---|---|---|
| E. coli strain | BL21(DE3) Star | Enhances protein stability |
| Expression vector | pCold I | Cold-shock promoter improves folding |
| Growth medium | LB broth | With 100 μg/mL ampicillin |
| Pre-induction temperature | 37°C | Until reaching OD600 = 0.6 |
| Induction | 1.0 mM IPTG | Final concentration |
| Post-induction temperature | 15°C | Critical for proper folding |
| Post-induction duration | 20 hours | Allows complete expression |
| Culture volume | 200 mL in 2L flask | Ensures adequate aeration |
| Harvesting | 1,500 × g, 20 min, 4°C | Preserves protein integrity |
The reduced temperature during induction (15°C) is particularly important as it slows protein synthesis, allowing for proper folding and reducing inclusion body formation .
How can different forms of Apolipophorin-3 be separated and characterized?
Different forms of apoLp-III can be separated and characterized using two-dimensional electrophoresis (IEF/SDS-PAGE) followed by immunoblotting with anti-apoLp-III antibodies . This approach enables separation based on both charge (resulting from post-translational modifications) and molecular weight.
The profile of apoLp-III forms changes over time in different tissues (hemolymph, hemocytes, and fat body) following immunization with various pathogens, including Gram-negative bacteria (E. coli), Gram-positive bacteria (Micrococcus luteus), yeast (Candida albicans), and filamentous fungi (Fusarium oxysporum) .
For detailed characterization:
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Mass spectrometry can identify specific modifications
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Immunological approaches using modification-specific antibodies can detect phosphorylation, glycosylation, or other post-translational modifications
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Functional assays can determine whether different forms exhibit varied lipid-binding or immune-activating properties
What purification strategies yield the highest purity recombinant apoLp-III?
For high-purity recombinant apoLp-III, a multi-step purification strategy is recommended:
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Initial capture: Metal affinity chromatography utilizing the His-tag incorporated into recombinant constructs achieves primary purification .
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Secondary purification: Size exclusion chromatography separates the target protein from contaminants of different molecular sizes.
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Final polishing: Ion exchange chromatography removes remaining impurities based on charge differences.
Quality assessment should include:
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SDS-PAGE with both Coomassie blue and silver staining to detect trace contaminants
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Western blotting with anti-apoLp-III antibodies to confirm identity
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Spectroscopic methods to assess protein folding
For applications requiring removal of the His-tag, specific proteases (such as Factor Xa) can be used if the expression vector includes an appropriate cleavage site between the tag and the protein sequence.
How does recombinant apoLp-III compare to native protein in immune stimulation?
Studies have demonstrated that recombinant G. mellonella apoLp-III retains the immune-stimulating capacity of the native protein . When injected into G. mellonella larvae, both recombinant and native forms induce significantly increased antibacterial activities against E. coli and enhanced lysozyme-like activities in hemolymph collected 24 hours post-injection .
Interestingly, Northern blot analysis reveals that neither recombinant nor native apoLp-III alters the transcription rate of endogenous apoLp-III mRNA compared to untreated larvae . This suggests that the immune-stimulating effect is mediated directly by the protein rather than by inducing endogenous apoLp-III production.
The functional equivalence of recombinant and native proteins indicates that:
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The core protein structure, rather than specific modifications, is responsible for immune-stimulating properties
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Bacterial expression systems are suitable for producing functionally active protein
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The addition of expression vector-derived sequences (His-tags, etc.) does not impair function
What is the relationship between Apolipophorin-3 levels and pathogen virulence?
Research has established a clear correlation between apoLp-III levels in G. mellonella hemolymph and pathogen virulence. Pathogens with different virulence levels induce distinct patterns of apoLp-III expression:
| Pathogen | Virulence Status | ApoLp-III Response | Time Course |
|---|---|---|---|
| C. albicans (wild-type) | Virulent | High induction | Peaks at 8-12h post-infection |
| C. albicans (kex2Δ mutant) | Avirulent | Failed induction | No significant increase |
| C. albicans (och1Δ mutant) | Attenuated | Failed induction | No significant increase |
| C. albicans (och1Δ+OCH1) | Restored virulence | Restored induction | Similar to wild-type |
| S. brasiliensis | Highly virulent | Highest induction | Peaks at 4h post-inoculation |
| S. schenckii | Moderately virulent | Moderate induction | Peaks at 4h post-inoculation |
| S. globosa | Least virulent | Lowest induction | Minimal increase at all timepoints |
This correlation makes apoLp-III levels a potential biomarker for pathogen virulence in the G. mellonella model . When comparing Sporothrix species, S. brasiliensis (the most virulent) stimulated the highest levels of apoLp-III, while S. globosa (the least virulent) induced the lowest levels .
How can recombinant apoLp-III be used to study insect immune responses?
Recombinant apoLp-III offers multiple approaches for studying insect immunity:
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As an immune stimulant: Purified recombinant protein can be injected to study apoLp-III-mediated immune activation without confounding factors from pathogen infection .
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For generating detection tools: It serves as an antigen for producing specific antibodies that enable immunodetection systems like ELISA or Western blotting to track apoLp-III dynamics during immune responses .
