Recombinant Xenopus laevis Phytanoyl-CoA hydroxylase-interacting protein-like (phyhipl)

What is PHYHIPL and why study it in Xenopus laevis?

Phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) is a protein that appears to play significant roles in cellular metabolism. Studying PHYHIPL in Xenopus laevis provides unique advantages due to this amphibian's position as a well-established model organism. Xenopus offers evolutionary proximity to higher vertebrates in terms of physiology, gene expression, and organ development, while maintaining experimental advantages such as year-round breeding capability, external development, and transparent embryos . The PHYHIPL gene has been implicated in critical cellular functions including metabolism regulation and potentially neuroprotective effects, making its study in a vertebrate model system particularly valuable .

What are the optimal expression systems for recombinant Xenopus PHYHIPL?

For recombinant expression of Xenopus PHYHIPL, researchers should consider several expression systems based on experimental needs:

• E. coli-based expression: Suitable for basic structural studies, using pET vector systems with BL21(DE3) strains. Optimal induction typically occurs with 0.5-1.0 mM IPTG at 18°C overnight to reduce inclusion body formation.

• Baculovirus expression system: Preferred for functional studies requiring post-translational modifications. This system more accurately replicates the native protein's modifications.

• Cell-free systems: Useful for rapid, small-scale expression of potentially toxic proteins.

• Xenopus oocyte expression: Particularly valuable when studying PHYHIPL in its native cellular context, using microinjection of mRNA.

The choice between these systems should be guided by research objectives, with consideration of protein folding requirements, post-translational modifications, and downstream applications.

How do you validate the expression and identity of recombinant Xenopus PHYHIPL?

Validation of recombinant Xenopus PHYHIPL requires a multi-faceted approach:

• SDS-PAGE and Western blotting: Primary verification using anti-PHYHIPL antibodies or against epitope tags (His, FLAG, etc.)

• Mass spectrometry analysis: For definitive protein identification and post-translational modification mapping

• Functional assays: To verify biological activity based on known metabolic functions of PHYHIPL

• Structural validation: Circular dichroism or thermal shift assays to confirm proper protein folding

Verification should include comparison with native PHYHIPL from Xenopus tissue extracts when possible. Protein identity confirmation via mass spectrometry is particularly crucial when antibody availability is limited.

What are the most effective purification protocols for recombinant Xenopus PHYHIPL?

Purification of recombinant Xenopus PHYHIPL typically employs a sequential multi-step approach:

• Initial capture: Affinity chromatography using His-tag or GST-tag systems (Ni-NTA or glutathione columns)

• Intermediate purification: Ion exchange chromatography (typically anion exchange with Q or DEAE resins)

• Polishing step: Size exclusion chromatography (Superdex 200 or similar)

Key buffer considerations include:

• Maintaining pH 7.0-8.0 throughout purification

• Including reducing agents (1-5 mM DTT or 0.5-2 mM TCEP)

• Adding 10% glycerol for stability

• Using protease inhibitor cocktails during initial extraction

For challenging preparations, detergent screening (with mild non-ionic detergents like 0.03% DDM or 0.1% Triton X-100) may improve solubility while maintaining functionality.

How do you overcome solubility issues with recombinant Xenopus PHYHIPL?

Solubility challenges with recombinant PHYHIPL can be addressed through:

• Expression optimization:

  • Reducing expression temperature to 16-18°C

  • Using specialized E. coli strains (Rosetta, ArcticExpress)

  • Co-expressing with chaperones (GroEL/GroES, trigger factor)

• Buffer optimization:

  • Screening various pH conditions (typically 6.5-8.5)

  • Testing different ionic strengths (150-500 mM NaCl)

  • Adding stabilizing agents (10-20% glycerol, 0.5-2 M urea, 50-200 mM arginine)

• Fusion tag approaches:

  • Using solubility-enhancing tags (MBP, SUMO, TRX)

  • Testing various tag positions (N-terminal vs. C-terminal)

• Refolding strategies:

  • Rapid dilution from denaturant

  • Step-wise dialysis

  • On-column refolding

Empirical screening of these conditions is essential as PHYHIPL solubility characteristics may differ from predictions based on primary sequence.

What methods are most effective for studying PHYHIPL's role in metabolism?

PHYHIPL appears to play important roles in cellular metabolism based on protein-protein interaction networks . Effective methods for studying these functions include:

• Metabolic flux analysis:

  • Isotope labeling (13C-glucose, 13C-glutamine) to track metabolic pathways

  • Seahorse XF analysis for measuring oxygen consumption and extracellular acidification rates

  • NMR-based metabolomics to identify altered metabolites

• Proteomics approaches:

  • Proximity labeling (BioID, APEX) to identify interacting proteins in Xenopus cells

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening with Xenopus cDNA libraries

• Subcellular localization:

  • Fluorescent protein fusions in Xenopus cell lines or embryos

  • Immunofluorescence in tissue sections

  • Subcellular fractionation and Western blotting

• Loss/gain-of-function studies:

  • CRISPR/Cas9 gene editing in Xenopus embryos

  • Morpholino knockdown

  • Overexpression via microinjection

These methodologies should be combined to build a comprehensive understanding of PHYHIPL's metabolic functions.

How can PHYHIPL's potential neuroprotective functions be assessed in Xenopus models?

Based on findings suggesting PHYHIPL may have neuroprotective functions in human studies , several approaches can be used to assess similar functions in Xenopus:

• Neurodevelopmental analysis:

  • PHYHIPL knockdown/knockout followed by neural marker analysis

  • Time-lapse imaging of neural development in PHYHIPL-modified embryos

  • Assessment of axon guidance and synapse formation

• Neuroprotection assays:

  • Exposure of PHYHIPL-overexpressing tadpoles to neurotoxins

  • Analysis of neuronal survival after oxidative stress induction

  • Evaluation of neurite outgrowth in primary neuronal cultures

• Electrophysiological assessment:

  • Patch-clamp recordings from Xenopus neurons with altered PHYHIPL expression

  • Field potential recordings from intact neural circuits

• Behavioral analyses:

  • Vision-guided behavior assays in tadpoles

  • Swimming performance metrics

  • Startle response quantification

These methods leverage the transparency of Xenopus tadpoles and the accessibility of their nervous system for in vivo imaging and manipulation .

How does Xenopus PHYHIPL compare structurally and functionally to its human ortholog?

Comparative analysis of Xenopus and human PHYHIPL provides valuable evolutionary insights:

The structural similarities between Xenopus and human PHYHIPL make the amphibian model valuable for studying functions potentially relevant to human health, particularly regarding neurological conditions and metabolic regulation .

What insights can comparative studies of PHYHIPL across vertebrate models provide?

Comparative studies of PHYHIPL across vertebrate models offer several key insights:

• Evolutionary conservation of function:

  • Analysis of conserved domains and motifs across species

  • Identification of selective pressure on different protein regions

  • Correlation between conservation patterns and known functional sites

• Species-specific adaptations:

  • Identification of unique structural features in Xenopus PHYHIPL

  • Correlation of these features with environmental or physiological adaptations

  • Analysis of expression differences across developmental stages compared to mammals

• Disease relevance:

  • Understanding how variations in PHYHIPL structure relate to disease susceptibility

  • Identification of conserved interacting partners across species

  • Assessment of functional conservation in metabolic and neurological contexts

The amphibian model provides a unique evolutionary perspective, representing a transitional form between aquatic and terrestrial vertebrates, making comparative PHYHIPL studies particularly valuable for understanding the evolution of metabolic regulation systems.

How can recombinant Xenopus PHYHIPL be used to study glioblastoma mechanisms?

PHYHIPL has been identified as a potentially protective gene in glioblastoma multiforme (GBM), where its downregulation correlates with poor survival outcomes . Recombinant Xenopus PHYHIPL offers several approaches to study this connection:

• Comparative oncology models:

  • Expression of Xenopus PHYHIPL in human GBM cell lines to assess tumor suppressor activity

  • Evaluation of cell cycle effects, apoptosis resistance, and metabolic reprogramming

  • Analysis of migration and invasion capabilities

• Pathway analysis:

  • Investigation of interactions with TNF and IL-17 signaling pathways, which have been implicated in PHYHIPL's role in GBM

  • Assessment of impacts on retrograde endocannabinoid signaling and cAMP pathways

  • Identification of conserved interactions that might explain tumor suppressor functions

• Metabolic studies:

  • Analysis of how PHYHIPL affects mitochondrial function in GBM models

  • Assessment of changes in energy metabolism pathways

  • Evaluation of oxidative stress responses

• Xenopus tadpole models:

  • Development of PHYHIPL-deficient tadpoles to study neural development perturbations

  • Application of rabies virus systems for circuit-level analysis of PHYHIPL effects

  • In vivo imaging of neural development in PHYHIPL-modified embryos

These approaches leverage both the recombinant protein and the unique advantages of the Xenopus model system for studying complex disease mechanisms.

What cutting-edge methodologies can be applied to study PHYHIPL neural circuit functions in Xenopus?

Recent advances in neuroscience techniques offer powerful approaches to study PHYHIPL's role in neural circuits using the Xenopus model:

• Recombinant rabies virus applications:

  • Retrograde labeling of PHYHIPL-expressing neurons in tadpole brain

  • Expression of transgenes in specific neuronal populations

  • Analysis of connectivity between PHYHIPL-expressing neurons

• Optogenetic approaches:

  • Expression of channelrhodopsin or halorhodopsin in PHYHIPL-positive neurons

  • Precise manipulation of neuronal activity in intact circuits

  • Correlation between circuit manipulation and behavioral outcomes

• Calcium imaging:

  • GCaMP expression in PHYHIPL neurons for activity monitoring

  • Whole-brain imaging in transparent tadpoles

  • Activity correlation with environmental stimuli or behaviors

• CRISPR-based approaches:

  • Precise genome editing to create PHYHIPL variants

  • CRISPRi/CRISPRa for temporal control of expression

  • Base editing for introduction of specific mutations

These approaches can leverage the transparency of Xenopus tadpoles and the accessibility of their nervous system for in vivo imaging and manipulation, providing insights that would be difficult to obtain in mammalian models .

What are the most promising future directions for PHYHIPL research in Xenopus?

Based on current knowledge and methodological capabilities, several promising research directions emerge:

• Integration of multi-omics approaches:

  • Combining transcriptomics, proteomics, and metabolomics to understand PHYHIPL's systems-level impacts

  • Correlation of expression patterns with developmental stages and tissue specialization

  • Identification of regulatory networks controlling PHYHIPL expression

• Developmental neurobiology applications:

  • Detailed analysis of PHYHIPL's role in neural circuit formation

  • Correlation with regenerative capabilities at different developmental stages

  • Investigation of metamorphosis-related changes in expression and function

• Translational research connections:

  • Development of PHYHIPL-based interventions for metabolic or neurological conditions

  • Screening platforms using Xenopus embryos for compounds affecting PHYHIPL function

  • Validation of GBM-related findings from human studies in Xenopus models

• Technical innovations:

  • Development of PHYHIPL-specific tools for Xenopus research

  • Creation of reporter lines for in vivo monitoring

  • Application of spatial transcriptomics to understand regional variation in expression

The versatility of the Xenopus model combined with advances in recombinant protein technology positions PHYHIPL research at the intersection of fundamental biology and translational applications, offering significant potential for future discoveries.