Recombinant Apis mellifera Phospholipase A2

What is Recombinant Apis mellifera Phospholipase A2?

Recombinant Apis mellifera Phospholipase A2 (BVPLA2) is a lipolytic enzyme originally found in honeybee venom that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. When produced recombinantly, this enzyme maintains the structural and functional properties of the native protein but can be generated in laboratory settings without bee venom extraction. BVPLA2 is also known by several synonyms including phosphatidylcholine 2-acylhydrolase and allergen Api m 1 .

The enzyme consists of a full-length mature protein with 134 amino acids (positions 34-167) and has significant biochemical importance due to its toxicity to insects and other invaders, causing hemolysis on erythrocytes . The recombinant version typically includes an affinity tag (commonly His-tag) to facilitate purification .

What is the molecular structure and physical properties of BVPLA2?

Recombinant BVPLA2 has the following key structural and physical characteristics:

• Molecular weight: Approximately 16-19.3 kDa (with variations depending on tags and glycosylation)

• Isoelectric point: Between 5.9 (for Apis mellifera lamarckii) and 7.01 (for some other Apis mellifera subspecies)

• Amino acid sequence: The enzyme contains the conserved sequence: IIYPGTLWCGHGNKSSGPNELGRFKHTDACCRTHDMCPDVMSAGESKHGLTNTASHTRLSCDCDDKFYDCLKNSADTISSYFVGKMYFNLIDTKCYKLEHPVTGCGERTEGRCLHYTVDKSKPKVYQWFDLRKY

• Structure: BVPLA2 is a monomeric protein with a 3D structure that can be modeled based on known PLA2 structures (such as d1poca)

When expressed in appropriate systems, the recombinant protein may exhibit post-translational modifications similar to the native protein, such as glycosylation observed in Tn cell expression systems .

How does BVPLA2 compare genetically to PLA2 from other bee species?

The genetic sequence of BVPLA2 shows high conservation among bee species but with some notable differences:

• The nucleotide sequence for Apis mellifera subspecies PLA2 is approximately 507 bp in length, encoding 167 amino acids

• Sequence homology:

  • 99% homology with Apis mellifera ligustica, with only one nucleotide difference at position 39

  • 96% homology with Apis cerana cerana, with 6 nucleotide differences

• The predicted amino acid sequence of Apis mellifera subspecies PLA2 is 99% identical to that of Apis mellifera ligustica

• The isoelectric point (pI) of some Apis mellifera subspecies PLA2 (7.01) is lower than those of Apis mellifera ligustica and Apis cerana cerana

These subtle genetic differences may contribute to species-specific enzymatic properties and may be relevant for researchers studying evolutionary aspects or seeking specific activity profiles.

What expression systems are suitable for producing recombinant BVPLA2?

Different expression systems offer varying advantages for BVPLA2 production:

For E. coli expression, recombinant BVPLA2 is typically produced with an N-terminal His-tag to facilitate purification . When expressed in Tn cells, the protein appears as a double band with molecular weights of 16 and 18 kDa due to glycosylation, which aligns with the pattern observed in native AmPLA2 .

The choice of expression system should be guided by the research requirements. If post-translational modifications (particularly glycosylation) are important for the study, insect cell expression is preferable despite lower yields. For biochemical studies where high yields are prioritized, E. coli expression may be more suitable.

What are the optimal purification methods for recombinant BVPLA2?

Effective purification of BVPLA2 can be achieved through various chromatographic techniques:

• For His-tagged recombinant BVPLA2 from E. coli:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices

  • Follow with size exclusion chromatography if higher purity is required

• For native-like purification (as demonstrated with Apis mellifera lamarckii):

  • Ion exchange chromatography using DEAE-cellulose column (equilibrated with 0.02 M Tris/HCl buffer, pH 7.8)

  • Elution with NaCl gradient (0-1 M), where BVPLA2 typically elutes in two peaks:

    • Major peak at 0.05 M NaCl

    • Minor peak at 0.1 M NaCl

  • Further purification of the major peak using size exclusion chromatography (Sephacryl S-300)

How can researchers assess BVPLA2 purity and activity after purification?

Multiple analytical methods can be employed to assess purity and activity:

Purity Assessment:

• SDS-PAGE: The purified protein should appear as a distinct band at approximately 16-19 kDa, with purity >85%

• Western blot analysis: Using anti-PLA2 antibodies to confirm identity

• Size exclusion chromatography: To confirm monomeric state and absence of aggregates

Activity Assessment:

• Phosphatidylcholine hydrolysis assay: The standard assay mixture contains:

  • 7.5 μmol Tris/HCl, pH 7.9

  • Phosphatidylcholine (15 μmol)

  • Triton X-100 (18 μmol)

  • CaCl₂ (5 μmol)

  • Phenol red (80 μmol)

  • Monitor decrease in absorbance at 558 nm after 1-hour incubation at 37°C

• Egg yolk hydrolysis assay: Has been used to measure a PLA2 activity of approximately 3.16 μmol/(min·mg) for recombinant AccPLA2

One unit of PLA2 activity is defined as the amount of enzyme needed to hydrolyze 1 μmol phosphatidylcholine per hour at 37°C .

