Recombinant Rat Uncharacterized protein C6orf47 homolog (G4)

What is known about the structure and function of Rat G4 protein?

Rat G4 (Uncharacterized protein C6orf47 homolog) remains largely uncharacterized in terms of its specific biological functions. Current research indicates it is homologous to the human C6orf47 protein, with counterparts in other species including Macaca mulatta (Rhesus monkey) and Mus musculus (house mouse) .

The protein's structure has not been fully elucidated through crystallography. Based on sequence analysis, it shares evolutionary conserved regions with other mammalian C6orf47 homologs, suggesting potential functional importance across species. Researchers should consider comparative genomic approaches when investigating this protein's function, particularly examining conserved domains across species.

Methodological approach: To study G4's function, implement a multi-faceted approach combining:

• Differential expression analysis in various tissues

• Knockdown/knockout studies followed by phenotypic characterization

• Protein-protein interaction studies using techniques like co-immunoprecipitation

• Expression profiling under various experimental conditions

How is G4 expression regulated by chemical compounds and environmental factors?

G4 expression appears to be highly responsive to various chemical compounds. According to the Rat Genome Database annotations, multiple chemicals demonstrate significant effects on G4 expression :

Methodological approach: When studying chemical regulation of G4:

• Design dose-response experiments with time-course measurements

• Use qPCR to validate expression changes at the mRNA level

• Employ ChIP-seq to investigate transcription factor binding and epigenetic modifications

• Consider investigating multiple cell types, as regulation may be tissue-specific

What methods are most effective for detecting G4 protein in biological samples?

The most validated method for G4 protein detection is enzyme-linked immunosorbent assay (ELISA). Commercial ELISA kits for rat G4 are available with the following specifications :

Methodological considerations:

• For accurate results, sample concentrations must be diluted to mid-range of the kit detection limits

• ELISA kits are optimized for detection of native samples rather than recombinant proteins

• Western blotting can serve as a complementary method for verification, though specific antibodies may need to be validated

• Mass spectrometry-based approaches can be used for unbiased detection and quantification

How does G4 protein compare to its homologs in other species?

G4 has several identified homologs across mammalian species :

While sequence conservation suggests functional importance, the precise biological roles remain to be fully elucidated across species. The differences in chromosomal location (e.g., chromosome 6 in humans versus chromosome 4 in macaques) reflect evolutionary genomic reorganization.

Methodological approach:

• Perform comparative genomic analysis to identify conserved domains

• Use phylogenetic analysis to reconstruct evolutionary relationships

• Consider the utility of model organisms based on sequence conservation

• Examine expression patterns in homologous tissues across species

What expression patterns does G4 show across rat tissues and developmental stages?

While comprehensive expression profiling of G4 across all rat tissues is not fully documented in the provided literature, research approaches should focus on:

• Quantitative assessment using qPCR across major tissues

• Developmental time course studies from embryonic to adult stages

• Comparison with known expression patterns of human C6orf47

Preliminary data suggests G4 gene expression may be altered during brain development and differentiation processes, though it wasn't specifically highlighted among the significantly regulated genes in sexually mature male rat brains .

Methodological approach:

• Design tissue-specific qPCR assays using validated primer sets

• Consider RNA-seq for unbiased transcriptomic profiling

• Implement in situ hybridization to determine cell-type specificity

• Use reporter constructs to monitor expression in real-time in vivo

What are the best approaches for producing recombinant Rat G4 protein?

Production of recombinant G4 protein can be approached using several expression systems:

Methodological protocol:

• Clone the G4 coding sequence into appropriate expression vectors, considering fusion tags for purification (His, GST, MBP)

• For E. coli expression:

  • Use BL21(DE3) or Rosetta strains to enhance expression

  • Optimize induction conditions (temperature, IPTG concentration, time)

  • Consider fusion partners to improve solubility

• For mammalian expression:

  • HEK293T or CHO cells offer robust expression capabilities

  • Consider stable cell line development for repeated production

• Purification strategy:

  • Implement multi-step chromatography (affinity, ion exchange, size exclusion)

  • Validate protein identity by mass spectrometry

  • Assess purity by SDS-PAGE and activity by functional assays

How can gene editing techniques be applied to study G4 function in vivo?

