Recombinant Danio rerio Uncharacterized protein C17orf85 homolog (zgc:55870), partial

What is the functional characterization status of zgc:55870 in Danio rerio?

The zgc:55870 gene in zebrafish encodes an uncharacterized protein homologous to human C17orf85. While its precise function remains to be fully elucidated, it shares homology with the family of uncharacterized proteins that includes Fam210ab (also known as C18orf19 homolog B in zebrafish). Based on sequence analysis and evolutionary conservation, it likely plays roles in developmental processes, though specific pathways remain unclear. Research on related uncharacterized proteins suggests potential involvement in cellular organization or metabolism.

Methodologically, researchers approaching this protein typically employ comparative genomics with human orthologs, expression pattern analysis during development, and knockout/knockdown studies to observe phenotypic effects. The protein's uncharacterized status presents significant opportunities for novel functional discoveries, particularly when studied alongside better-characterized family members like Fam210ab that have established expression patterns in zebrafish models .

What expression systems are most effective for recombinant zebrafish proteins like zgc:55870?

Several expression systems can be utilized for recombinant production of zebrafish proteins like zgc:55870, each with distinct advantages depending on research objectives. Based on established protocols for similar uncharacterized zebrafish proteins, researchers can consider:

Cell-free expression systems provide rapid protein production without the complexities of cellular growth, offering advantages for initial characterization studies or when the protein might be toxic to host cells . E. coli systems offer cost-effectiveness and high yields but may struggle with complex folding requirements of eukaryotic proteins. For more authentic post-translational modifications, yeast, baculovirus-insect cell, and mammalian cell systems provide progressively more sophisticated eukaryotic expression environments .

For uncharacterized proteins like zgc:55870, a strategic approach involves testing multiple systems simultaneously, starting with both prokaryotic (E. coli) and eukaryotic (typically insect cells) hosts to determine which provides functional protein. The expression vector design should incorporate affinity tags to facilitate purification, with consideration given to tag positioning (N- or C-terminal) to minimize interference with protein function.

What purification strategies yield highest quality recombinant zgc:55870 protein?

Purifying recombinant zgc:55870 requires a systematic approach that maintains protein integrity while achieving high purity. The purification workflow typically begins with careful cell lysis optimized for the expression system used. For zebrafish recombinant proteins, affinity chromatography using tags like His, GST, or FLAG provides effective initial capture, with commercial proteins typically achieving ≥85% purity as determined by SDS-PAGE .

For research applications requiring higher purity, a multi-step purification strategy is recommended, incorporating secondary techniques such as ion exchange chromatography to separate based on charge differences, or size exclusion chromatography to remove aggregates and distinguish monomeric forms. When expressing zgc:55870 in eukaryotic systems, additional considerations for glycosylation heterogeneity may necessitate specialized purification approaches.

Quality control through analytical techniques including SDS-PAGE, mass spectrometry, and dynamic light scattering provides critical assessment of purity, identity, and homogeneity. For completely uncharacterized proteins like zgc:55870, pilot expression and purification trials should test multiple buffer compositions to identify conditions that maximize stability and solubility, with particular attention to pH, salt concentration, and stabilizing additives.

What are the structural characteristics of the zgc:55870 protein based on bioinformatic predictions?

While experimental structural data for zgc:55870 remains limited, bioinformatic analysis provides valuable insights into its structural characteristics. Sequence analysis indicates that zgc:55870, like other C17orf85 homologs, likely contains domains and motifs that suggest specific cellular functions. Structural prediction algorithms indicate potential globular domains, though particular structural elements require experimental validation.

Secondary structure predictions typically reveal a mixture of alpha-helical regions and beta-sheets, with potentially disordered regions that could be involved in protein-protein interactions. Comparative modeling with proteins of similar sequence, such as the Fam210 family members, suggests structural similarities that might indicate related functions . The uncharacterized protein likely contains conserved residues that maintain structural integrity across species.

Methodologically, researchers should approach structural predictions by combining multiple computational tools (PSIPRED, SWISS-MODEL, I-TASSER) to build consensus models. These predictions can guide experimental design, particularly for site-directed mutagenesis of putative functional residues or the design of truncation constructs for expression studies. Domain-focused expression may prove valuable when working with difficult-to-express proteins like zgc:55870, allowing structural and functional characterization of individual protein regions.