What are the optimal conditions for BVPLA2 enzymatic activity?

The optimal conditions for BVPLA2 activity have been characterized as:

• Optimal pH: 8.0

• Optimal temperature: 45°C

• Metal ion requirements: Ca²⁺ is typically essential for activity

• Storage stability:

  • Liquid form: Stable for up to 6 months at -20°C/-80°C

  • Lyophilized form: Stable for up to 12 months at -20°C/-80°C

  • Working aliquots stable at 4°C for up to one week

Researchers should note that repeated freeze-thaw cycles significantly reduce enzyme activity and should be avoided .

Which factors enhance or inhibit BVPLA2 catalytic activity?

BVPLA2 activity is influenced by various factors:

Activators:

• Metal ions: Cu²⁺, Ni²⁺, Fe²⁺, Ca²⁺, and Co²⁺ exhibit complete activating effects

• Ca²⁺ is particularly important for the catalytic mechanism

Inhibitors:

• Metal ions: Zn²⁺ and Mn²⁺ have inhibitory effects

• Chemical agents: NaN₃, PMSF (phenylmethylsulfonyl fluoride), N-Methylmaleimide, and EDTA demonstrate inhibitory effects

Understanding these activation and inhibition patterns is crucial for designing experiments that accurately measure BVPLA2 activity and for studies aiming to modulate its function.

What physiological and biochemical activities does BVPLA2 exhibit beyond its enzymatic function?

Beyond its primary phospholipase activity, BVPLA2 demonstrates several biologically significant properties:

Anti-platelet aggregation activity:

• 8 μg of purified PLA2 from Apis mellifera lamarckii venom can prevent platelet aggregation by reducing ADP-stimulated platelets aggregation by 60%

Anti-coagulation activity:

• BVPLA2 prolongs prothrombin time (PT)

• 6 μg of the enzyme can completely prevent coagulation in experimental settings

Allergenic properties:

• BVPLA2 is known as allergen Api m 1, making it significant in immunological research

These additional activities make BVPLA2 potentially valuable for research in thrombosis, hemostasis, and immunology.

How can recombinant BVPLA2 be used in anticoagulation and anti-platelet research?

Researchers investigating anticoagulation and anti-platelet mechanisms can utilize BVPLA2 through the following methodological approaches:

• Platelet aggregation inhibition studies:

  • Prepare platelet-rich plasma (PRP) from fresh blood

  • Add varying concentrations of purified BVPLA2 (starting with 2-10 μg)

  • Induce aggregation with ADP or other agonists

  • Measure aggregation percentage reduction compared to control

• Coagulation pathway analysis:

  • For extrinsic pathway: Perform prothrombin time (PT) assays with different BVPLA2 concentrations

  • For intrinsic pathway: Consider activated partial thromboplastin time (aPTT) assays

  • Determine minimum concentration for complete anticoagulation (approximately 6 μg in experimental models)

• Mechanism elucidation:

  • Investigate whether anticoagulant effects are directly linked to phospholipid hydrolysis or involve other mechanisms

  • Compare wild-type BVPLA2 with catalytically inactive mutants to separate enzymatic from non-enzymatic effects

These methodologies can contribute to developing new anticoagulant therapeutics or understanding mechanisms of hemostasis.

What molecular cloning strategies are recommended for BVPLA2 genetic engineering?

Based on successful approaches documented in the literature:

• RNA extraction and cDNA synthesis:

  • Extract total RNA from honey bee venom glands

  • Immediate processing is crucial - grind stingers in liquid nitrogen

  • Use standard cDNA synthesis protocols with oligo(dT) primers

• PCR amplification:

  • Design primers based on conserved regions of published PLA2 sequences

  • Expected product size: approximately 507 bp

  • Optimize PCR conditions for high-fidelity amplification

• Cloning strategy:

  • Initial cloning into a T/A cloning vector (e.g., pGEM T-easy)

  • Subcloning into expression vectors using appropriate restriction sites (e.g., BamHI and XhoI)

  • Verification by restriction digestion and sequencing

• Expression vector considerations:

  • For E. coli: pET vectors with N-terminal His-tag

  • For insect cells: Bacmid-based baculovirus expression systems

  • Include appropriate secretion signals if extracellular expression is desired

Researchers should verify sequence integrity before expression, as even single nucleotide changes can potentially affect enzyme properties.

How can site-directed mutagenesis be used to study or enhance BVPLA2 properties?

