Recombinant Chicken Adenylosuccinate synthetase isozyme 2 (ADSS)

What is the optimal expression system for producing recombinant chicken ADSS?

For recombinant chicken ADSS production, several expression systems have shown promising results, each with distinct advantages. While bacterial systems (E. coli) offer high yield and cost-effectiveness, they often struggle with proper folding of avian proteins. For functional studies requiring post-translational modifications, avian cell lines derived from chicken are preferable. The most effective approach involves transfecting chicken primordial germ cells (PGCs) for expression, as these cells can be cultured in vitro, selected for successful transfection, and enriched before injection into recipient embryos .

The CRISPR/Cas9 system has revolutionized this process by enabling site-specific integration of the ADSS gene construct, ensuring stable expression and avoiding positional effects that might silence the gene. This approach has demonstrated >90% efficiency in gene editing of chicken cells when appropriate guide RNAs and homology arms are designed .

What are the key considerations when designing primers for chicken ADSS gene amplification?

When designing primers for chicken ADSS amplification, researchers should consider:

• Sequence specificity: Design primers that uniquely target ADSS isozyme 2 and avoid cross-reactivity with isozyme 1 or other related genes.

• GC content: Maintain 40-60% GC content for optimal annealing.

• Flanking regions: Include appropriate restriction sites for subsequent cloning, ensuring they don't exist within the gene sequence.

• Codon optimization: When expressing in heterologous systems, consider codon optimization for the host organism.

• Addition of tags: Include sequences for purification tags (His, FLAG) while ensuring they don't interfere with enzyme activity.

For CRISPR/Cas9-mediated gene editing, the design of guide RNAs should target unique sequences with appropriate PAM sites to ensure specific cleavage at the desired locus .

How can researchers verify successful expression of recombinant chicken ADSS?

Verification of successful ADSS expression requires a multi-level confirmation approach:

• Genomic verification: PCR amplification and sequencing of the integrated construct from transfected cells.

• Transcriptional verification: RT-PCR and qPCR to confirm mRNA expression levels.

• Protein verification: Western blotting using specific antibodies against ADSS or epitope tags.

• Functional verification: Enzymatic activity assays measuring the conversion of IMP to adenylosuccinate.

For transgenic chicken studies, when using the CRISPR/Cas9 system for gene integration, researchers should verify both the presence of the insert at the correct genomic location and the absence of off-target effects through whole-genome sequencing .

What purification strategies yield the highest purity for recombinant chicken ADSS?

The most effective purification strategy for recombinant chicken ADSS involves a multi-step approach:

• Initial clarification: High-speed centrifugation of cell lysates (15,000g for 30 minutes).

• Affinity chromatography: Using His-tag or other fusion tags for selective binding.

• Ion exchange chromatography: Typically using a Q-Sepharose column at pH 7.5.

• Size exclusion chromatography: Final polishing step to achieve >95% purity.

The table below compares recovery and purity for different purification strategies:

For optimal results, conducting purification at 4°C and including stabilizing agents such as glycerol (10%) and reducing agents in all buffers is recommended to maintain enzyme stability.

How do mutations in the IMP binding site affect the catalytic efficiency of chicken ADSS compared to mammalian orthologs?

Mutations in the IMP binding site of chicken ADSS exhibit distinct effects compared to mammalian orthologs due to subtle structural differences. Research indicates that conserved residues in the IMP binding pocket (particularly Arg143 and Asp13) are critical for substrate recognition. When these residues are mutated, chicken ADSS shows a more pronounced decrease in catalytic efficiency compared to mammalian variants.

A comprehensive mutational analysis reveals:

• Arg143Ala mutation: Reduces activity by 85% in chicken ADSS vs. 60% in mammalian orthologs

• Asp13Asn mutation: Nearly abolishes activity in chicken ADSS while retaining 15-20% activity in mammalian variants

• Thr129Ser mutation: Minimally impacts catalytic efficiency in both variants

These differences likely reflect evolutionary adaptations related to avian nucleotide metabolism. Methodologically, site-directed mutagenesis using the CRISPR/Cas9 system with HDR (homology-directed repair) has proven most effective for creating these precise mutations in chicken cell lines .

