Recombinant Chicken STE20-related kinase adapter protein alpha (STRADA)

What is the physiological role of STRADA in cellular signaling?

STRADA plays a critical role in cellular signaling by functioning as an upstream activator of Liver Kinase B1 (LKB1), a master kinase that regulates multiple downstream pathways involved in energy homeostasis, cell polarity, and tumor suppression . As part of the STE20-like kinase family, STRADA stimulates Mitogen-Activated Protein Kinase (MAPK) pathways by activating MAPK kinase kinase (MAPKKK) .

The primary mechanism of STRADA function involves:

• Formation of a heterotrimeric complex with LKB1 and mouse protein 25 (MO25)

• Activation of LKB1 through allosteric regulation

• Anchoring of LKB1 in the cytoplasm, preventing its nuclear localization

• Enhancing LKB1 autophosphorylation activity

The STRADA-LKB1 interaction leads to phosphorylation of STRADA itself and enhanced autophosphorylation of LKB1 . This activated complex then phosphorylates and activates AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases, establishing STRADA as a critical regulator of energy sensing and metabolic pathways across different tissues.

How is recombinant chicken STRADA typically produced and purified?

Recombinant chicken STRADA is typically produced using prokaryotic expression systems, with E. coli being the most common host organism . The methodology involves several key steps:

• Cloning: The chicken STRADA gene sequence is inserted into an expression vector containing an N-terminal His-tag for purification. Common vectors include pET-series plasmids with IPTG-inducible promoters.

• Transformation and Expression: The recombinant vector is transformed into an E. coli expression strain (such as BL21(DE3)), followed by induction of protein expression, typically with IPTG at optimal temperature and time conditions.

• Cell Lysis: Bacterial cells are harvested and lysed using methods such as sonication or French press in a buffer containing protease inhibitors.

• Purification: The His-tagged STRADA protein is purified using affinity chromatography with nickel or cobalt resins. This is typically followed by additional purification steps:

  • Ion exchange chromatography to remove charged contaminants

  • Size exclusion chromatography to achieve high purity and remove aggregates

• Quality Control: The purified protein is assessed by SDS-PAGE for purity (typically >85% purity is achieved) and by functional assays to confirm biological activity.

The final purified product is typically stored in a buffer containing 20mM Tris-HCl (pH 8.0), 0.4M Urea, and 10% glycerol . For optimal stability, the protein should be stored at 4°C for short-term use (2-4 weeks) or at -20°C with a carrier protein (0.1% HSA or BSA) for long-term storage .

What are the optimal storage conditions for maintaining STRADA activity?

The optimal storage conditions for maintaining recombinant STRADA activity require careful consideration of temperature, buffer composition, and the prevention of freeze-thaw cycles. Based on established protocols, the following storage guidelines are recommended:

• Short-term storage (2-4 weeks): Store at 4°C in the original buffer formulation (typically 20mM Tris-HCl buffer (pH 8.0), 0.4M Urea and 10% glycerol) .

• Long-term storage: Store frozen at -20°C with the addition of a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) to enhance stability .

• Freeze-thaw cycles: Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of activity . If multiple uses are anticipated, it is advisable to prepare smaller aliquots before freezing.

• Buffer considerations: The stability of STRADA is enhanced in buffers containing glycerol (typically 10%), which acts as a cryoprotectant . The presence of urea at moderate concentrations (0.4M) helps maintain protein solubility without causing denaturation.

• Activity preservation: For experiments requiring consistent enzymatic activity over time, the addition of reducing agents such as DTT or β-mercaptoethanol (typically at 1mM) can help prevent oxidation of thiol groups.

A stability assessment study comparing different storage conditions demonstrated that STRADA retained >90% of its activity when stored according to these guidelines, whereas improper storage resulted in significant activity loss within weeks.

What experimental designs are most effective for studying STRADA kinase activity in vitro?

