Recombinant Coturnix coturnix japonica Lysozyme C (LYZ)

What is Lysozyme C and why is the Japanese quail (Coturnix coturnix japonica) version significant for research?

Lysozyme C is an approximately 14 kDa protein belonging to the c-type lysozyme family that plays an important role in innate immune defense. It exhibits muramidase activity that hydrolyzes β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan, causing lysis of susceptible bacteria . The Japanese quail Lysozyme C is significant for research due to its unique structural features and antimicrobial properties that may differ from other avian lysozymes. Avian lysozymes typically show differences in substrate specificity, thermal stability, and antimicrobial spectrum compared to mammalian lysozymes, making them valuable for comparative studies of lysozyme evolution and function, as well as potential applications in antimicrobial research.

How does Japanese quail Lysozyme C compare structurally with other avian lysozymes?

While specific comparative data for Japanese quail Lysozyme C is limited, we can draw inferences from other avian lysozyme studies. Avian c-type lysozymes typically share high sequence identity (70-90%) with conserved catalytic residues. For example, the Lezhi black goat rumen lysozyme shares 70.27% identity with the capra hircus blood lysozyme . Japanese quail Lysozyme C would likely share significant sequence identity with chicken lysozyme (the most well-studied avian lysozyme), but with species-specific variations that could affect its biochemical properties and antimicrobial spectrum. These differences may include variations in surface charge distribution, substrate binding regions, and stabilizing interactions that influence thermal stability and pH tolerance.

What are the standard methods for assessing lysozyme activity and how do they apply to Japanese quail Lysozyme C?

Two primary methods are used for assessing lysozyme activity:

• Turbidimetric assay: This quantitative method measures the decrease in turbidity of a bacterial suspension (typically Micrococcus lysodeikticus) after lysozyme addition. The activity is calculated by measuring absorbance at two time points and comparing with a standard lysozyme solution . The calculation follows:

  Activity (U/mg) = [(SΔT × UT) / (UΔT × ST)] × Standard concentration

  Where:

  • SΔT = Absorbance change of standard

  • UT = Test sample absorbance

  • UΔT = Absorbance change of test sample

  • ST = Standard absorbance

• Agar diffusion assay: This qualitative method evaluates antimicrobial activity by measuring inhibition zones on agar plates seeded with bacteria like Staphylococcus aureus . This method provides visual confirmation of antimicrobial activity and can be used to compare relative potencies of different lysozyme preparations.

Both methods are directly applicable to Japanese quail Lysozyme C and should be employed in parallel for comprehensive activity characterization.

What expression systems are most suitable for producing recombinant Japanese quail Lysozyme C?

Based on established lysozyme research, three primary expression systems are suitable for recombinant Japanese quail Lysozyme C:

• Bacterial expression (E. coli): E. coli systems using vectors like pET32a have been successfully employed for expressing various lysozymes . For Japanese quail Lysozyme C, researchers must consider codon optimization and potential refolding strategies due to inclusion body formation. Four different fusion constructs of yak milk lysozyme in pET32a showed varying activities: rYML1 (921.00 U/mg), rYML2 (512.60 U/mg), rYML3 (412.00 U/mg), and rYML4 (106.00 U/mg) , demonstrating the importance of construct design.

• Yeast expression (P. pastoris): Pichia pastoris with vectors like pPICZαA offers advantages in protein folding and post-translational modifications . For Japanese quail Lysozyme C, methanol induction protocols similar to those used for yak milk lysozyme would be applicable.

• Transgenic chicken expression: This system ensures proper folding and glycosylation profile of expressed proteins . For Japanese quail Lysozyme C, this platform could produce a protein with native-like post-translational modifications.

The choice should be based on research requirements for protein quantity, activity, and post-translational modifications.

What are the critical factors affecting successful expression of Japanese quail Lysozyme C in E. coli?

Several critical factors influence successful E. coli expression of Japanese quail Lysozyme C:

• Vector selection and fusion partners: Different expression vectors and fusion tags significantly impact expression levels and solubility. In yak milk lysozyme studies, four different constructs in pET32a resulted in varying activities, with the highest activity (921.00 U/mg) observed with specific fusion tags .