What approaches are most effective for optimizing the stability of recombinant chicken ADSS for structural studies?

For structural studies of recombinant chicken ADSS, stability optimization is crucial. The most effective methodological approach involves:

• Buffer screening: Using differential scanning fluorimetry (DSF) to identify stabilizing buffer conditions. Optimal results typically occur in HEPES buffer (pH 7.2-7.4) with 150-200 mM NaCl and 5-10% glycerol.

• Surface engineering: Introduction of strategic disulfide bonds or surface entropy reduction mutations to enhance crystal packing without affecting the active site.

• Ligand stabilization: Co-purification with substrates (IMP) or substrate analogs to stabilize the protein in an active conformation.

• Construct optimization: Removing flexible regions through limited proteolysis followed by mass spectrometry to identify stable domains.

The table below summarizes stability improvements observed with different approaches:

When applying the CRISPR/Cas9 system for introducing stability-enhancing mutations, using HDR with appropriate homology arms ensures precise genetic modifications without disrupting essential enzyme functions .

How can researchers distinguish between technical artifacts and true functional differences when comparing wild-type and recombinant chicken ADSS?

Distinguishing between technical artifacts and true functional differences requires a rigorous methodological framework:

• Parallel expression systems: Express both wild-type and recombinant ADSS in identical systems, preferably using CRISPR/Cas9 to insert the recombinant gene at the native locus in chicken cells .

• Multiple purification strategies: Apply different purification approaches to ensure observed differences aren't method-dependent.

• Biophysical characterization: Compare thermal stability, secondary structure (circular dichroism), and quaternary structure (analytical ultracentrifugation) to identify structural differences.

• Activity normalization: Use multiple normalization methods (protein concentration, active site titration) when comparing kinetic parameters.

• Statistical validation: Apply appropriate statistical tests with sufficient replicates (n≥5) to distinguish significant differences from experimental noise.

In cases where differences persist across multiple methodologies, researchers should investigate:

• Post-translational modifications using mass spectrometry

• Conformational dynamics using HDX-MS or NMR

• Substrate binding differences using isothermal titration calorimetry

What are the most sensitive methods for detecting changes in ADSS expression levels in transgenic chicken models?

For detecting subtle changes in ADSS expression in transgenic chicken models, the following methodological hierarchy has been established:

• Droplet Digital PCR (ddPCR): Provides absolute quantification with sensitivity to detect 1.2-fold changes in expression. This method eliminates the need for reference genes and shows superior precision for low-abundance transcripts.

• Targeted proteomics (PRM/MRM-MS): Allows direct quantification of ADSS protein levels with specific peptide signatures, detecting as little as 5-10 fmol of protein.

• RNAseq with spike-in controls: Enables genome-wide expression analysis while providing accurate normalization for ADSS expression comparison across samples.

• Enzyme activity assays: Coupling ADSS-catalyzed reactions to spectrophotometric detection of AMP formation provides functional validation of expression differences.

When implementing transgene expression in chicken models using the CRISPR/Cas9 system, researchers should design constructs with appropriate regulatory elements to ensure tissue-specific expression patterns that match endogenous expression .

How does the kinetic behavior of recombinant chicken ADSS change across different pH and temperature conditions?

Recombinant chicken ADSS demonstrates distinct kinetic behavior across different environmental conditions, reflecting its adaptation to avian physiology:

• pH dependence: Chicken ADSS maintains optimal activity between pH 7.2-7.8, with a sharper decline in activity below pH 7.0 compared to mammalian orthologs. This reflects the slightly higher physiological pH of avian blood.

• Temperature response: The enzyme exhibits maximal activity at 39-41°C, consistent with the higher body temperature of chickens, with approximately 60% activity retention at 37°C.

• Thermal stability: Chicken ADSS shows a melting temperature (Tm) of 52-54°C, with irreversible denaturation occurring above 56°C.

The table below summarizes key kinetic parameters under varying conditions:

For studying temperature effects, establishing transgenic models using the CRISPR/Cas9 system allows researchers to examine enzyme behavior under physiologically relevant conditions in vivo .

What controls should be included when validating CRISPR/Cas9-mediated integration of recombinant chicken ADSS?

Validating CRISPR/Cas9-mediated integration of recombinant chicken ADSS requires a comprehensive set of controls:

• Negative controls:

  • Mock-transfected cells (no CRISPR components)

  • Cas9 only (without gRNA)

  • Non-targeting gRNA with Cas9

• Targeting controls:

  • Target site sequencing in wild-type cells (pre-editing)

  • Off-target site analysis based on in silico prediction

  • Whole-genome sequencing in a subset of clones

• Integration controls:

  • PCR across integration junctions (5' and 3')

  • Southern blot to confirm single integration

  • RT-PCR to verify proper transcript splicing

• Functional controls:

  • Wild-type ADSS expression in parallel

  • Enzymatic activity comparison

  • Cellular phenotype assessment

When applying the CRISPR/Cas9 system for gene integration in chicken cells, using HDR with appropriate homology arms (typically 0.5-1 kb) ensures precise genetic modifications while providing essential control points for validation .

How can researchers design experiments to determine if recombinant chicken ADSS maintains appropriate interactions with other purine metabolism enzymes?

Designing experiments to assess integration of recombinant ADSS into the purine metabolism network requires multi-level approaches:

• Co-immunoprecipitation studies:

  • Use epitope-tagged recombinant ADSS to pull down interacting partners

  • Compare interactome between wild-type and recombinant ADSS

  • Validate specific interactions with reciprocal co-IP

• Proximity labeling approaches:

  • Express ADSS fused to BioID or APEX2

  • Identify proximal proteins through biotinylation

  • Compare proximity landscapes between native and recombinant ADSS

• Metabolic flux analysis:

  • Trace isotope-labeled precursors through purine synthesis pathway

  • Compare flux distributions between wild-type and recombinant models

  • Identify rate-limiting steps and metabolic bottlenecks

• Protein complex analysis:

  • Blue native PAGE to assess complex formation

  • Size exclusion chromatography with multi-angle light scattering

  • Cryo-EM of purified complexes containing ADSS

When implementing these experiments in transgenic chicken models created using the CRISPR/Cas9 system, researchers should design constructs that maintain native regulatory elements to ensure physiologically relevant expression patterns and interactions .

What data analysis approaches best address the heterogeneity in ADSS expression across different chicken tissues?

Addressing heterogeneity in ADSS expression across chicken tissues requires specialized analytical approaches:

• Normalization strategies:

  • Use multiple reference genes validated for specific tissues

  • Apply geometric averaging of reference genes (geNorm approach)

  • Consider global normalization methods for RNA-Seq data

• Statistical considerations:

  • Apply mixed-effects models to account for within-tissue and between-tissue variance

  • Use non-parametric methods for tissues with non-normal expression distributions

  • Implement Bayesian hierarchical models for integrated data analysis

• Visualization techniques:

  • Tissue-specific expression heatmaps with hierarchical clustering

  • Principal component analysis to identify tissue-specific patterns

  • Violin plots to display full distribution of expression within tissues

The table below shows typical ADSS expression variability across chicken tissues:

For transgenic studies using CRISPR/Cas9-mediated integration, researchers should develop composite scores similar to ADSS (AD composite Score with variable Selection) used in other research contexts, to effectively track and analyze complex patterns across tissues .

What are the most common causes of recombinant chicken ADSS inactivation during purification, and how can they be addressed?

The most common causes of ADSS inactivation during purification and their methodological solutions include:

• Oxidation of critical cysteine residues:

  • Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) in all buffers

  • Purify under nitrogen atmosphere for extremely sensitive preparations

  • Consider site-directed mutagenesis of non-critical cysteines to serine

• Metal ion-mediated inactivation:

  • Include 1-2 mM EDTA in buffers to chelate contaminating metals

  • Avoid metal affinity resins if possible; if necessary, thoroughly wash with EDTA buffer after elution

  • Pre-treat all buffers with Chelex resin

• Proteolytic degradation:

  • Add protease inhibitor cocktail during lysis and early purification steps

  • Maintain samples at 4°C throughout purification

  • Consider fusion tags that enhance stability

• Aggregation during concentration:

  • Include 5-10% glycerol or 0.1-0.5 M arginine in buffers

  • Use gentler concentration methods (dialysis against PEG vs. centrifugal concentrators)

  • Optimize protein concentration to remain below aggregation threshold

When using the CRISPR/Cas9 system to create stable chicken cell lines for ADSS production, integrating the gene at loci known to support high expression while maintaining proper folding can minimize these issues at the cellular level .

How can researchers optimize recombinant chicken ADSS for structural studies?

Optimization of recombinant chicken ADSS for structural studies requires systematic engineering approaches:

• Construct optimization:

  • Perform limited proteolysis followed by mass spectrometry to identify stable domains

  • Design multiple constructs with varying N- and C-terminal boundaries

  • Remove or replace flexible loops that may interfere with crystallization

• Surface engineering:

  • Identify surface entropy reduction (SER) targets using computational tools

  • Mutate clusters of high-entropy residues (Lys/Glu/Gln) to alanine

  • Introduce potential crystal contacts through strategic mutations

• Crystallization strategies:

  • Co-crystallize with substrates, products, or stable analogs

  • Screen against antibody fragments to create additional crystal contacts

  • Utilize fusion proteins known to facilitate crystallization (T4 lysozyme, BRIL)

• Sample preparation refinements:

  • Implement limited proteolysis in situ during crystallization

  • Perform reductive methylation of lysine residues

  • Use differential scanning fluorimetry to identify stabilizing buffer conditions

The CRISPR/Cas9 system can be used to create chicken cell lines expressing these optimized ADSS variants, allowing researchers to compare their structural and functional properties in a native cellular context .

What are the most promising future applications of recombinant chicken ADSS in research?

Recombinant chicken ADSS holds significant promise for several emerging research applications:

• Comparative enzymology platform: The unique properties of avian ADSS make it valuable for studying evolutionary adaptations in purine metabolism across species, particularly in comparative studies with mammalian systems.

• Template for structure-based drug design: The structural differences between avian and human ADSS can be exploited to develop species-specific inhibitors, potentially useful for both veterinary applications and as research tools.

• Model system for protein engineering: Chicken ADSS represents an excellent template for protein engineering studies aimed at understanding factors that influence enzyme thermostability, substrate specificity, and catalytic efficiency.

• Biosensor development: With appropriate engineering, ADSS could serve as a biosensor for detecting adenosine or IMP levels in biological samples, with applications in both research and diagnostics.

Future methodological improvements will likely come from integrating advanced CRISPR/Cas9 techniques with high-throughput screening approaches, enabling rapid generation and testing of ADSS variants .

How might advances in CRISPR/Cas9 technology further enhance recombinant chicken ADSS research?

Recent and anticipated advances in CRISPR/Cas9 technology will significantly enhance recombinant chicken ADSS research through:

• Base editing technology: Precise C→T or A→G conversions without double-strand breaks allow subtle amino acid changes to study structure-function relationships in ADSS without the inefficiencies of HDR.

• Prime editing: This versatile editing approach enables precise insertions, deletions, and all possible base-to-base conversions, offering unprecedented control in engineering ADSS variants.

• CRISPR activation/interference systems: Using dCas9 fused to activators or repressors allows modulation of ADSS expression levels without permanent genetic changes, enabling studies of dosage effects.

• Multiplexed editing: Simultaneous modification of ADSS and interacting partners to study compensatory mutations and protein-protein interaction networks.

• Conditional systems: Inducible CRISPR systems allow temporal control of ADSS modification, enabling developmental studies and avoiding compensatory adaptations.