Recombinant Mouse RING finger protein 222 (Rnf222)

What is Mouse RNF222 and what are its basic molecular properties?

Mouse RNF222 (Ring finger protein 222) is a protein encoded by the Rnf222 gene (Gene ID: 320040) in Mus musculus with UniProt ID Q8CEF8 . As a RING finger protein, it likely possesses E3 ubiquitin ligase activity, although its specific substrates and functional roles are still being characterized. The protein contains the characteristic RING domain which coordinates zinc ions and mediates protein-protein interactions critical for ubiquitination processes.

When expressed recombinantly, RNF222 is typically produced as a full-length or partial-length protein with various affinity tags (commonly His-tag) to facilitate purification . The recombinant protein is available in either liquid form or as a lyophilized powder with purity typically exceeding 80%, with endotoxin levels below 1.0 EU per μg as determined by the LAL method .

How should recombinant mouse RNF222 protein be stored and handled in the laboratory?

For optimal stability and activity, recombinant mouse RNF222 requires proper storage conditions:

• Short-term storage (up to 2 weeks): Store at +4°C in PBS buffer

• Long-term storage: Store at -20°C to -80°C

When working with the protein:

• Minimize freeze-thaw cycles to prevent protein degradation

• Thaw aliquots on ice and keep samples cold during handling

• For reconstitution of lyophilized powder, use sterile, cold PBS buffer

• After reconstitution, centrifuge briefly to collect the solution at the bottom of the tube

• The protein is typically stable for at least one week when stored at 4°C, though activity may gradually decrease

What are the typical research applications for recombinant mouse RNF222?

Recombinant mouse RNF222 can be utilized in various research contexts:

• Protein-protein interaction studies: Identifying binding partners through pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens

• Ubiquitination assays: Examining potential E3 ligase activity and substrate specificity

• Structural biology: Crystallization trials or NMR studies to determine protein structure

• Antibody production: Generating and validating antibodies against mouse RNF222

• Enzymatic activity assays: Characterizing potential catalytic functions

When designing experiments, researchers should consider the specific tag used in the recombinant protein and its potential influence on protein folding, activity, and interactions. For interaction studies, it's advisable to use multiple approaches to validate findings, as single methodologies may yield false positives or negatives.

How can I design experiments to investigate the potential E3 ligase activity of RNF222?

To study the E3 ligase activity of RNF222, consider implementing the following experimental approach:

In vitro ubiquitination assay protocol:

• Prepare reaction mixture containing:

  • Recombinant RNF222 (200-500 ng)

  • E1 ubiquitin-activating enzyme (100-200 ng)

  • E2 ubiquitin-conjugating enzyme panel (test multiple E2s initially)

  • Ubiquitin (1-5 μg)

  • ATP regeneration system (2 mM ATP, 10 mM creatine phosphate, 3.5 U/ml creatine kinase)

  • Reaction buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 2 mM DTT)

• Incubate at 30°C for 1-2 hours

• Stop reaction with SDS-PAGE sample buffer

• Analyze by western blotting using anti-ubiquitin antibodies

Drawing parallels from research on other RING finger proteins, such as the MuRF2-mediated ubiquitination of PPARγ1 at lysine 222 , can provide methodological insights. MuRF2 studies have demonstrated successful approaches to identifying specific ubiquitination sites using computational prediction, immunoprecipitation, ubiquitination assays, and cycloheximide chase experiments .

What CRISPR/Cas9 strategies can be used to investigate RNF222 function in cellular models?

CRISPR/Cas9 technology offers powerful approaches to study RNF222 function through gene knockout or modification. Based on established protocols, researchers should consider:

• Design multiple guide RNAs targeting RNF222: At least two gRNA constructs are recommended to increase success probability . The Zhang laboratory at the Broad Institute has designed efficient gRNAs targeting the RNF222 gene with minimal off-target effects .

• Vector selection considerations:

  • Choose vectors containing appropriate selection markers

  • Ensure vectors include all required elements: U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator

• Validation strategies:

  • Genomic PCR and sequencing to confirm mutations

  • Western blotting to verify protein knockdown

  • RT-qPCR to assess mRNA levels

How do structure prediction tools aid in understanding RNF222 function and interactions?