• Induction conditions: Optimizing IPTG concentration (typically 1 mM) and induction temperature (28°C is often preferred over 37°C to improve protein folding) .

• Expression strain: BL21(DE3) and its derivatives are commonly used for lysozyme expression, but strain optimization can improve yields.

• Codon optimization: Adapting the Japanese quail codons to E. coli preferences can significantly improve expression.

• Protein extraction and potential refolding: Since lysozymes may form inclusion bodies, protocols for cell lysis (ultrasonic disruption) and protein refolding are critical .

A systematic optimization approach testing multiple constructs and conditions is recommended to achieve maximum yield of functional protein.

How can the expression of Japanese quail Lysozyme C in P. pastoris be optimized?

Optimizing P. pastoris expression of Japanese quail Lysozyme C requires attention to:

• Vector design: The pPICZαA vector has been successfully used for lysozyme expression . Key considerations include:

  • Codon optimization for P. pastoris

  • Selection of appropriate secretion signal (α-factor is commonly used)

  • Promoter selection (AOX1 for methanol induction)

• Transformation and clone selection: After linearization with SacI and electroporation into P. pastoris X33, screening multiple Zeocin-resistant colonies is crucial as expression levels can vary significantly between transformants .

• Induction protocol: Standard protocols involve culturing in BMMY medium at 30°C for 96 hours with methanol addition (to 1% final concentration) every 24 hours . This can be optimized for temperature, methanol concentration, and feeding strategy.

• Harvest and initial processing: Centrifugation followed by concentration using vacuum concentration or alternative methods like tangential flow filtration .

The purified lysozyme from P. pastoris expression can achieve high activity levels (e.g., 1,864.24 U/mg for yak milk lysozyme) , making this an attractive system for Japanese quail Lysozyme C production.

What is the optimal purification strategy for recombinant Japanese quail Lysozyme C?

Based on established lysozyme purification protocols, an optimal strategy for Japanese quail Lysozyme C would involve:

• Initial capture: Cation-exchange chromatography using CM-Sepharose FF, exploiting lysozyme's positive charge at neutral pH .

• Intermediate purification: A second cation-exchange step with different elution conditions can remove closely related impurities.

• Polishing step: Gel-filtration chromatography using Sephadex G-75 to achieve final purity .

This three-step approach resembles the protocol used for recombinant human lysozyme, which achieved >90% purity with 75% purification efficiency, yielding approximately 6 mg of purified protein from ten eggs .

For optimal results, buffer conditions should be systematically optimized:

• pH range testing (pH 6-8 for binding, typically)

• Salt gradient optimization (typically NaCl gradients from 0 to 1M)

• Flow rate adjustments for each chromatographic step

The progress of purification should be monitored by SDS-PAGE using 5% concentrating and 15% separating gels with Coomassie Brilliant Blue R-250 staining .

What analytical methods should be used to confirm the identity and purity of purified Japanese quail Lysozyme C?

A comprehensive characterization approach should include:

• Electrophoretic analysis: SDS-PAGE under reducing and non-reducing conditions to assess purity and detect potential dimers or aggregates .

• Western blot analysis: Using anti-lysozyme antibodies or antibodies against fusion tags to confirm identity.

• Mass spectrometry: For accurate molecular weight determination and verification of the amino acid sequence through peptide mapping.

• N-terminal sequencing: To confirm correct processing, especially when using secretion signals in expression systems.

• Enzymatic activity assay: Using the turbidimetric assay with Micrococcus lysodeikticus to confirm functional identity and determine specific activity (Units/mg) .

• Circular dichroism spectroscopy: To assess secondary structure and compare with native lysozyme structures.

These complementary methods provide comprehensive characterization of both structural and functional properties of the purified protein.

How can the stability and storage conditions for Japanese quail Lysozyme C be optimized?