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For pathway mapping: Using labeled recombinant apoLp-III in binding studies can identify molecular partners in immune pathways.
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In structure-function analysis: Modified versions (through site-directed mutagenesis) help identify domains critical for immune function.
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As a quantitative standard: Purified recombinant protein provides calibration standards for quantitative assays measuring endogenous apoLp-III levels .
What are reliable methods for quantifying Apolipophorin-3 in insect hemolymph?
Several methods are available for apoLp-III quantification, each with specific advantages:
| Method | Sensitivity | Specificity | Throughput | Key Applications |
|---|---|---|---|---|
| ELISA | High | High | High | Quantifying total apoLp-III in hemolymph samples |
| Western blotting | Medium | High | Low | Distinguishing apoLp-III forms by size |
| 2D-electrophoresis/immunoblotting | Medium | Very high | Very low | Separating apoLp-III forms by charge and size |
| Mass spectrometry | Very high | Very high | Low | Identifying specific modifications |
A recently developed ELISA-based method has proven effective for quantifying apoLp-III in the hemolymph of G. mellonella larvae challenged with various pathogenic agents . This approach allows for rapid quantification that correlates with pathogen virulence .
How can ELISA be optimized for Apolipophorin-3 detection?
An optimized ELISA protocol for apoLp-III detection includes:
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Plate preparation: Sensitize clear flat-bottom 96-well plates with serial dilutions of polyclonal anti-apoLp-III antibodies in 50 mM Tris-HCl, 150 mM NaCl, 1% BSA, pH 7.4; incubate for 2 hours at 37°C .
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Blocking: After washing three times with PBS-Tween 20 (0.05%), block with 300 μL of 1% porcine skin gelatin in TBS per well overnight at 37°C .
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Sample application: Apply 100 μL of hemolymph sample per well and incubate at room temperature for 2 hours .
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Detection: After washing, add 100 μL of anti-apoLp-III antibody (1:400 dilution) per well, incubate for 2 hours at room temperature, then add 50 μL of goat anti-rabbit IgG-HRP (1:2,000 dilution) and incubate for 1 hour .
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Development: Use 3,3',5,5' tetramethylbenzidine (TMB) as a substrate for color development .
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Standards: Include a standard curve using purified recombinant apoLp-III at known concentrations.
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Controls: Include both naïve (untouched) larvae and PBS-injected larvae as negative controls.
This method has been validated with hemolymph from larvae infected with E. coli, C. albicans, and various Sporothrix species, demonstrating reliable quantification that correlates with pathogen virulence .
What controls should be included when studying immune effects of recombinant apoLp-III?
A comprehensive experimental design should include the following controls:
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Naïve control: Untouched larvae provide baseline values for apoLp-III levels and immune parameters .
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Injection control: PBS-injected larvae control for injection trauma effects, which themselves can stimulate immune responses .
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Protein specificity control: Unrelated proteins at equivalent concentrations demonstrate effects specific to apoLp-III.
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Dose-response series: Multiple apoLp-III concentrations establish dose-dependent relationships.
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Time-course measurements: Samples collected at multiple timepoints (2h, 4h, 8h, 12h, 24h, 48h) track the temporal dynamics of immune responses .
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Positive controls: Known immune stimulants such as lipopolysaccharide provide comparison standards.
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Purification control: Elution buffer processed through the same purification protocol controls for potential contaminants.
How should time-course experiments be designed to study apoLp-III dynamics?
Optimized time-course experiments should include:
Research has shown that apoLp-III levels increase significantly at early post-inoculation times (8h and 12h) in larvae challenged with E. coli or C. albicans, then decrease to control levels by 24h post-inoculation . This pattern differs somewhat with Sporothrix species, where levels peak earlier (4h post-inoculation) for S. brasiliensis and S. schenckii .
For comprehensive analysis, hemolymph, hemocytes, and fat body samples should be collected at each timepoint to track tissue-specific changes in apoLp-III levels and forms .
What factors affect apoLp-III expression in response to pathogens?
Multiple factors influence apoLp-III dynamics during immune responses:
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Pathogen species and strain: Different pathogens elicit distinct patterns of apoLp-III expression. For example, Sporothrix species induce different levels of apoLp-III corresponding to their virulence .
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Pathogen virulence factors: C. albicans mutants lacking virulence factors (kex2Δ, och1Δ) fail to induce apoLp-III at the levels seen with virulent strains .
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Temporal dynamics: ApoLp-III levels typically increase early after infection (peaking at 4-12h depending on the pathogen) and return to baseline by 24h .
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Physical trauma: Even PBS injection causes a significant increase in apoLp-III levels compared to untouched larvae, indicating sensitivity to physical trauma .
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Tissue compartmentalization: ApoLp-III profile changes occur in the hemolymph, hemocytes, and fat body after immunization, with tissue-specific patterns .
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Pathogen cell wall components: Specific molecular patterns like β-glucans, peptidoglycans, and lipopolysaccharides likely contribute to differential apoLp-III responses.
Understanding these factors is essential for proper experimental design and interpretation of results when studying apoLp-III dynamics in immune responses.