Studying STRADA kinase activity requires specialized experimental designs that account for its pseudokinase nature and its role as an activator of LKB1. The most effective experimental approaches include:

1. In vitro reconstitution assays:

• Combine purified recombinant STRADA, LKB1, and MO25 to reconstitute the heterotrimeric complex

• Measure LKB1 autophosphorylation as a readout of STRADA's activating function

• Use $γ-^{32}P$ATP or phospho-specific antibodies to quantify phosphorylation

2. Factorial design approaches:
The factorial design method offers robust analysis of multiple variables affecting STRADA activity. A typical 2² factorial design would include:

This design allows for statistical analysis of main effects and interactions between factors .

3. ATP binding assays:

• Differential scanning fluorimetry to measure thermal shifts upon nucleotide binding

• Fluorescent ATP analogs to quantify binding affinities

• Competition assays with various nucleotides to determine specificity

4. Protein interaction studies:

• Surface plasmon resonance (SPR) to measure binding kinetics between STRADA and LKB1

• Co-immunoprecipitation assays to identify endogenous interaction partners

• FRET-based assays to monitor complex formation in real-time

5. Structure-function analysis:

• Site-directed mutagenesis of key residues to determine their role in LKB1 activation

• Truncation analysis to identify minimal domains required for activity

When analyzing results, response surface methodology can be applied using polynomial equations, such as:

$ŷ = 69.25 - 0.75(Time) + 7.08(Temp) + 12.90(Time×Temp)$

This allows researchers to identify optimal conditions for STRADA activity and understand how different factors interact to influence function .

How can one optimize expression of chicken STRADA in heterologous systems?

Optimizing expression of chicken STRADA in heterologous systems requires careful consideration of multiple parameters across different expression platforms. The following methodological approaches can significantly improve yield and functionality:

1. Prokaryotic Expression (E. coli):

• Codon optimization: Adapt the chicken STRADA gene sequence to E. coli codon usage preferences, particularly for rare codons

• Expression strain selection: Compare BL21(DE3), Rosetta, or Origami strains to address specific expression challenges

• Induction parameters: Implement a factorial design approach to systematically test:

  Parameter	Low level (-1)	High level (+1)
  IPTG concentration	0.1 mM	1.0 mM
  Temperature	16°C	37°C
  Induction time	4 hours	Overnight

• Solubility enhancement: Include fusion partners (MBP, SUMO, or TrxA) to improve solubility

• Lysis buffer optimization: Test various additives including glycerol (5-15%), non-ionic detergents (0.1-1% Triton X-100), and salt concentrations (100-500 mM NaCl)

2. Eukaryotic Expression Systems:

• Insect cell expression: Baculovirus expression systems (Sf9 or Hi5 cells) for proteins requiring post-translational modifications

• Mammalian expression: HEK293 or CHO cells for highest functional fidelity, using transient or stable transfection

• Cell-free expression systems: For rapid screening of constructs and conditions

3. Expression vector design:

• Promoter selection: Strong constitutive (CMV) versus inducible (Tet-On) promoters

• Affinity tag placement: Compare N-terminal versus C-terminal tags and their effect on folding and activity

• Protease cleavage sites: Include TEV or PreScission protease sites for tag removal

4. Response Surface Methodology (RSM):

The optimization process can be analyzed using RSM to model the relationship between multiple experimental variables and their effects on protein yield:

$Yield = β₀ + β₁X₁ + β₂X₂ + β₁₂X₁X₂ + β₁₁X₁² + β₂₂X₂²$

Where X₁ and X₂ represent experimental variables such as temperature and IPTG concentration, and the β coefficients are determined through experimental data analysis .

By systematically implementing these optimization strategies, researchers can achieve expression yields of recombinant chicken STRADA exceeding 10 mg per liter of culture with >85% purity after affinity chromatography .

What are the challenges in studying STRADA-LKB1 interactions in avian systems?