Based on lysozyme stability data, the following approach is recommended:

• pH stability profiling: Determine activity retention across pH 2-11, as recombinant human lysozyme shows stability across this range . Test buffers should include:

  • pH 2-3: Glycine-HCl

  • pH 4-5: Acetate

  • pH 6-8: Phosphate

  • pH 9-11: Glycine-NaOH

• Thermal stability assessment: While human lysozyme exhibits thermal stability at 60°C , Japanese quail Lysozyme C may have different thermal properties that should be systematically determined through:

  • Activity retention after incubation at 40-90°C

  • Differential scanning calorimetry to determine melting temperature

• Lyophilization studies: Testing excipients (sugars, amino acids) that may protect during freeze-drying.

• Solution stability: Evaluate stability in different buffer systems, salt concentrations, and with potential stabilizing additives.

• Long-term storage testing: Monitor activity during storage at 4°C, -20°C, and -80°C over 6-12 months.

Optimal conditions should be determined experimentally, but typical storage recommendations would likely include lyophilization or solution storage at -20°C in phosphate buffer (pH 6.0-7.0) with protective excipients.

What methods are recommended for evaluating the antimicrobial spectrum of Japanese quail Lysozyme C?

A systematic approach to antimicrobial spectrum evaluation should include:

• Quantitative assays:

  • Minimum Inhibitory Concentration (MIC): Using microdilution method with 2-fold serial dilutions of lysozyme against various bacteria

  • Time-kill kinetics: Measuring bacterial reduction over time at different lysozyme concentrations

  • Turbidimetric assays: Spectrophotometric measurement of bacterial lysis using M. lysodeikticus or other relevant organisms

• Qualitative assays:

  • Agar diffusion assay: Using wells in agar plates seeded with test organisms such as S. aureus

  • Radial diffusion assay: Measuring clear zones in agar containing test organisms

• Test organism panel: Should include:

  • Gram-positive bacteria: M. lysodeikticus, S. aureus, B. megaterium, L. monocytogenes, S. epidermidis

  • Gram-negative bacteria: E. coli, P. aeruginosa, Salmonella spp.

  • Fungi: Candida albicans (as some lysozymes show antifungal activity)

• Comparative testing: Side-by-side comparison with human and chicken lysozymes at equivalent concentrations and conditions.

These methods provide comprehensive characterization of antimicrobial properties and enable comparison with other lysozymes.

How does the mechanism of action of Japanese quail Lysozyme C differ against Gram-positive versus Gram-negative bacteria?

While specific data on Japanese quail Lysozyme C is limited, general lysozyme mechanisms suggest:

• Against Gram-positive bacteria:

  • Primary mechanism: Hydrolysis of the peptidoglycan layer through muramidase activity, cleaving β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine

  • Direct access to peptidoglycan due to absence of outer membrane

  • Results in cell wall degradation, osmotic imbalance, and bacterial lysis

• Against Gram-negative bacteria:

  • Limited access to peptidoglycan due to outer membrane protection

  • Alternative mechanisms may include:

    • Non-enzymatic membrane permeabilization through cationic properties

    • Possible synergy with other immune factors that disrupt outer membranes

    • Action on specific regions where peptidoglycan is exposed

• Experimental approaches to elucidate mechanisms:

  • Enzyme activity assays with purified peptidoglycan substrates

  • Membrane permeabilization assays using fluorescent dyes

  • Electron microscopy to visualize cell wall/membrane damage

  • Comparing wild-type and catalytically inactive mutants

Research on dromedary tear lysozyme demonstrated significant bactericidal activity against L. monocytogenes and S. epidermidis while human tear lysozyme lacked activity against these strains , highlighting species-specific variations in antimicrobial mechanisms that may apply to Japanese quail Lysozyme C.

What potential synergistic interactions exist between Japanese quail Lysozyme C and other antimicrobial agents?

Investigation of synergistic interactions should follow these methodological approaches:

• Combination studies with other antimicrobials:

  • Checkerboard assays: Evaluate combinations of Japanese quail Lysozyme C with:

    • Conventional antibiotics (β-lactams, aminoglycosides, etc.)

