Recombinant Danio rerio Uncharacterized protein C17orf62 homolog (zgc:91940)

What is zgc:91940 and what are its alternative names?

Zgc:91940 is an uncharacterized protein C17orf62 homolog from Danio rerio (zebrafish). It has several alternative names in the scientific literature, including:

• cybc1 (Cytochrome b-245 chaperone 1 homolog)

• eros (Essential for reactive oxygen species protein)

• Uncharacterized protein C17orf62 homolog

The protein is identified in UniProt database with the accession number Q6DGA7 .

What is the known function of zgc:91940?

Based on current research, zgc:91940 functions as a chaperone protein essential for the stable expression of the CYBA and CYBB subunits within the cytochrome b-245 heterodimer. This role is crucial for maintaining the functional integrity of the cytochrome b-245 complex, which is involved in reactive oxygen species (ROS) production and cellular redox regulation.

What are the recommended storage conditions for recombinant zgc:91940?

For optimal stability and activity retention of recombinant zgc:91940, follow these storage guidelines:

Repeated freeze-thaw cycles should be strictly avoided as they can significantly diminish protein activity. When preparing working stocks, the addition of 5-50% glycerol (final concentration) is recommended for long-term storage .

How should zgc:91940 be reconstituted for experimental use?

For optimal reconstitution of lyophilized zgc:91940:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended default: 50%)

• Prepare small aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability during the reconstitution process .

What expression systems are used to produce recombinant zgc:91940?

Recombinant zgc:91940 is typically expressed in E. coli expression systems. The full-length protein (amino acids 1-206) is often fused to an N-terminal His tag to facilitate purification through affinity chromatography. The expression in E. coli allows for high yield production of the functional protein while maintaining its structural integrity .

How can zgc:91940 be utilized in zebrafish model systems for neurological research?

Zebrafish (Danio rerio) has emerged as a valuable vertebrate model for neurological research due to genetic, organ, and cellular similarities to humans. When investigating zgc:91940's role in neurological systems:

• CRISPR/Cas9 gene editing can be employed to create zgc:91940 knockdown or knockout zebrafish lines

• Behavioral assays can then assess potential neurological phenotypes:

  • Open field tests measuring thigmotaxis (edge preference) as an anxiety indicator

  • Novel object approach tests to evaluate boldness/exploration behaviors

  • Social investigation tests examining conspecific interactions

These approaches can reveal how zgc:91940, through its role in cytochrome b-245 function and ROS regulation, may impact neurological development and behavior. Analysis should include comprehensive parameters such as time spent in different zones, distance traveled, and erratic swimming patterns .

What considerations should be made when designing qRT-PCR experiments to study zgc:91940 expression?

When designing qRT-PCR experiments to study zgc:91940 expression in zebrafish:

• Select appropriate reference genes based on experimental conditions:

  • For tissue-specific studies: sep15 and metap1 are recommended as top stable genes

  • For developmental stage experiments: ube2a and tmem50a show highest stability

  • For chemical treatment studies: rpl13a and rp1p0 demonstrate superior stability

• Avoid conventional housekeeping genes like eef1a1l1, b2m, hrpt1l and actb1, which show greater expression variability across conditions

• Design primers specific to zgc:91940 (Q6DGA7) with optimal characteristics:

  • Amplicon size: 70-150 bp

  • GC content: 40-60%

  • Melting temperature: 58-62°C

  • Minimal secondary structure

• Validate primer efficiency using standard curves with serial dilutions of cDNA

This approach ensures reliable quantification of zgc:91940 expression across different experimental conditions in zebrafish studies .

What is the relationship between zgc:91940 and reactive oxygen species (ROS) production in zebrafish immune function?

Zgc:91940 (also known as eros or cybc1) plays a critical role in the regulation of ROS production in zebrafish through its chaperone function for the cytochrome b-245 complex. To investigate this relationship:

• Knockdown studies can be performed using morpholinos or CRISPR/Cas9 targeting zgc:91940

• ROS production can be measured using:

  • Chemiluminescence assays with luminol

  • Fluorescent probes like CM-H2DCFDA or DHE

  • Hydrogen peroxide-specific probes like Amplex Red

• Immune challenges can be introduced to assess functional consequences:

  • Bacterial infection models

  • Chemical inducers of inflammation

  • Wound healing assays

Expected outcomes in zgc:91940-deficient models include:

• Decreased ROS production in response to stimuli

• Compromised neutrophil respiratory burst activity

• Potential increased susceptibility to certain pathogens

This research is particularly relevant for understanding innate immune function in zebrafish as a model for human immune disorders related to NADPH oxidase deficiencies.