What challenges exist in studying uncharacterized proteins like zgc:55870 in zebrafish models?

Studying uncharacterized proteins like zgc:55870 in zebrafish presents multiple significant challenges at both technical and conceptual levels. Without established functional knowledge, researchers must develop experimental approaches without the benefit of precedent or validated protocols specific to the protein. Hypothesis generation relies heavily on bioinformatic predictions and comparative genomics, which may have limitations for novel proteins.

From a technical perspective, expression and purification of uncharacterized proteins often require extensive optimization due to unknown properties affecting solubility, stability, or toxicity to expression hosts. Similar challenges are seen with related zebrafish proteins, necessitating testing across multiple expression systems including cell-free, bacterial, and eukaryotic hosts . Additionally, the lack of validated reagents such as specific antibodies hampers detection and characterization efforts, often requiring researchers to develop and validate their own tools.

Phenotypic assessment following genetic manipulation presents another layer of complexity; without knowing protein function, determining appropriate assays for knockout or knockdown studies becomes challenging. Researchers must employ broad phenotypic screens covering multiple biological processes and consider temporal and spatial expression patterns. Integration of multiple approaches (genomic, transcriptomic, proteomic) can help overcome these limitations by providing converging evidence for protein function.

How can CRISPR-Cas9 technology be optimized for functional studies of zgc:55870?

CRISPR-Cas9 technology offers powerful approaches for investigating the function of uncharacterized proteins like zgc:55870 in zebrafish, but requires careful optimization for maximum effectiveness. When designing a CRISPR-based study for zgc:55870, researchers should first conduct thorough bioinformatic analysis to identify optimal target sites with minimal off-target potential. For uncharacterized genes, targeting conserved domains identified through cross-species comparison increases the likelihood of disrupting functional regions.

Guide RNA design should incorporate recent advances in CRISPR efficiency prediction, with multiple guides tested to identify those with highest editing efficiency. For knockout studies, targeting early exons creates frameshift mutations that typically result in complete loss of function through nonsense-mediated decay. Alternatively, for more nuanced functional analysis, precise editing can introduce specific mutations in predicted functional domains or create in-frame deletions.

Given the potential redundancy in zebrafish genes due to genome duplication, researchers should consider generating multiple mutant lines and compound mutants if paralogs exist. Methodologically, verification of editing efficiency requires both genotyping and measurement of protein levels when antibodies are available. For uncharacterized proteins like zgc:55870, where specific antibodies may not be commercially available, epitope tagging through homology-directed repair provides an alternative approach for protein detection.

Phenotypic analysis should be comprehensive, including developmental timing, morphology, behavior, and tissue-specific effects, with particular attention to stages and tissues where expression data indicates the protein is abundant. Advanced approaches like single-cell RNA-seq of mutant embryos can reveal cell-type specific effects that might be missed in whole-organism studies.

What strategies are effective for developing specific antibodies against uncharacterized zebrafish proteins like zgc:55870?

Developing specific antibodies against uncharacterized zebrafish proteins requires strategic planning to overcome several technical challenges. For proteins like zgc:55870, antigen design represents the first critical decision point. In the absence of structural information, researchers typically use bioinformatic approaches to identify immunogenic regions with high surface probability and low sequence similarity to other zebrafish proteins, reducing cross-reactivity risk.

Multiple antigen approaches increase success probability: full-length recombinant protein provides comprehensive epitope coverage, while synthetic peptides corresponding to predicted antigenic regions offer targeted specificity. When designing peptide antigens, focusing on regions that diverge from closely related proteins while remaining conserved across vertebrates often yields antibodies with both specificity and cross-species utility.

The choice of host animal significantly impacts antibody quality. As demonstrated with other zebrafish proteins, rabbits are commonly used for polyclonal antibody generation against zebrafish antigens . Polyclonal approaches typically offer higher success rates for uncharacterized proteins, though monoclonal development provides long-term reproducibility advantages.