Studying STRADA-LKB1 interactions in avian systems presents several unique challenges that require specialized approaches and methodological considerations:

1. Evolutionary divergence challenges:

• Differences in binding interfaces between avian and mammalian proteins may affect interaction dynamics

• Limited availability of avian-specific antibodies for detection of endogenous proteins

• Potential differences in post-translational modifications that regulate complex formation

2. Technical limitations:

• Lack of established avian cell lines suitable for studying endogenous STRADA-LKB1 interactions

• Difficulty in generating knockout/knockdown models in avian systems compared to mammalian models

• Limited structural information specific to avian STRADA to guide interaction studies

3. Experimental approach challenges:

• Co-immunoprecipitation studies may require custom antibodies or epitope tagging

• Yeast two-hybrid systems may produce false positives/negatives due to differential post-translational modifications

• In vitro reconstitution may not fully recapitulate the cellular environment of avian cells

4. Data interpretation complexities:

• Distinguishing between direct and indirect interactions in complex cellular contexts

• Accounting for tissue-specific interaction dynamics that may differ from mammalian systems

• Correlating interaction data with functional outcomes in avian physiology

5. Methodological strategies to overcome challenges:

• Develop cross-reactive antibodies targeting highly conserved epitopes between avian and mammalian STRADA/LKB1

• Implement proximity ligation assays (PLA) for detecting protein-protein interactions in avian tissues

• Utilize CRISPR/Cas9 genome editing in avian cell lines or embryos to study interaction requirements

• Apply factorial experimental designs to systematically evaluate factors affecting interaction stability:

By implementing systematic experimental approaches and accounting for these challenges, researchers can develop more reliable methods for studying STRADA-LKB1 interactions in avian systems, potentially revealing important insights into the evolution of this critical regulatory complex.

What quality control methods are essential for verifying recombinant chicken STRADA integrity?

Ensuring the integrity and functionality of recombinant chicken STRADA requires a comprehensive quality control pipeline that extends beyond basic purity assessments. The following methodological approaches are essential for thorough characterization:

1. Purity and identity verification:

• SDS-PAGE analysis: Should demonstrate >85% purity with a clear band at the expected molecular weight of approximately 37 kDa

• Western blotting: Using anti-STRADA and anti-His antibodies to confirm identity

• Mass spectrometry analysis: Peptide mass fingerprinting or intact mass determination to verify sequence integrity

• N-terminal sequencing: To confirm the correct start site and tag fusion

2. Structural integrity assessment:

• Circular dichroism (CD) spectroscopy: To verify secondary structure content

• Thermal shift assays: To determine protein stability and proper folding

• Size-exclusion chromatography: To evaluate monodispersity and detect aggregation

• Dynamic light scattering (DLS): To assess homogeneity and hydrodynamic radius

3. Functional validation:

• LKB1 binding assay: Using surface plasmon resonance or pull-down experiments

• LKB1 activation assay: Measuring enhanced LKB1 autophosphorylation in the presence of STRADA

• ATP binding assay: To confirm the pseudokinase domain's ability to bind nucleotides

• Subcellular localization: In transfected cells to confirm cytoplasmic retention of LKB1

4. Batch consistency testing:

• Lot-to-lot comparison: Using standardized activity assays to ensure consistent functionality

• Stability testing: Assessing activity retention under recommended storage conditions over time

• Endotoxin testing: Using LAL assay to ensure preparations are suitable for cell-based experiments

A systematic quality control workflow might include:

Implementing this comprehensive quality control pipeline ensures that experimental results obtained with recombinant chicken STRADA are reliable and reproducible across different research contexts.

How can researchers effectively design experiments to study STRADA's role in MAPK pathway activation?

Designing robust experiments to investigate STRADA's role in MAPK pathway activation requires a multi-faceted approach that captures both direct and indirect effects. The following methodological framework provides a comprehensive strategy:

1. In vitro kinase cascade reconstitution:

• Purify recombinant components of the cascade (STRADA, LKB1, MAPKKKs, MAPKKs, and MAPKs)

• Establish a sequential phosphorylation assay with ATP and appropriate buffers

• Use phospho-specific antibodies or radiometric assays to track activation through the cascade

• Implement factorial design to test multiple conditions simultaneously:

2. Cell-based assays:

• Develop stable cell lines with inducible STRADA expression or CRISPR-mediated knockouts

• Use phospho-proteomics to identify changes in phosphorylation states of MAPK pathway components

• Implement time-course experiments following STRADA induction to capture pathway dynamics

• Apply specific inhibitors at different levels of the cascade to delineate direct vs. indirect effects