How can I assess the functional activity of recombinant zgc:91940 in vitro?

To assess the functional activity of recombinant zgc:91940 in vitro:

• Chaperone activity assay:

  • Co-express zgc:91940 with CYBA and CYBB subunits in a cell-free system

  • Measure the proper folding and assembly of the cytochrome b-245 complex using:

    • Western blot analysis for protein levels

    • Blue native PAGE for complex formation

    • Circular dichroism to assess proper protein folding

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with CYBA and CYBB

  • Use surface plasmon resonance to measure binding kinetics

  • Employ FRET or BiFC for real-time interaction visualization

• Functional reconstitution:

  • Reconstitute the cytochrome b-245 complex in liposomes

  • Measure electron transfer activity using cytochrome c reduction

  • Assess ROS production with chemiluminescence or fluorescent probes

These methods provide complementary approaches to characterize the chaperone function of zgc:91940 and its impact on cytochrome b-245 activity .

What are the best experimental designs to study zgc:91940 in zebrafish embryonic development?

For studying zgc:91940 in zebrafish embryonic development:

• Temporal expression profiling:

  • Collect embryos at key developmental stages (0, 3, 6, 12, 24, 48, 72 hpf)

  • Perform qRT-PCR using validated reference genes (ube2a and tmem50a)

  • Complement with whole-mount in situ hybridization to localize expression

• Loss-of-function studies:

  • Generate transient knockdown using morpholinos

  • Create stable mutant lines using CRISPR/Cas9

  • Design rescue experiments with mRNA injection to confirm specificity

• Phenotypic analysis:

  • Document morphological changes using bright-field and fluorescence microscopy

  • Assess physiological parameters:

    • Heart rate and blood flow

    • ROS levels using live imaging with fluorescent probes

    • Immune cell development and function

• Molecular pathway analysis:

  • Perform RNA-seq on wild-type and zgc:91940-deficient embryos

  • Validate differential expression using qRT-PCR

  • Conduct pathway enrichment analysis to identify affected biological processes

This comprehensive approach allows for detailed characterization of zgc:91940's role during zebrafish development .

How can high-throughput approaches be used to identify potential interaction partners of zgc:91940?

To identify potential interaction partners of zgc:91940 using high-throughput approaches:

• Yeast two-hybrid screening:

  • Use zgc:91940 as bait against a zebrafish cDNA library

  • Validate positive interactions with secondary screens

  • Confirm with reciprocal bait-prey configurations

• Proximity-dependent biotin identification (BioID):

  • Generate zgc:91940-BirA* fusion construct

  • Express in zebrafish cells or transgenic zebrafish

  • Purify biotinylated proteins and identify by mass spectrometry

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged zgc:91940 in zebrafish cells

  • Perform immunoprecipitation under native conditions

  • Identify co-purified proteins by LC-MS/MS

  • Use SILAC or TMT labeling for quantitative comparison

• Protein microarray analysis:

  • Probe zebrafish protein arrays with purified zgc:91940

  • Identify binding partners through fluorescent detection

  • Validate interactions using orthogonal methods

These complementary approaches can reveal the interactome of zgc:91940, providing insights into its functional role in various cellular processes .

What are common challenges when working with zgc:91940 and how can they be addressed?

Common challenges when working with zgc:91940 and potential solutions include:

Implementing these solutions can significantly improve experimental outcomes when working with zgc:91940 in various research settings .

How can the purity and integrity of recombinant zgc:91940 be assessed?

To assess the purity and integrity of recombinant zgc:91940:

• SDS-PAGE analysis:

  • Run samples on 12-15% polyacrylamide gels

  • Confirm the presence of a single band at the expected molecular weight (~23 kDa including His-tag)

  • Assess purity visually or using densitometry (should be >90%)

• Western blot verification:

  • Use anti-His antibodies to detect the tagged protein

  • If available, use specific antibodies against zgc:91940

  • Confirm the absence of degradation products

• Mass spectrometry:

  • Perform intact protein MS to verify molecular weight

  • Use peptide mapping to confirm sequence coverage

  • Identify any post-translational modifications

• Functional assays:

  • Verify chaperone activity using in vitro assays

  • Confirm ability to bind known interacting partners

  • Assess stability under experimental conditions

These methods provide complementary information about protein quality and should be used in combination for comprehensive assessment .