Rigorous validation is essential, particularly for uncharacterized proteins. Western blotting against both recombinant protein and zebrafish tissue lysates establishes basic reactivity, while immunoprecipitation followed by mass spectrometry confirms target specificity. When available, testing in knockout or knockdown models provides definitive validation by demonstrating signal reduction or elimination. For proteins like zgc:55870 without established function, antibodies enable critical experiments including expression pattern determination, subcellular localization, and protein interaction studies.

How can protein-protein interaction studies reveal the function of zgc:55870?

Protein-protein interaction (PPI) studies provide powerful approaches for elucidating the function of uncharacterized proteins like zgc:55870. By identifying binding partners with known functions, researchers can gain insights into biological pathways and cellular processes involving the protein of interest.

Affinity purification coupled with mass spectrometry (AP-MS) offers a comprehensive approach for identifying interaction partners. For zgc:55870, this would typically involve expressing the tagged protein in zebrafish embryos or relevant cell lines, purifying complexes under conditions that maintain native interactions, and identifying co-purifying proteins by mass spectrometry. Methodologically, quantitative comparison between specific pulldowns and controls using stable isotope labeling or label-free quantification distinguishes true interactors from background proteins.

Proximity-based labeling techniques such as BioID or APEX provide complementary approaches that can capture transient or weak interactions missed by traditional co-immunoprecipitation. These methods involve expressing zgc:55870 fused to an enzyme that biotinylates nearby proteins, allowing subsequent purification and identification of proximity partners regardless of binding strength or stability.

For validation of key interactions, orthogonal methods including co-immunoprecipitation, fluorescence resonance energy transfer (FRET), or bimolecular fluorescence complementation (BiFC) provide confirmation and can reveal spatial and temporal dynamics of interactions in living zebrafish embryos. Network analysis of interaction data often reveals functional clusters that place uncharacterized proteins within known biological pathways, generating testable hypotheses about protein function.

What approaches should be used to determine the developmental expression pattern of zgc:55870?

Determining the developmental expression pattern of zgc:55870 requires a multi-faceted approach combining temporal and spatial analysis. RNA-based methods provide the foundation for expression analysis, with quantitative PCR offering precise measurement of transcript levels across developmental stages. This should be complemented by whole-mount in situ hybridization (WISH) to visualize spatial distribution patterns in intact embryos, revealing tissue-specific expression domains that may suggest functional roles.

For protein-level analysis, immunohistochemistry using specific antibodies against zgc:55870 provides spatial resolution of protein expression, though this depends on antibody availability. When antibodies are unavailable, transgenic reporter lines expressing fluorescent proteins under the zgc:55870 promoter offer an alternative approach, enabling real-time visualization of expression in live embryos. BAC (Bacterial Artificial Chromosome) transgenesis incorporating the extended genomic context ensures that regulatory elements controlling developmental expression are included.

Single-cell RNA sequencing provides unprecedented resolution of expression patterns, revealing cell type-specific expression that may be missed in whole-tissue analyses. This approach is particularly valuable for uncharacterized proteins like zgc:55870, as co-expression analysis with known markers can suggest functional associations. For temporal regulation, examining expression under various experimental conditions or perturbations can reveal regulatory mechanisms and potential functional contexts.

Methodologically, incorporating multiple approaches strengthens confidence in expression data, with each technique offering different advantages in terms of sensitivity, spatial resolution, or throughput. For developmental biologists, correlating expression patterns with key developmental processes and comparing patterns between zgc:55870 and better-characterized homologs like Fam210ab can generate functional hypotheses for further investigation.

How should researchers interpret phenotypic data from zgc:55870 knockout or knockdown studies?

Researchers should first establish knockout or knockdown efficiency through molecular validation, confirming reduction at both RNA and protein levels when possible. For CRISPR-generated mutants, detailed genotypic characterization is essential to understand the precise molecular lesion, as in-frame mutations might retain partial function while complete knockouts would eliminate all function.

When analyzing phenotypes, developmental timing must be carefully considered, as uncharacterized proteins may function at specific developmental windows. Comparison with expression data helps correlate phenotypes with spatiotemporal expression patterns, strengthening causal relationships. For zebrafish-specific considerations, researchers should account for potential genetic compensation by related genes, which can mask phenotypes in stable knockout lines but may be less prevalent in acute knockdowns.