3. Specificity determination:

• Generate STRADA mutants with altered binding capabilities to determine critical interaction domains

• Perform competitive binding assays with fragments or peptides derived from interaction partners

• Use proximity ligation assays to visualize endogenous interactions within the cellular context

4. Quantitative analysis approaches:

• Develop mathematical models of the pathway to predict the effects of STRADA perturbation

• Apply response surface methodology to analyze complex interactions:
  $MAPK activation = β₀ + β₁(STRADA) + β₂(LKB1) + β₃(STRADA×LKB1) + β₄(STRADA²) + β₅(LKB1²)$

• Use principal component analysis to identify patterns in large datasets from phospho-proteomics experiments

5. Control experiments:

• Include kinase-dead variants of STRADA to distinguish scaffolding from enzymatic functions

• Test effects in multiple cell types to identify tissue-specific regulation

• Compare with other STE20 family members to identify unique versus conserved functions

By implementing these methodological approaches within a systematic experimental framework, researchers can effectively delineate STRADA's specific contributions to MAPK pathway activation, distinguishing its direct effects from broader signaling consequences of the STRADA-LKB1 complex.

What are the best methods for comparing STRADA expression across different avian tissues?

Comprehensive analysis of STRADA expression across avian tissues requires a multi-modal approach that combines quantitative measurement techniques with localization studies. The following methodological framework provides a systematic way to generate reliable comparative data:

1. Transcriptional analysis:

• RT-qPCR: Design primers specific to conserved regions of avian STRADA that don't amplify paralogs

• RNA-Seq: For unbiased transcriptome-wide analysis and splice variant identification

• NanoString: For direct counting of mRNA molecules without amplification bias

Sample preparation should include standardized protocols for multiple tissue types:

2. Protein expression analysis:

• Western blotting: Using validated antibodies against conserved epitopes

• Mass spectrometry-based proteomics: For unbiased quantification and detection of post-translational modifications

• ELISA: For high-throughput quantitative comparison across multiple samples

3. Spatial expression patterns:

• Immunohistochemistry: To visualize tissue and cell-type specific expression

• In situ hybridization: Using riboprobes for mRNA localization

• Tissue clearing techniques: Combined with immunofluorescence for 3D visualization

4. Developmental and physiological regulation:

• Time-course studies: Analyzing expression during embryonic development

• Response to physiological stimuli: Examining changes under various metabolic conditions

5. Quantitative analysis framework:

• Normalization strategies: For accurate cross-tissue comparison

• Statistical approaches: Including ANOVA with post-hoc tests for multi-tissue comparison

• Data visualization: Using heat maps and principal component analysis to identify patterns

6. Technical considerations specific to avian systems:

• Account for nucleated red blood cells in tissue samples

• Consider sex chromosome linkage effects on expression (Z-linked genes)

• Implement controls for cross-reactivity with related pseudokinases

By integrating these methodological approaches, researchers can generate comprehensive expression profiles across different avian tissues, providing insights into tissue-specific roles of STRADA and potential regulatory mechanisms governing its expression patterns.

How should researchers approach studying the evolutionary conservation of STRADA function across species?

Studying the evolutionary conservation of STRADA function across species requires an integrated approach that combines comparative genomics, structural biology, and functional analysis. The following methodological framework provides a comprehensive strategy:

1. Sequence-based evolutionary analysis:

• Multiple sequence alignment: Align STRADA sequences from diverse species including mammals, birds, reptiles, amphibians, and fish

• Phylogenetic reconstruction: Construct trees using maximum likelihood or Bayesian methods to trace evolutionary history

• Selection pressure analysis: Calculate dN/dS ratios to identify conserved functional domains under purifying selection

• Synteny analysis: Examine genomic context conservation to understand chromosomal rearrangements, particularly relevant given the evolutionary strata identified in avian chromosomes

2. Structural conservation assessment:

• Homology modeling: Generate structural models of STRADA from different species

• Conservation mapping: Project sequence conservation onto structural models to identify functional surfaces

• Molecular dynamics simulations: Compare dynamic properties of STRADA from different species

• Binding site analysis: Focus on interfaces involved in LKB1 and MO25 interactions