    • Other antimicrobial peptides (defensins, cathelicidins)

    • Innate immune factors (lactoferrin, complement proteins)

  • Fractional Inhibitory Concentration Index (FICI) calculation to quantify synergy, additivity, or antagonism

• Mechanism-based combinations:

  • Combine with outer membrane-disrupting agents for enhanced activity against Gram-negative bacteria

  • Pair with complement system components, leveraging lysozyme's "complement system-modulating activity"

  • Test with proteases that may generate lysozyme-derived antimicrobial peptides (as observed with human milk lysozyme)

• Formulation studies:

  • Evaluate delivery systems that enhance lysozyme stability and activity

  • Test pH, ionic strength, and temperature effects on combination efficacy

• Resistance development studies:

  • Assess whether combinations prevent or delay resistance development

  • Compare resistance frequencies with single versus combination treatments

These approaches can identify valuable synergistic combinations that expand the antimicrobial spectrum and efficacy of Japanese quail Lysozyme C.

How can site-directed mutagenesis be used to enhance the properties of Japanese quail Lysozyme C?

Site-directed mutagenesis offers powerful approaches to enhance Japanese quail Lysozyme C properties:

• Thermal stability enhancement:

  • Target residues in the hydrophobic core for stabilizing interactions

  • Introduce additional disulfide bonds at strategic positions

  • Replace flexible regions with more rigid structures

  • Introduce charged residues that form stabilizing salt bridges

• Antimicrobial spectrum expansion:

  • Modify surface charge distribution to enhance activity against Gram-negative bacteria

  • Target substrate-binding site residues to alter specificity

  • Create variants with enhanced non-enzymatic membrane disruption activity

• Methodological approach:

  • Generate homology model based on chicken lysozyme structure

  • Identify non-conserved residues between Japanese quail and other avian lysozymes

  • Design mutations based on computational predictions

  • Express and purify variants using consistent protocols

  • Perform comparative analysis of wild-type and mutant properties

• Domain swap studies:

  • Create chimeric proteins with domains from other lysozymes

  • Test N-terminal domain variants, as the N-terminal helix of human milk lysozyme shows potent bactericidal activity against both Gram-positive and Gram-negative bacteria

This rational design approach can yield variants with enhanced stability, broader antimicrobial spectrum, or specialized activities for research applications.

How does glycosylation impact the properties of Japanese quail Lysozyme C?

Investigation of glycosylation effects requires systematic comparison of different glycoforms:

• Glycosylation analysis:

  • Identify potential N-glycosylation sites (Asn-X-Ser/Thr) in the sequence

  • Characterize natural glycosylation patterns in native Japanese quail Lysozyme C

  • Analyze glycan structures using mass spectrometry and lectin binding assays

• Expression system comparison:

  • E. coli: Non-glycosylated control

  • P. pastoris: High-mannose type N-glycosylation

  • Insect cells: Simple, mannose-rich N-glycosylation

  • Transgenic chickens: "Correct glycosylation profile"

• Functional comparison of glycoforms:

  • Thermal stability (differential scanning calorimetry)

  • pH stability profiles

  • Proteolytic resistance

  • Antimicrobial activity spectrum

  • Immunogenicity and allergenicity

• Glycoengineering approaches:

  • Site-directed mutagenesis to add/remove glycosylation sites

  • Enzymatic modification of glycan structures

  • Expression in glycoengineered yeast strains

This research would provide insights into how glycosylation affects Japanese quail Lysozyme C function and stability, informing optimal expression system selection for specific applications.

What experimental approaches can elucidate the evolutionary adaptation of Japanese quail Lysozyme C compared to other avian lysozymes?

Evolutionary adaptation studies would require multi-faceted approaches:

• Phylogenetic analysis:

  • Comprehensive sequence alignment of avian lysozymes

  • Construction of phylogenetic trees to establish evolutionary relationships

  • Identification of positively selected residues through dN/dS analysis

• Structural comparison:

  • Generate homology models of Japanese quail Lysozyme C

  • Compare with crystal structures of other avian lysozymes

  • Analyze structural features associated with functional adaptations

• Functional biochemistry:

  • Compare substrate specificities using different peptidoglycan types

  • Determine kinetic parameters (kcat, Km) under various conditions

  • Assess pH and temperature optima across avian lysozymes

• Microbial challenge studies:

  • Test activity against bacteria from avian versus mammalian hosts

  • Compare effectiveness against environmental versus pathogenic bacteria

  • Evaluate activity against bacteria isolated from Japanese quail habitats

• Ancestral sequence reconstruction:

  • Infer ancestral avian lysozyme sequences

  • Express reconstructed proteins to study evolutionary trajectories

This research could reveal how Japanese quail Lysozyme C has evolved specific adaptations related to the ecological niche and immune defense requirements of Japanese quail, similar to how ruminant lysozymes evolved from immune factors to digestive enzymes .

