Recombinant Danio rerio Chitinase domain-containing protein 1 (chid1)

What is the genomic and evolutionary background of Danio rerio chid1?

Danio rerio chid1 belongs to the glycoside hydrolase family 18, which includes chitinases and non-enzymatic chitinase-like proteins. Unlike most vertebrate species where CHID1 evolved without gene duplication, zebrafish represents a notable exception. Phylogenetic analyses have revealed that the CHID1 gene underwent duplication specifically in Danio rerio, while the domain architectures of CHID1 orthologs remain identical across species . The zebrafish chid1 gene is cataloged in the NCBI Danio rerio Annotation Release 104, which includes 26,603 protein-coding genes across the zebrafish genome .

How does the structure of Danio rerio chid1 compare to mammalian orthologs?

Danio rerio chid1 shares structural similarities with its mammalian counterparts, particularly in the chitinase domain. The protein is characterized by its extracellular localization and contains a catalytic domain similar to that found in other chitinase family proteins. The human ortholog (CHID1, UniProt: Q13231) contains binding sites for chitin, heparin, and hyaluronic acid . Comparative analyses of zebrafish and human chitinase domains reveal conserved functional regions, though species-specific variations exist in non-catalytic regions. The protein's extracellular localization suggests it functions in the extracellular matrix or potentially in secreted form, similar to human CHI3L1 .

What expression systems are most effective for producing recombinant Danio rerio chid1?

Based on established protocols for similar chitinase domain-containing proteins, E. coli expression systems are commonly used for recombinant production of zebrafish chid1. The bacterial expression approach typically involves:

• Cloning the DNA sequence encoding Danio rerio chid1 into a suitable expression vector (similar to methods used for human CHID1)

• Transformation into an E. coli strain optimized for recombinant protein expression

• Induction of protein expression using IPTG or auto-induction media

• Purification via affinity chromatography using tags such as His-tag

For more complex applications requiring post-translational modifications, mammalian or insect cell expression systems may be preferable, though with potentially lower yields compared to bacterial systems.

What purification strategies yield the highest purity and activity for recombinant chid1?

A multi-step purification protocol is recommended for obtaining high-purity recombinant Danio rerio chid1:

• Initial capture using affinity chromatography (His-tag purification is common)

• Secondary purification using ion exchange chromatography

• Final polishing step using size exclusion chromatography

Standard quality control metrics should include:

Researchers should maintain the purified protein in a Tris-based buffer with 50% glycerol for optimal stability, similar to conditions used for other recombinant chitinase-domain proteins .

What are the known biochemical properties and activities of Danio rerio chid1?

While specific biochemical characterization of Danio rerio chid1 is limited in the literature, inferences can be made based on related chitinase-domain proteins. The protein likely binds chitin, heparin, and hyaluronic acid, similar to human CHI3L1 . Its presumed functions include:

• Potential involvement in extracellular matrix remodeling

• Possible role in inflammatory responses and tissue repair

• Interaction with glycosaminoglycans in the extracellular matrix

To assess enzymatic activity, researchers should employ:

• Fluorogenic substrates (4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside)

• Colorimetric assays using p-nitrophenyl-N-acetyl-β-D-glucosaminide

• Turbidimetric assays with colloidal chitin suspensions

How can I determine if my recombinant Danio rerio chid1 is properly folded and functional?

Multiple complementary approaches should be used to confirm proper folding and functionality:

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

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

• Size exclusion chromatography: To confirm monomeric state and absence of aggregation

• Binding assays: Using chitin beads or glycosaminoglycan arrays to verify substrate binding

• Activity assays: Using appropriate substrates mentioned in 3.1

For functional validation, compare the recombinant protein to a positive control like human CHID1, with the caveat that substrate specificity may differ between species orthologs.

What are effective strategies for generating chid1 knockout zebrafish models?

Based on successful CRISPR/Cas9 editing protocols in zebrafish , the following approach is recommended for generating chid1 knockout models:

• Guide RNA design: Target conserved regions within the coding sequence, particularly within the chitinase domain. Multiple gRNAs (2-3) targeting different exons can increase knockout efficiency.

• Microinjection protocol:

  • Inject 2-4 cell stage zebrafish embryos with a mixture of:

    • Cas9 protein (300-500 pg) or Cas9 mRNA (150-300 pg)

    • Guide RNAs (25-50 pg each)

    • Phenol red (tracer dye)

• Screening protocol:

  • At 24 hours post-fertilization, extract genomic DNA from a subset of injected embryos

  • Perform PCR across the target region followed by T7 endonuclease assay or direct sequencing

  • Expected efficiency ranges from 20-80% for F0 embryos

• Establishing stable lines:

  • Raise F0 founders to adulthood

  • Outcross with wild-type fish and screen F1 offspring

  • Incross F1 heterozygotes to obtain homozygous knockouts in F2

Note that transmission rates to F1 may be lower than initial targeting efficiency, as observed in other zebrafish knockout studies (0.01-0.025%) .

What phenotyping approaches are most informative for characterizing chid1 mutant zebrafish?

A comprehensive phenotyping strategy should include:

• Molecular confirmation:

  • PCR genotyping and sequencing to confirm mutations

  • qPCR and Western blotting to verify loss of chid1 expression

• Developmental analysis:

  • Morphological assessment at key developmental stages

  • Survival and growth rate monitoring

  • Assessment of tissue integrity using histological methods

• Functional assays:

  • Evaluation of inflammatory responses using transgenic lines like Tg(nkx2.2a:mEGFP)

  • Assessment of wound healing and tissue repair capabilities

  • Examination of sensitivity to pathogens or immune challenges

• Compensatory mechanisms:

  • Analysis of expression changes in related chitinase family genes

  • Assessment of genetic compensation similar to that observed in vitellogenin knockout studies

How can chid1 be used to study evolutionary conservation of chitinase functions across species?

Zebrafish chid1 provides an excellent model for evolutionary studies of chitinase functions due to its unique gene duplication pattern . A comprehensive approach would include:

• Comparative genomic analysis:

  • Alignment of chitinase domains across species from fish to mammals

  • Identification of conserved versus divergent regions

  • Phylogenetic analysis to map evolutionary relationships

• Cross-species functional complementation:

  • Generate knockout zebrafish and attempt rescue with orthologs from other species

  • Assess if human CHID1 can rescue zebrafish chid1 knockout phenotypes

  • Evaluate if zebrafish chid1 can functionally substitute for mammalian orthologs in cell culture

• Domain swap experiments:

  • Create chimeric proteins with domains from different species

  • Test functionality of these chimeras in appropriate systems

  • Map species-specific versus universally conserved functional regions

This approach can be modeled after successful cross-species functional studies such as those performed with Nanog, where zebrafish knockout could be rescued by murine orthologs but not by more distant homologs .

How can recombinant Danio rerio chid1 be used for high-throughput screening of potential inhibitors?

A robust screening platform would include:

• Assay development:

  • Establish fluorescence-based activity assays using synthetic substrates

  • Develop binding assays using surface plasmon resonance or thermal shift

  • Create cell-based assays using zebrafish cell lines or embryos

• Screening workflow:

  • Primary screen: Enzymatic or binding assays with compound libraries

  • Secondary validation: Orthogonal assays to confirm hits

  • Tertiary validation: Testing in zebrafish embryos or cell models

• Data analysis framework:

  • Dose-response curve generation

  • Structure-activity relationship analysis

  • Comparison with effects on mammalian orthologs

This approach could yield valuable tool compounds for studying chid1 function or potential therapeutic leads for related human conditions where chitinase activity is implicated.

How does chid1 expression change during key developmental stages in zebrafish?

Based on expression patterns of other developmentally regulated zebrafish genes, researchers should investigate chid1 expression through:

• Temporal expression analysis:

  • Perform qPCR at defined developmental stages (2-4 cell, shield, somitogenesis, 24 hpf, 48 hpf, 72 hpf, and 5 dpf)

  • Western blot analysis at key time points

  • RNA-seq analysis to place chid1 in broader gene expression networks

• Spatial expression analysis:

  • Whole-mount in situ hybridization to map tissue-specific expression

  • Fluorescent reporter constructs using the chid1 promoter

  • Single-cell RNA-seq to identify cell types expressing chid1

• Regulatory analysis:

  • Promoter analysis to identify developmental transcription factor binding sites

  • Treatment with developmental pathway modulators to assess regulation

  • ChIP-seq for key developmental transcription factors at the chid1 locus

This multi-faceted approach would reveal not only when and where chid1 is expressed during development but also provide insights into its regulation and potential functions.