Site-directed mutagenesis offers powerful opportunities to study structure-function relationships and enhance BVPLA2 properties:

• Key residues for targeted mutations:

  • Catalytic dyad/triad residues to study mechanism

  • Metal binding sites (particularly Ca²⁺ binding residues)

  • Surface residues to modify stability or reduce immunogenicity

  • Interface residues that might influence oligomerization

• Mutation strategies for specific outcomes:

  • Enhance thermal stability: Target surface residues for introducing stabilizing interactions

  • Modify substrate specificity: Alter residues in the substrate-binding pocket

  • Reduce immunogenicity: Modify known epitopes while preserving catalytic function

  • Enhance expression: Address potential codon usage issues or problematic sequences

• Analytical methods to assess mutant properties:

  • Thermal shift assays to quantify stability changes

  • Comparative kinetic analyses (Km, kcat, kcat/Km) against various substrates

  • Structural studies (X-ray crystallography or NMR) for detailed insights

Combining bioinformatics analysis with structural modeling can help identify the most promising residues for mutation to achieve specific research objectives.

What are common challenges in working with recombinant BVPLA2 and how can they be addressed?

Researchers commonly encounter several challenges when working with BVPLA2:

• Expression issues:

  • Problem: Low yield or insoluble protein in E. coli

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Use specialized strains (BL21(DE3)pLysS, Rosetta)

    • Consider fusion partners (MBP, SUMO) to enhance solubility

    • Switch to insect cell expression system for better folding

• Protein stability concerns:

  • Problem: Activity loss during storage/handling

  • Solutions:

    • Store in 5-50% glycerol buffers

    • Aliquot to avoid freeze-thaw cycles

    • For long-term storage, lyophilize with 6% trehalose as a stabilizer

    • Include Ca²⁺ in storage buffers when appropriate

• Purification difficulties:

  • Problem: Co-purifying contaminants

  • Solutions:

    • For His-tagged protein, include imidazole wash steps to reduce non-specific binding

    • Consider multi-step purification (ion exchange followed by size exclusion)

    • If glycosylation heterogeneity is an issue, consider deglycosylation enzymes or E. coli expression

• Activity measurement inconsistencies:

  • Problem: Variable activity results

  • Solutions:

    • Standardize substrate preparation (especially for phospholipid substrates)

    • Ensure consistent Ca²⁺ concentrations

    • Control temperature precisely during assays

    • Include appropriate controls in each experiment

How can researchers accurately measure BVPLA2 activity in complex biological samples?

Accurate activity measurement in complex samples requires specialized approaches:

• Activity assay optimization:

  • Use fluorescent or colorimetric substrates for increased sensitivity

  • Include negative controls with known PLA2 inhibitors (e.g., EDTA)

  • Create a standard curve with commercial PLA2 of known activity

• Specific protocol for complex matrices:

  • Sample preparation: Centrifuge samples at high speed to remove particulates

  • Pre-incubate samples with non-ionic detergents (e.g., Triton X-100) to disperse lipids

  • Consider sample pre-fractionation to reduce interference

• Recommended assay for complex biological samples:

  • Reaction mixture: 7.5 μmol Tris/HCl (pH 7.9), phosphatidylcholine (15 μmol), Triton X-100 (18 μmol), CaCl₂ (5 μmol), phenol red (80 μmol)

  • Record initial optical density at 558 nm as blank

  • Add sample and incubate for 1 hour at 37°C

  • Measure the decrease in absorbance at 558 nm

  • Calculate activity using a standard curve

• Confirmatory approaches:

  • Western blot to verify BVPLA2 presence alongside activity measurements

  • Specific inhibition tests to confirm that measured activity is due to BVPLA2 rather than other phospholipases

What controls should be included in experiments utilizing recombinant BVPLA2?

Proper experimental design for BVPLA2 studies should include these essential controls:

• Enzymatic activity controls:

  • Positive control: Commercial PLA2 with established activity

  • Negative control: Heat-inactivated BVPLA2 (95°C for 10 minutes)

  • Inhibition control: BVPLA2 treated with known inhibitors (EDTA, PMSF)

  • Ca²⁺-free control: Reaction mixture without calcium to demonstrate dependence

• Expression and purification controls:

  • Empty vector control: Cells transformed with expression vector lacking the BVPLA2 gene

  • Mock purification: Apply purification protocol to control culture to identify non-specific contaminants

  • Western blot with anti-PLA2 antibodies to confirm identity

• Application-specific controls:

  • For anticoagulation studies: Include standard anticoagulants (heparin, warfarin) as reference

  • For platelet aggregation: Include known inhibitors (aspirin, clopidogrel) for comparison

  • For immunological studies: Include other bee venom components to test specificity

• Sample integrity controls:

  • Fresh vs. stored enzyme comparison to assess stability

  • Different buffer compositions to optimize reaction conditions

  • Substrate-only controls to account for spontaneous hydrolysis

Implementing these controls will enhance data reliability and facilitate troubleshooting if unexpected results occur.

What emerging applications of recombinant BVPLA2 show the most promise?

Based on current research findings, recombinant BVPLA2 shows significant potential in several research areas:

• The anticoagulant and anti-platelet properties suggest applications in cardiovascular research and potential therapeutic development for thrombotic disorders .

• The well-characterized structural and functional properties make BVPLA2 a valuable model system for studying enzyme mechanisms, protein engineering, and structure-function relationships .

• As an important allergen (Api m 1), recombinant BVPLA2 offers opportunities for immunological research, allergy diagnostics, and potential desensitization therapies .