What considerations should be made when designing experiments to study zgc:91940 function in different zebrafish tissues?

When designing tissue-specific experiments to study zgc:91940 function in zebrafish:

• Tissue-specific expression analysis:

  • Use validated reference genes specifically stable in zebrafish tissues (sep15 and metap1)

  • Employ qRT-PCR with tissue-specific cDNA libraries

  • Complement with in situ hybridization for spatial resolution

• Tissue-specific genetic manipulation:

  • Utilize Gal4-UAS or Cre-loxP systems for conditional expression

  • Generate tissue-specific promoter constructs (e.g., mpeg1 for macrophages, lyz for neutrophils)

  • Consider temporal control using heat-shock or chemical-inducible promoters

• Functional assessment approaches:

  • For immune tissues: bacterial infection assays, inflammation models

  • For brain: behavioral assays, including thigmotaxis and novel object tests

  • For other tissues: specific functional readouts relevant to tissue type

• Experimental controls:

  • Include both positive and negative controls for each tissue type

  • Use appropriate tissue-specific markers to confirm targeting

  • Consider potential developmental compensation in genetic models

These considerations ensure robust experimental design when investigating zgc:91940's tissue-specific functions in zebrafish .

How might zgc:91940 function be relevant to human disease models?

Zgc:91940 (cybc1/eros) function may be relevant to human disease models in several ways:

• Immune disorders:

  • Chronic granulomatous disease (CGD) involves defects in NADPH oxidase complex

  • Human CYBC1 mutations have been identified in CGD patients

  • Zebrafish zgc:91940 models can help understand disease mechanisms and test potential therapeutics

• Neurodegenerative conditions:

  • ROS dysregulation is implicated in numerous neurodegenerative diseases

  • Zebrafish models can assess how zgc:91940 dysfunction affects neurological parameters:

    • Behavioral changes (anxiety, exploration)

    • Neuronal survival and function

    • Microglial activation and neuroinflammation

• Developmental disorders:

  • The zebrafish model allows investigation of zgc:91940's role in early development

  • Potential links to human developmental conditions can be explored

  • Craniofacial development studies in zebrafish may reveal connections to human disorders

These areas represent promising avenues for translational research using zgc:91940 zebrafish models to better understand human disease mechanisms .

What emerging technologies might enhance zgc:91940 research in zebrafish?

Emerging technologies that could enhance zgc:91940 research in zebrafish include:

• Advanced gene editing approaches:

  • Prime editing for precise genetic modifications

  • Base editing for specific nucleotide changes

  • Inducible CRISPR systems for temporal control of gene editing

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell-specific expression patterns

  • CRISPR-QTL mapping to understand regulatory relationships

  • Spatial transcriptomics to visualize zgc:91940 expression in tissue context

• Advanced microscopy techniques:

  • Lightsheet microscopy for whole-organism imaging with minimal phototoxicity

  • Super-resolution microscopy for subcellular localization

  • Intravital microscopy for real-time in vivo observation

• Functional proteomics:

  • Proximity labeling techniques (TurboID, APEX) for in vivo interactome analysis

  • Targeted protein degradation approaches (e.g., AID, dTAG) for acute protein depletion

  • Optical control of protein function using optogenetic approaches

These technologies offer powerful new ways to investigate zgc:91940 function in zebrafish with unprecedented precision and detail .

How might comparative studies of zgc:91940 across species inform our understanding of its evolutionary significance?

Comparative studies of zgc:91940 across species can provide valuable insights into its evolutionary significance:

• Phylogenetic analysis:

  • Compare zgc:91940 sequences across vertebrate and invertebrate species

  • Identify conserved domains and motifs crucial for function

  • Determine evolutionary rates to identify selective pressures

• Functional conservation studies:

  • Test if human CYBC1 can rescue zgc:91940 deficiency in zebrafish

  • Compare expression patterns and cellular localization across species

  • Assess functional interactions with cytochrome b-245 components in different organisms

• Structural biology approaches:

  • Determine protein structures across multiple species

  • Identify structurally conserved regions that suggest functional importance

  • Model evolutionary changes and their impact on protein-protein interactions

• Molecular ecology studies:

  • Examine zgc:91940 variants in wild zebrafish populations

  • Correlate genetic variations with environmental factors

  • Assess potential adaptive significance in different ecological niches

These comparative approaches can reveal how zgc:91940 function has been conserved or diversified throughout evolution, providing context for its fundamental biological roles .