CRISPR-Cas9 gene editing presents a powerful approach to investigate G4 function in rat models:

Methodological workflow:

• gRNA design:

  • Design several guide RNAs targeting different exons of G4

  • Validate guides in vitro before in vivo application

  • Consider potential off-target effects using computational predictions

• Delivery methods:

  • For cell lines: plasmid transfection, lentiviral vectors

  • For in vivo: adeno-associated virus (AAV), electroporation, or direct pronuclear injection for germline modifications

• Verification of editing:

  • PCR-based genotyping

  • Sanger sequencing of targeted regions

  • Western blotting to confirm protein knockout

  • qPCR to assess transcript levels

• Phenotypic characterization:

  • Systematic profiling of physiological parameters

  • Molecular analysis (transcriptomics, proteomics)

  • Context-specific functional assays based on expression patterns

• Complementary approaches:

  • Consider conditional knockout systems (Cre-loxP)

  • Implement rescue experiments to confirm specificity

  • Use knockin strategies for reporter or tagged versions

How do post-translational modifications affect G4 protein function?

While specific post-translational modifications (PTMs) of G4 have not been comprehensively characterized in the provided literature, this represents an important area for investigation.

Methodological approach:

• PTM profiling by mass spectrometry:

  • Purify G4 protein from different tissues/conditions

  • Perform MS/MS analysis with PTM-specific enrichment strategies

  • Common modifications to investigate: phosphorylation, glycosylation, ubiquitination, acetylation

• Site-directed mutagenesis:

  • Generate mutants at predicted modification sites

  • Assess functional consequences in appropriate assays

• Modification-specific antibodies:

  • Develop or acquire antibodies recognizing specific PTMs

  • Use for western blotting and immunoprecipitation

• Enzyme inhibitor studies:

  • Use inhibitors of kinases, phosphatases, acetyltransferases, etc.

  • Determine effects on G4 function and localization

• Disease context:

  • Investigate if alterations in PTMs are associated with pathological conditions

  • Compare PTM patterns between normal and disease states

What are the technical challenges in working with recombinant G4 protein?

Researchers working with recombinant G4 protein should be aware of several technical challenges:

Specific technical considerations:

• Solubility issues:

  • G4 may form aggregates or inclusion bodies in bacterial expression systems

  • Optimization strategies: lower induction temperature, reduced IPTG concentration, solubility tags

• Stability concerns:

  • The half-life of purified G4 may be limited

  • Buffer optimization is critical (pH, salt concentration, additives)

  • Consider storage in aliquots with cryoprotectants

• Detection limitations:

  • Commercial antibodies may have specificity issues

  • ELISA kits are optimized for native proteins rather than recombinant forms

  • Validation across multiple techniques is recommended

• Functional assays:

  • Due to limited characterization, establishing functional assays is challenging

  • Consider comparative approaches with homologs from other species

  • Leverage interacting partners to develop indirect functional readouts

• Crystallization challenges:

  • Uncharacterized proteins often present difficulties in crystallization

  • Consider alternative structural approaches (NMR, cryo-EM)

  • Experiment with various truncation constructs to identify stable domains

How is G4 involved in disease mechanisms and pathways?

Current understanding of G4's involvement in disease mechanisms is limited, but several research approaches can help elucidate potential roles:

Methodological approach:

• Expression analysis in disease models:

  • Compare G4 expression in normal versus disease tissues

  • Use quantitative techniques (qPCR, western blot, immunohistochemistry)

• Genetic association studies:

  • Investigate if variations in G4 are associated with specific conditions

  • Examine SNPs or structural variants in patient cohorts

• Pathway analysis:

  • Based on interaction partners, determine if G4 participates in known disease pathways

  • Consider systems biology approaches to position G4 in cellular networks

• Knockdown/knockout consequences:

  • Evaluate if G4 depletion causes disease-relevant phenotypes

  • Analyze in context of specific tissue types or developmental stages

• Therapeutic potential:

  • Assess if G4 could serve as a biomarker or therapeutic target

  • Investigate potential for antibody or small molecule targeting

The involvement of C6orf47 in human Lynch syndrome suggests potential roles for G4 in DNA mismatch repair pathways , though direct evidence for the rat homolog requires further investigation.

What is known about the epigenetic regulation of G4 expression?

The search results indicate that G4 expression may be regulated by methylation changes, as demonstrated by the effects of aflatoxin B1 and B2, which decrease methylation of the G4 promoter .

Methodological approaches:

• Methylation analysis:

  • Bisulfite sequencing of the G4 promoter region

  • Compare methylation patterns across tissues and conditions

  • Investigate the effects of DNA methyltransferase inhibitors

• Histone modification profiling:

  • ChIP-seq for various histone marks (H3K4me3, H3K27ac, H3K27me3)

  • Determine correlation between modifications and expression levels

• Chromatin accessibility:

  • ATAC-seq or DNase-seq to assess open chromatin regions

  • Identify potential regulatory elements affecting G4 expression

• Transcription factor binding:

  • ChIP-seq for relevant transcription factors

  • Perform reporter assays with mutated binding sites

• Non-coding RNA interactions:

  • Investigate if miRNAs or lncRNAs regulate G4 expression

  • Perform RNA immunoprecipitation to identify RNA-protein interactions