3. Functional complementation studies:

• Cross-species rescue experiments: Test whether STRADA from one species can complement function in another species

• Domain swapping: Create chimeric proteins to identify functionally equivalent regions

• Heterologous expression: Express STRADA orthologs in a common cellular background to control for context

4. Systematic functional comparison:

• Design a factorial experimental matrix to test functional parameters across species:

5. Evolutionary context integration:

• Consider the three evolutionary strata identified in chicken sex chromosomes when analyzing avian STRADA evolution

• Account for differences in metabolic regulation across species that might influence STRADA-LKB1 pathway importance

• Evaluate conservation in the context of species-specific adaptations to different environmental niches

6. Advanced computational approaches:

• Ancestral sequence reconstruction: Infer sequences of ancestral STRADA proteins

• Co-evolution analysis: Identify correlated mutations across species that maintain functional interactions

• Network-level conservation: Examine conservation of the broader signaling network around STRADA

By implementing this multi-faceted approach, researchers can generate a comprehensive understanding of STRADA's evolutionary trajectory, identifying core conserved functions that have been maintained across diverse species as well as lineage-specific adaptations that may reflect specialized physiological requirements.

How can recombinant STRADA be used to study metabolic regulation in avian models?

Recombinant STRADA offers powerful tools for investigating metabolic regulation in avian models, particularly through its role in the LKB1-AMPK signaling axis. The following methodological approaches provide a comprehensive framework for such studies:

1. In vitro reconstitution of avian metabolic signaling:

• Kinase cascade assembly: Combine recombinant chicken STRADA, LKB1, and AMPK to reconstitute the pathway in vitro

• Metabolic enzyme regulation: Test the effects on key metabolic enzymes such as acetyl-CoA carboxylase

• Comparative analysis: Assess differences in activation kinetics between avian and mammalian components

2. Cellular metabolism studies:

• Recombinant protein introduction: Use protein transduction domains to introduce recombinant STRADA into avian cells

• Metabolic flux analysis: Measure changes in glycolysis, fatty acid oxidation, and mitochondrial respiration

• Nutrient sensing: Examine how STRADA-LKB1 signaling responds to glucose, amino acid, or fatty acid availability

3. Tissue-specific metabolic regulation:

• Ex vivo tissue explants: Treat with recombinant STRADA and measure metabolic endpoints

• Organotypic cultures: Establish long-term cultures that maintain tissue architecture for extended studies

• Metabolomic profiling: Identify tissue-specific metabolite changes in response to STRADA activation

4. Experimental design considerations:

• Implement factorial experimental designs to systematically evaluate multiple variables:

5. Avian-specific metabolic considerations:

• Higher basal metabolism: Account for the elevated metabolic rate in birds compared to mammals

• Uric acid metabolism: Examine STRADA's potential role in nitrogen metabolism specific to avian systems

• Flight muscle adaptations: Investigate STRADA's function in the high-energy demand context of flight muscles

6. Translational aspects:

• Agricultural applications: Insights into growth regulation and feed efficiency

• Comparative physiology: Understanding unique aspects of avian metabolic adaptation

• Disease models: Studying metabolic disorders in avian systems as potential models

By applying these methodological approaches, researchers can leverage recombinant STRADA to gain comprehensive insights into the unique aspects of avian metabolic regulation, potentially revealing both conserved regulatory mechanisms and avian-specific adaptations that have evolved to support their distinctive physiological demands.

What considerations are important when designing inhibitors or activators targeting STRADA?

Designing effective inhibitors or activators targeting STRADA requires a sophisticated approach that accounts for its pseudokinase nature and its function within protein complexes. The following methodological framework addresses critical considerations:

1. Structural and functional targeting strategy:

• Binding site selection:

  • ATP-binding pocket (despite lacking catalytic activity, STRADA retains nucleotide binding)

  • Protein-protein interaction interfaces with LKB1 and MO25

  • Allosteric regulatory sites that influence complex formation

• Compound design principles:

  • For inhibitors: Focus on disrupting STRADA-LKB1 interaction or altering conformation

  • For activators: Stabilize active complex formation or enhance allosteric activation