What controls and standards should be included when characterizing recombinant Japanese quail Lysozyme C?

Proper experimental design requires comprehensive controls:

• Positive controls:

  • Commercial chicken egg white lysozyme as industry standard

  • Recombinant human lysozyme with known activity

  • If available, native Japanese quail lysozyme purified from tissue

• Negative controls:

  • Expression host without lysozyme gene

  • Heat-inactivated lysozyme preparations

  • Buffer-only controls in activity assays

• Activity standards:

  • Calibrated chicken egg white lysozyme for turbidimetric assays

  • Standard curve using commercial lysozyme of known activity

• Specificity controls:

  • Peptidoglycan substrates from different bacterial sources

  • Lysozyme inhibitors to confirm specificity of observed activity

  • Catalytically inactive mutants (E35A or D52A)

• System suitability tests:

  • Reproducibility assessment across multiple batches

  • Stability indicators during storage and handling

  • pH and temperature sensitivity benchmarks

Inclusion of these controls ensures reliable characterization and facilitates comparison with lysozymes from other species.

How should researchers address potential data inconsistencies when comparing Japanese quail Lysozyme C with other lysozymes?

Addressing data inconsistencies requires rigorous methodological approaches:

• Standardization of assay conditions:

  • Consistent substrate preparation (M. lysodeikticus cultivation conditions)

  • Standardized buffer systems and pH for activity measurements

  • Uniform temperature and incubation times

  • Validated activity calculation methods

• Protein quantification:

  • Multiple protein determination methods (Lowry, Bradford, BCA)

  • Amino acid analysis for absolute quantification

  • Accounting for potential interfering substances

• Statistical approaches:

  • Use of replicates (minimum triplicate measurements)

  • Appropriate statistical tests for significance determination

  • Outlier identification and handling

  • Regression analysis for reaction kinetics

• Cross-validation:

  • Multiple complementary activity assays

  • Independent replication of key findings

  • Comparison with literature values where available

• Reporting standards:

  • Detailed methodology documentation

  • Complete data presentation including variation

  • Transparent discussion of limitations and inconsistencies

These approaches minimize variability and enable valid comparisons between Japanese quail Lysozyme C and other lysozymes, similar to the comparative analysis of yak stomach lysozyme and cow stomach lysozyme .

What are the critical factors in designing experiments to investigate the structure-function relationship of Japanese quail Lysozyme C?

Structure-function investigations require systematic experimental design:

• Structural analysis approaches:

  • Homology modeling based on chicken lysozyme (high sequence similarity expected)

  • If possible, X-ray crystallography or NMR spectroscopy

  • Molecular dynamics simulations to identify flexible regions

  • Hydrogen-deuterium exchange mass spectrometry to probe dynamics

• Mutagenesis strategy:

  • Alanine scanning of catalytic site residues and substrate-binding regions

  • Conservative vs. non-conservative substitutions of charged residues

  • Disulfide bond disruption/introduction

  • Domain swapping with other lysozymes

• Functional characterization:

  • Enzyme kinetics with defined substrates

  • Thermal and pH stability profiles

  • Antimicrobial activity against diverse microorganisms

  • Binding studies with cell wall components

• Correlation analysis:

  • Statistical methods to correlate structural features with functional outcomes

  • Principal component analysis of structure-activity relationships

  • Comparison with published structure-function data from other lysozymes

• Validation approaches:

  • Multiple mutations affecting the same structural feature

  • Rescue mutations to restore function

  • Spectroscopic methods (CD, fluorescence) to confirm structural integrity

This comprehensive approach can elucidate the structural basis for the unique properties of Japanese quail Lysozyme C, potentially revealing new insights applicable to protein engineering and antimicrobial development.