Methodologically, rescue experiments represent the gold standard for establishing specificity—re-introduction of wild-type zgc:55870 should reverse observed phenotypes. Structure-function analysis through rescue with mutated versions can identify critical domains or residues. When working with uncharacterized proteins, researchers should also consider cellular phenotypes that might precede or underlie morphological changes, including alterations in cell proliferation, migration, differentiation, or subcellular organization.

What quality control metrics should be applied when working with recombinant zgc:55870?

Quality control for recombinant zgc:55870 requires a comprehensive approach encompassing multiple analytical techniques to ensure protein identity, purity, integrity, and functionality. For initial purity assessment, SDS-PAGE with densitometry analysis provides quantitative measurement, with commercial standards typically requiring ≥85% purity . This should be complemented by more sensitive techniques such as capillary electrophoresis or high-performance liquid chromatography to detect minor contaminants.

Protein identity confirmation is critical for uncharacterized proteins, where mass spectrometry provides definitive verification through peptide mass fingerprinting or tandem MS sequencing. For recombinant zgc:55870, coverage of at least 70% of the amino acid sequence through MS analysis ensures correct protein identity. Western blotting with specific antibodies provides additional confirmation when available.

Structural integrity assessment through circular dichroism spectroscopy reveals secondary structure content, while thermal shift assays measure protein stability under various buffer conditions. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) evaluates sample homogeneity and detects aggregation. For proteins expressed in eukaryotic systems, glycosylation analysis through mass spectrometry or specialized staining provides critical information about post-translational modification consistency.

Methodologically, implementing standardized quality control workflows with defined acceptance criteria ensures batch-to-batch reproducibility. Researchers should establish protein-specific reference standards and retain samples from successful preparations as benchmarks for future batches. Documentation of all quality control results with raw data preservation enables retrospective analysis if downstream applications reveal unexpected issues.

What techniques are most effective for studying post-translational modifications of zgc:55870?

Studying post-translational modifications (PTMs) of uncharacterized proteins like zgc:55870 requires specialized approaches that can identify, localize, and quantify these critical regulatory elements. Mass spectrometry-based proteomics forms the foundation of comprehensive PTM analysis, with several complementary workflows offering different advantages.

Bottom-up proteomics, involving enzymatic digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides the most comprehensive coverage for PTM discovery. Database searching with variable modifications enables identification of common PTMs like phosphorylation, glycosylation, acetylation, and ubiquitination. For uncharacterized proteins like zgc:55870, searches should include a wide array of possible modifications to capture unexpected PTMs that might provide functional clues.

Enrichment strategies significantly enhance detection sensitivity for specific modifications: immobilized metal affinity chromatography (IMAC) or titanium dioxide enrichment for phosphopeptides, lectin affinity for glycopeptides, and antibody-based enrichment for acetylation or ubiquitination. These approaches allow detection of low-abundance modifications that might be missed in global analyses.

Top-down proteomics, analyzing intact proteins without digestion, offers advantages for understanding PTM combinations and stoichiometry, though technical challenges limit its application to smaller proteins or domains. For zgc:55870, a domain-based approach might be most practical, expressing and analyzing individual domains separately.

Bioinformatic prediction should guide experimental design, identifying potential modification sites based on consensus sequences and evolutionary conservation. For expression system selection, researchers should consider that different systems vary in their capacity for authentic modifications—mammalian cells providing the most vertebrate-like PTMs compared to simpler systems like E. coli or cell-free expression .

How can researchers overcome solubility challenges when expressing zgc:55870?

Overcoming solubility challenges for uncharacterized proteins like zgc:55870 requires a multifaceted strategy addressing issues at both the expression and purification stages. When initial expression attempts yield insoluble protein, researchers should first conduct bioinformatic analysis to identify potential aggregation-prone regions or hydrophobic patches that might contribute to poor solubility.