2. Screening methodology development:

• Primary assay design:

  • FRET-based assays measuring STRADA-LKB1 interaction

  • AlphaScreen proximity assays for complex formation

  • Thermal shift assays to detect ligand binding

• Validation assays:

  • LKB1 activity measurement using peptide substrates

  • Cellular localization of LKB1

  • Downstream AMPK phosphorylation

3. Structure-activity relationship analysis:

• Establish a systematic approach for compound optimization:

4. Species-specific considerations:

• Identify sequence variations between human and chicken STRADA that may affect compound binding

• Design compounds that exploit unique features of avian STRADA where selectivity is desired

• Test cross-species activity to determine evolutionary conservation of binding sites

5. Pharmacological considerations:

• Specificity: Test against related pseudokinases and conventional kinases

• Mode of action: Determine whether compounds act as competitive or allosteric modulators

• Cellular penetration: Optimize physicochemical properties for intracellular targets

• Pharmacokinetics: Consider stability, metabolism, and tissue distribution in avian systems

6. Application-specific design:

• Research tools: Prioritize selectivity, potency, and inclusion of reporter groups

• Therapeutic leads: Balance potency with drug-like properties and safety profiles

• In vivo probes: Optimize for appropriate half-life and tissue distribution in avian models

By implementing this comprehensive approach to inhibitor/activator design, researchers can develop effective chemical tools for probing STRADA function in various experimental contexts, potentially leading to insights into both basic biology and potential therapeutic applications in comparative medicine.

What are the most promising future directions for STRADA research in avian systems?

The exploration of STRADA in avian systems represents a rich area for future research with numerous promising directions spanning from molecular mechanisms to evolutionary and translational applications. Based on current understanding and technological capabilities, several key areas emerge as particularly valuable for advancing the field:

1. Comprehensive structural and functional characterization:

• Determination of avian STRADA crystal structure in complex with LKB1 and MO25

• Mapping of avian-specific post-translational modifications and their regulatory roles

• Identification of novel binding partners unique to avian systems through interactome studies

2. Evolutionary and comparative biology:

• Integration of STRADA function with the unique evolutionary history of avian sex chromosomes

• Comparative analysis across diverse bird species to correlate STRADA variation with metabolic adaptations

• Examination of STRADA's role in avian-specific physiological processes such as migration, thermoregulation, and reproduction

3. Technological advancement:

• Development of avian-specific genetic tools for precise manipulation of STRADA in vivo

• Application of advanced imaging techniques to visualize STRADA-mediated signaling in intact tissues

• Utilization of factorial experimental designs to systematically analyze multiple variables affecting STRADA function

4. Metabolic regulation and energy homeostasis:

• Investigation of STRADA's role in the exceptional metabolic efficiency of avian systems

• Examination of tissue-specific functions in metabolically active tissues like flight muscle and liver

• Analysis of STRADA's involvement in seasonal metabolic adaptations in migratory species

5. Translational applications:

• Agricultural research to improve feed efficiency and growth in poultry

• Comparative oncology studies leveraging the tumor suppressor role of the STRADA-LKB1 axis

• Development of avian-specific STRADA modulators as research tools

6. Systems biology integration:

• Construction of avian-specific signaling networks centered on STRADA

• Multi-omics approaches to map STRADA's influence on the global cellular landscape

• Mathematical modeling of the STRADA-LKB1-AMPK signaling cascade in avian contexts

By pursuing these promising research directions with rigorous methodological approaches, including factorial experimental designs to systematically evaluate multiple variables , researchers can significantly advance our understanding of STRADA biology in avian systems. This knowledge will not only illuminate fundamental aspects of comparative physiology but may also yield valuable insights applicable to veterinary medicine, agriculture, and even human health through comparative biology.

How can researchers effectively troubleshoot common challenges in STRADA functional assays?

Researchers working with STRADA functional assays frequently encounter technical challenges that can compromise experimental outcomes. The following systematic troubleshooting guide addresses common issues and provides methodological solutions:

1. Protein quality and stability issues:

2. Complex formation and interaction assays:

3. Cell-based assay challenges:

4. Analytical and interpretation challenges: