Recombinant Xenopus laevis Peroxisomal membrane protein PEX16 (pex16)
Description
Introduction to PEX16 Protein
Peroxisomal membrane protein PEX16, also known as Peroxin-16 or Peroxisomal biogenesis factor 16, is a critical component of the peroxisomal membrane in eukaryotic cells. In Xenopus laevis, PEX16 is essential for the biogenesis of peroxisomes, organelles that perform various metabolic functions including fatty acid oxidation, hydrogen peroxide metabolism, and bile acid synthesis . The recombinant form of this protein enables detailed study of its structure, function, and role in developmental processes. While significant research has focused on peroxisomal proteins in mammalian systems, studies in Xenopus laevis provide unique insights into the developmental regulation of peroxisome formation in vertebrates .
The recombinant form of Xenopus laevis PEX16 protein consists of the full-length sequence (amino acids 1-340) with an N-terminal histidine tag to facilitate purification and detection . This recombinant protein maintains the functional domains present in native PEX16, making it suitable for various research applications including structural studies, functional assays, and antibody production.
Physical and Biochemical Properties
The recombinant PEX16 protein has been extensively characterized to understand its biochemical properties. These properties are summarized in the following table:
| Property | Specification |
|---|---|
| Species | Xenopus laevis |
| Expression System | E. coli |
| Tag | N-terminal His |
| Protein Length | Full Length (1-340 amino acids) |
| Physical Form | Lyophilized powder |
| Purity | Greater than 90% (determined by SDS-PAGE) |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Molecular Weight | Approximately 38 kDa (calculated from amino acid sequence) |
| UniProt ID | Q6INN0 |
The high purity level (>90%) ensures reliable results in downstream applications . The lyophilized form provides stability during shipping and storage, while the histidine tag facilitates protein detection and purification through affinity chromatography.
Expression System and Methodology
The recombinant Xenopus laevis PEX16 protein is typically produced using E. coli expression systems . This bacterial expression platform offers several advantages for recombinant protein production, including rapid growth, high protein yields, and cost-effectiveness. The gene encoding the full-length Xenopus laevis PEX16 protein is cloned into an appropriate expression vector with an N-terminal histidine tag sequence. Following transformation into E. coli cells, the bacteria are cultured under optimized conditions to induce protein expression.
After cultivation, the bacterial cells are harvested and lysed to release the recombinant protein. Purification typically involves immobilized metal affinity chromatography (IMAC), exploiting the affinity of the histidine tag for metal ions such as nickel or cobalt. Additional purification steps may include ion-exchange chromatography or size exclusion chromatography to achieve the high purity level (>90%) observed in the final product .
Role in Peroxisome Biogenesis
PEX16 plays a critical role in peroxisome biogenesis as an integral membrane protein that facilitates the insertion of other proteins into the peroxisomal membrane. In Xenopus laevis, as in other vertebrates, PEX16 functions as an early peroxisome biogenesis factor that is essential for the formation of new peroxisomes . Mutations or deficiencies in PEX16 can lead to severe defects in peroxisome formation and function, highlighting its importance in cellular metabolism.
Research using recombinant PEX16 has contributed to understanding the molecular mechanisms of peroxisome membrane protein import and assembly. Studies suggest that PEX16 acts as a receptor or docking factor for other peroxisomal membrane proteins, facilitating their integration into the peroxisomal membrane .
Developmental Expression Pattern
The temporal and spatial expression of PEX16 during Xenopus laevis development provides insights into the regulatory mechanisms governing peroxisome biogenesis. RT-PCR analyses have revealed that PEX16, along with other peroxisomal genes (PEX5, ACO, PEX19, PMP70, and catalase), exhibits elevated RNA levels relatively late during embryogenesis .
Peroxisome formation and function appear to be particularly important in late dorsal-anterior structures during development. This pattern suggests that peroxisomal metabolism becomes increasingly significant as embryos transition from relying on stored yolk proteins to active feeding and independent metabolism . The coordinated expression of functionally related peroxisomal genes indicates a programmed developmental regulation of peroxisome biogenesis that aligns with the changing metabolic needs of the developing embryo.
Interestingly, there has been limited prior research on PEX16 gene regulation compared to other peroxisomal genes such as catalase . This gap in knowledge presents an opportunity for further investigation using the recombinant PEX16 protein as a research tool.
Current Research Applications
The recombinant Xenopus laevis PEX16 protein serves as a valuable tool for various research applications:
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Structural Studies: The high-purity recombinant protein enables detailed structural analyses using techniques such as X-ray crystallography or nuclear magnetic resonance spectroscopy.
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Antibody Production: Recombinant PEX16 can be used to generate specific antibodies for immunodetection, immunoprecipitation, and immunohistochemistry applications.
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Protein-Protein Interaction Studies: The recombinant protein facilitates investigations of interactions between PEX16 and other peroxisomal proteins.
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Functional Assays: In vitro assays using recombinant PEX16 help elucidate its role in peroxisome membrane assembly and protein import.
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Developmental Research: As a tool for studying peroxisome biogenesis during embryonic development, recombinant PEX16 contributes to our understanding of metabolic regulation in vertebrate development .
Future Research Directions
Several promising research directions could utilize recombinant Xenopus laevis PEX16 protein:
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Detailed Characterization of Gene Regulation: Given the limited prior work on PEX16 gene regulation, investigations into the transcriptional and post-transcriptional regulatory mechanisms could provide valuable insights .
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Comparative Studies Across Species: Comparison of Xenopus PEX16 with its mammalian counterparts could reveal evolutionary conservation and divergence in peroxisome biogenesis mechanisms.
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Metabolic Transitions During Development: Further exploration of how peroxisome formation correlates with metabolic transitions during development, particularly as embryos deplete yolk stores and commence feeding .
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Relationship to Human Peroxisomal Disorders: Research using Xenopus laevis PEX16 could contribute to understanding human peroxisomal biogenesis disorders, many of which result from mutations in peroxin genes including PEX16.
Product Specs
Please note: We prioritize shipping the format currently in stock. However, if you have specific requirements for the format, please specify them during order placement, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please contact us in advance as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: xla:432288
UniGene: Xl.47100
Q&A
What is PEX16 and what is its fundamental role in peroxisome biogenesis?
PEX16 is an integral peroxisomal membrane protein essential for peroxisome organization and biogenesis. It functions as a critical peroxin that mediates the trafficking and insertion of peroxisomal membrane proteins (PMPs). Studies have shown that PEX16, together with PEX3 and PEX19, is specifically involved in the early stages of peroxisome formation, particularly in the targeting and import of PMPs .
PEX16 has a molecular weight of approximately 39-40 kDa and functions as a central component in the machinery responsible for peroxisome assembly . In human systems, PEX16 plays a crucial role in the endoplasmic reticulum (ER)-dependent trafficking pathway for peroxisomal membrane proteins, serving as a docking factor that recruits other peroxins, particularly PEX3, to the peroxisomal membrane .
Why is Xenopus laevis a valuable model organism for studying peroxisomal proteins?
Xenopus laevis offers several distinct advantages as a model organism for studying peroxisomal proteins:
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Developmental accessibility: Xenopus embryos develop externally, allowing for easy experimental manipulation and observation from early embryonic stages .
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Large embryo size: The relatively large size of Xenopus eggs and embryos facilitates microinjection techniques for introducing constructs or performing knockdown experiments .
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Abundant material: Female Xenopus can produce thousands of embryos, providing ample experimental material .
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Evolutionary significance: As an amphibian, Xenopus occupies an important phylogenetic position for comparative studies between mammals and lower vertebrates .
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Established techniques: Well-developed protocols exist for molecular manipulation in Xenopus, including microinjection, in situ hybridization, and immunohistochemistry .
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Temporal separation of events: The clear temporal progression of peroxisome biogenesis during Xenopus development allows researchers to study distinct phases of this process .
How is PEX16 expressed during Xenopus laevis embryogenesis?
The expression pattern of PEX16 during Xenopus embryogenesis follows a specific temporal and spatial pattern:
Research has demonstrated that peroxisome biogenesis occurs relatively late during Xenopus embryogenesis, primarily in dorsal-anterior structures . This temporal pattern correlates with the depletion of yolk reserves and the transition to active metabolism, suggesting a developmental regulation of peroxisome formation that coincides with metabolic changes .
What methods are most effective for studying PEX16 function in Xenopus laevis?
Several methodological approaches have proven effective for investigating PEX16 function in Xenopus:
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RT-PCR analysis: Used to elucidate the temporal expression patterns of peroxisomal genes including PEX16 during embryogenesis .
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Microinjection techniques: Effective for introducing mRNA constructs (such as HA-tagged PEX16) or morpholinos for gain-of-function or loss-of-function studies .
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Immunohistochemistry: Using antibodies against peroxisomal markers like PMP70 and catalase to visualize peroxisome formation and distribution in embryonic tissues .
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Fluorescent protein tagging: GFP-SKL (containing a peroxisomal targeting sequence) can be microinjected to visualize peroxisomes in vivo during development .
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Western blot analysis: For quantifying PEX16 protein levels and assessing changes during development or experimental manipulations .
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RNA-seq and transcriptome analysis: For comprehensive gene expression profiling during development .
How does PEX16 function in Xenopus compare to its role in other model organisms?
PEX16 function shows both conservation and divergence across species:
Research in Drosophila has revealed that PEX16 requirements for peroxisome biogenesis differ between mammals and insects. While PEX16 is essential for peroxisome biogenesis in humans, Drosophila pex16 mutants still maintained a small number of peroxisome-like granules, suggesting that the role of PEX16 orthologs may have diverged evolutionarily .
What techniques are used to produce and validate recombinant Xenopus laevis PEX16?
The production and validation of recombinant Xenopus laevis PEX16 involves several technical approaches:
Production Methods:
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Bacterial expression systems: E. coli-based expression systems can be used to produce recombinant PEX16 protein with appropriate tags (His, Myc, DDK) for purification .
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Mammalian cell expression: HEK293 cells can be transfected with Xenopus PEX16 expression constructs for production of properly folded protein .
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In vitro transcription/translation: Cell-free systems can be employed for production of recombinant PEX16 protein.
Validation Methods:
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Western blotting: Using anti-PEX16 antibodies to confirm protein expression and size (approximately 39-40 kDa) .
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Immunofluorescence microscopy: To verify proper subcellular localization of the recombinant protein .
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Functional complementation assays: Testing whether recombinant Xenopus PEX16 can rescue peroxisome biogenesis defects in PEX16-deficient cells .
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Protein-protein interaction studies: Co-immunoprecipitation experiments to verify interactions with known PEX16 partners such as PEX3 and PEX19 .
How can mutations in PEX16 be studied using Xenopus laevis as a model system?
Xenopus laevis provides several advantages for studying PEX16 mutations:
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Microinjection of mutant constructs: Synthetic mRNA encoding mutant forms of PEX16 can be microinjected into embryos to study dominant-negative effects .
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Rescue experiments: Wild-type PEX16 mRNA can be co-injected with morpholinos targeting endogenous PEX16 to validate specificity .
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Humanized models: Human PEX16 variants can be expressed in Xenopus to study their function, similar to approaches used in Drosophila .
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Phenotypic analysis: Effects of PEX16 mutations on peroxisome biogenesis can be assessed through immunohistochemistry, metabolic assays, and developmental phenotyping .
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CRISPR/Cas9 genome editing: Though technically challenging in the tetraploid Xenopus laevis, CRISPR-based approaches can be used to introduce specific mutations in the endogenous PEX16 gene .
Recent studies in Drosophila have demonstrated the utility of cross-species rescue experiments to evaluate the functional consequences of human PEX16 variants. Similar approaches in Xenopus could provide valuable insights into the pathogenicity of human PEX16 mutations .
How does PEX16 contribute to peroxisome biogenesis during Xenopus development?
PEX16 plays a critical role in the developmental regulation of peroxisome biogenesis in Xenopus:
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Temporal regulation: PEX16 expression increases during late embryogenesis, coinciding with the appearance of functional peroxisomes .
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Spatial distribution: PEX16 and peroxisomes are predominantly localized to dorsal-anterior structures in developing Xenopus embryos .
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Metabolic transition: The timing of PEX16 expression correlates with the depletion of maternal yolk reserves and the transition to active metabolism, suggesting a coordinated metabolic regulation .
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Interaction with other peroxins: PEX16 functions in concert with other peroxins (such as PEX3 and PEX19) to facilitate the assembly of functional peroxisomes .
Research has demonstrated that peroxisomes are not detected in early Xenopus embryos (stage 10) but become visible in the somites by stage 20, correlating with the expression patterns of peroxisomal genes including PEX16 .
What are the challenges in studying PEX16 in the tetraploid Xenopus laevis compared to diploid species?
Studying PEX16 in Xenopus laevis presents several unique challenges due to its tetraploid genome:
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Gene duplication: As an allotetraploid species with ~18 chromosomes (compared to 10 in the diploid X. tropicalis), X. laevis likely contains two homeologous copies of the PEX16 gene .
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Redundancy: Functional redundancy between homeologs can complicate loss-of-function studies, as disruption of one copy may be compensated by the other .
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Genetic manipulation: The presence of duplicated genes makes genetic approaches such as CRISPR/Cas9 editing more challenging, as both homeologs may need to be targeted simultaneously .
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Expression dynamics: The two homeologs may exhibit different temporal or spatial expression patterns, or undergo subfunctionalization .
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Longer generation time: X. laevis has a longer generation time (1-2 years) compared to the diploid X. tropicalis (4-6 months), making genetic studies more time-consuming .
Research on allotetraploidy in Xenopus has shown that while many genes retain both homeologous copies, they often undergo asymmetric activation during embryogenesis . This may also apply to PEX16, potentially complicating the interpretation of experimental results.
What experimental approaches can be used to study the interaction between PEX16 and other peroxins in Xenopus laevis?
Several experimental approaches can be employed to investigate protein-protein interactions involving PEX16:
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Co-immunoprecipitation (Co-IP): Using antibodies against PEX16 or epitope-tagged versions to pull down interacting proteins from Xenopus embryo lysates or A6 cell extracts .
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Yeast two-hybrid assays: To identify direct protein-protein interactions between PEX16 and other peroxins.
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Bimolecular fluorescence complementation (BiFC): By expressing PEX16 and potential interaction partners fused to complementary fragments of fluorescent proteins in Xenopus embryos or cells.
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Proximity labeling approaches: Such as BioID or APEX2, where PEX16 is fused to a proximity-dependent labeling enzyme to identify proteins in its vicinity.
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Co-localization studies: Using fluorescently tagged proteins and confocal microscopy to assess spatial proximity in Xenopus cells .
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In vitro binding assays: Using purified recombinant proteins to assess direct interactions.
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Simultaneous manipulation: Co-injecting morpholinos or mRNAs targeting multiple peroxins to assess genetic interactions and functional redundancy .
How can recombinant Xenopus laevis PEX16 be used to study peroxisomal disorders?
Recombinant Xenopus laevis PEX16 offers several applications for studying peroxisomal disorders:
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Disease modeling: Expression of mutant forms of PEX16 associated with human peroxisomal biogenesis disorders in Xenopus embryos can provide insights into pathogenic mechanisms .
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Functional complementation: Testing whether Xenopus PEX16 can rescue peroxisome biogenesis defects in human patient-derived cells with PEX16 mutations .
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Comparative studies: Analysis of functional conservation between Xenopus and human PEX16 can illuminate evolutionarily conserved mechanisms of peroxisome biogenesis .
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Drug screening: Xenopus embryos expressing mutant PEX16 could serve as a platform for screening compounds that might restore peroxisome function.
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Structure-function analysis: Using domain swapping between human and Xenopus PEX16 to identify critical functional regions of the protein .
Recent research in Drosophila has demonstrated the value of cross-species studies for understanding human PEX gene mutations. Similar approaches with Xenopus PEX16 could provide valuable insights into the pathogenicity of human variants .
What is known about the temporal and spatial expression pattern of PEX16 during Xenopus development?
The expression of PEX16 during Xenopus development follows specific temporal and spatial patterns:
| Developmental Stage | PEX16 Expression | Peroxisome Formation | Tissue Distribution |
|---|---|---|---|
| Early cleavage (stages 1-9) | Minimal expression | Not detected | N/A |
| Gastrula (stage 10) | Low expression | Not detected by PMP70 or catalase staining | N/A |
| Neurula (stages 15-20) | Increasing expression | Beginning to appear | Primarily in dorsal-anterior structures |
| Tailbud/tadpole (stages 21+) | High expression | Clearly visible peroxisome structures | Predominant in somites and developing organs |
Research on peroxisome biogenesis in Xenopus has shown that peroxisomes become detectable in stage 20 embryos, particularly in somitic tissues, correlating with increased expression of peroxisomal proteins including PEX16 . This temporal pattern suggests that peroxisome biogenesis is developmentally regulated and coincides with the transition from yolk-dependent metabolism to active metabolism in developing embryos .
What are the emerging technologies that could advance our understanding of Xenopus laevis PEX16?
Several cutting-edge technologies hold promise for advancing PEX16 research in Xenopus:
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CRISPR/Cas9 genome editing: Despite challenges in the tetraploid genome, CRISPR technologies are being adapted for Xenopus laevis, enabling precise genetic manipulation of PEX16 .
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Single-cell RNA sequencing: This approach could reveal cell-type specific expression patterns of PEX16 during development .
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Advanced imaging techniques: Super-resolution microscopy and live-cell imaging can provide insights into PEX16 dynamics and peroxisome biogenesis in real-time.
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Cryo-electron microscopy: Could potentially resolve the structure of PEX16 and its interactions with other peroxins.
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Organoid models: Xenopus organoid systems could allow for the study of PEX16 function in tissue-specific contexts.
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Comparative genomics: With the sequenced genomes of both X. laevis and X. tropicalis, comparative analyses can reveal evolutionary patterns in PEX16 function .
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Proteomics approaches: Such as BioID or APEX proximity labeling, combined with mass spectrometry, could identify novel PEX16 interaction partners .
Recent advances in RNA-seq approaches in Xenopus, including subgenome-specific analysis in X. laevis, provide powerful tools for studying the expression dynamics of duplicated genes like PEX16 .
How does PEX16 expression correlate with metabolic changes during Xenopus development?
The correlation between PEX16 expression and metabolic transitions during Xenopus development reveals important insights:
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Temporal coordination: The appearance of peroxisomes coincides with the depletion of maternal yolk reserves and the transition to active metabolism .
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Metabolic enzyme expression: The expression of peroxisomal metabolic enzymes (such as catalase) correlates with PEX16 expression, suggesting coordinated regulation .
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Tissue-specific patterns: Peroxisome formation is particularly prominent in metabolically active tissues like the developing somites .
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Transcriptional regulation: PEX16 expression may be coordinated with peroxisome proliferator-activated receptors (PPARs), which are known regulators of peroxisome biogenesis and lipid metabolism .
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Maternal-to-zygotic transition: The timing of peroxisome biogenesis correlates with the broader maternal-to-zygotic transition in gene expression .
Research in Xenopus A6 cells has shown that manipulation of peroxisome biogenesis factors can alter the expression of metabolic genes, suggesting a complex regulatory network linking peroxisome formation with metabolic adaptation .