Expression system optimization represents a primary approach, with multiple systems tested in parallel. While E. coli provides cost-effective high-yield production, eukaryotic systems including insect cells and mammalian cells often improve folding of complex proteins . For zgc:55870, a comparative approach testing expression in multiple systems would identify optimal conditions for soluble protein production.

Fusion tags can dramatically enhance solubility, with solubility-enhancing partners like maltose-binding protein (MBP), NusA, or SUMO generally outperforming simple affinity tags. Strategic tag placement at either N- or C-terminus can impact effectiveness, necessitating testing of multiple constructs. For uncharacterized proteins, domain-based expression targeting individual structural domains often yields more soluble protein than full-length constructs.

Expression condition optimization through temperature reduction (typically 15-25°C instead of 37°C), slower induction, and specialized media formulations can significantly improve soluble yields. Co-expression with molecular chaperones like GroEL/ES or trigger factor enhances correct folding, particularly in bacterial systems.

For proteins recalcitrant to soluble expression, refolding approaches from inclusion bodies provides an alternative, though refolding protocols require extensive optimization. Modern high-throughput screening of refolding conditions using fractional factorial designs can efficiently identify optimal refolding parameters from hundreds of possible combinations of pH, ionic strength, additives, and redox conditions.

What are the most reliable approaches for determining the subcellular localization of zgc:55870?

Immunofluorescence microscopy using specific antibodies against zgc:55870 provides an alternative approach that detects endogenous protein without overexpression artifacts. This technique requires carefully validated antibodies and appropriate controls, including competitive blocking with recombinant protein and testing in knockout or knockdown backgrounds to confirm specificity.

Biochemical fractionation offers complementary evidence through physical separation of subcellular compartments followed by Western blot detection of zgc:55870 in specific fractions. This approach provides quantitative distribution data but requires careful validation of fraction purity using established markers for each cellular compartment.

For uncharacterized proteins, co-localization studies with well-characterized markers for specific organelles or structures provide functional context. Advanced techniques including super-resolution microscopy or proximity labeling (BioID, APEX) offer enhanced spatial resolution or detection of proteins in close proximity, revealing functional neighborhoods within cells.

Bioinformatic prediction of localization signals (nuclear localization signals, mitochondrial targeting sequences, transmembrane domains) should guide experimental design but requires experimental validation, as prediction algorithms may miss non-canonical signals. For comprehensive analysis, researchers should examine localization across different cell types and developmental stages, as proteins may shuttle between compartments in response to signals or during different cellular processes.

What experimental design is optimal for identifying small molecule modulators of zgc:55870 function?

Identifying small molecule modulators of uncharacterized proteins like zgc:55870 presents unique challenges requiring careful experimental design. The process begins with developing functional assays that can detect protein activity or interactions, despite limited knowledge of biological function. For enzymes, activity-based assays measuring substrate conversion provide direct readouts, while for proteins with unknown enzymatic activity, thermal shift assays detecting ligand-induced stabilization offer an alternative approach independent of functional knowledge.

High-throughput screening approaches should be designed with appropriate statistical controls and validation steps. Primary screens typically employ single-concentration testing against diverse compound libraries, with hits confirmed through dose-response studies and orthogonal assays. For zgc:55870, researchers might develop multiple assay formats including biochemical, cellular, and zebrafish-based systems to comprehensively evaluate compound effects across different contexts.

Structure-based approaches provide complementary strategies when structural information becomes available. Homology modeling based on related proteins can guide virtual screening efforts to identify potential binding sites and select compounds for experimental testing. Fragment-based screening using NMR or crystallography can identify chemical starting points for optimization even without prior knowledge of binding sites.

For in vivo validation, zebrafish embryos provide an excellent model system to evaluate compound effects in a developmental context. Transgenic lines expressing fluorescently tagged zgc:55870 can reveal compound-induced changes in protein localization, while phenotypic assays in zgc:55870 mutants can identify compounds that mimic or rescue loss-of-function phenotypes.

Methodologically, counter-screening against related proteins helps establish selectivity, while mechanism of action studies using proteomics, transcriptomics, or cellular imaging clarify how compounds affect protein function. For uncharacterized proteins, small molecule modulators not only serve as potential therapeutic leads but also as chemical probes to elucidate biological function through targeted perturbation.