Advanced structural bioinformatics approaches can significantly enhance our understanding of RNF222 function:

• Template-based structure prediction: Tools like StarFunc combine template-based and deep learning approaches for protein function prediction . These methods can:

  • Perform fast Foldseek-based structure prefiltering

  • Select related templates for full-length TM-align alignment

  • Provide insights into structural features and potential functional domains of RNF222

• Structure-function analysis workflow:

  • Identify conserved domains through sequence analysis

  • Generate structural models using homology modeling or AI-based approaches

  • Predict binding pockets and functional sites

  • Compare with structures of well-characterized RING finger proteins to infer functional capabilities

The RING domain of RNF222 likely adopts a cross-brace arrangement coordinating two zinc ions, similar to other RING finger proteins. This structural feature is critical for recruiting E2 ubiquitin-conjugating enzymes and facilitating ubiquitin transfer to substrates.

What are the critical considerations when designing experiments to identify RNF222 substrates?

Identifying physiological substrates of E3 ubiquitin ligases like RNF222 presents significant challenges. A comprehensive experimental approach should include:

• Proximity-based labeling:

  • Express BioID or TurboID-fused RNF222 in relevant cell types

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Filter candidates based on enrichment scores and biological relevance

• Ubiquitinome analysis:

  • Compare global ubiquitination patterns in RNF222 knockout vs. wild-type cells

  • Utilize diGly-lysine antibodies to enrich ubiquitinated peptides

  • Perform quantitative proteomics to identify differentially ubiquitinated proteins

• Validation criteria:

  • Direct interaction with RNF222 (co-IP, GST-pulldown)

  • Increased ubiquitination in presence of RNF222

  • Reduced ubiquitination in RNF222 knockout/knockdown

  • Identification of specific ubiquitination sites

Drawing on approaches used for other RING finger proteins, researchers should consider methodologies similar to those used in characterizing MuRF2-mediated ubiquitination, which successfully identified lysine 222 as the key ubiquitination site in PPARγ1 .

How can post-translational modifications of RNF222 be investigated and their impact on function assessed?

Post-translational modifications (PTMs) often regulate E3 ligase activity. To study PTMs of RNF222:

• PTM identification workflow:

  • Immunoprecipitate endogenous or tagged RNF222 from tissues/cells

  • Perform mass spectrometry analysis to identify PTMs

  • Focus on phosphorylation, SUMOylation, acetylation, and auto-ubiquitination

• Functional impact assessment:

  • Generate site-specific mutants (e.g., phospho-mimetic or phospho-deficient)

  • Compare E3 ligase activity using in vitro ubiquitination assays

  • Assess protein stability through cycloheximide chase experiments

  • Examine subcellular localization through immunofluorescence

  • Analyze protein interaction changes using co-IP or proximity labeling

• Kinase identification (for phosphorylation):

  • In vitro kinase assays with candidate kinases

  • Inhibitor studies in cell culture

  • Bioinformatic prediction of kinase recognition motifs

Understanding PTM regulation of RNF222 may reveal mechanisms for controlling its activity in different cellular contexts and physiological conditions.

What control experiments are critical when working with recombinant RNF222?

When designing experiments with recombinant RNF222, include these essential controls:

• Negative controls:

  • Catalytically inactive mutant (mutation in RING domain)

  • Heat-denatured protein

  • Empty vector or irrelevant protein with matching tag

• Positive controls:

  • Well-characterized RING E3 ligase (if studying ubiquitination)

  • Known interaction partners of similar RING proteins (for interaction studies)

• Validation controls:

  • Multiple independent batches of recombinant protein

  • Different expression systems (bacterial, insect, mammalian)

  • Alternative affinity tags to rule out tag interference

• Specificity controls:

  • Competitive binding with untagged protein

  • Dose-dependent effects

  • Substrate specificity assessment with multiple potential targets

These controls help distinguish true biological activities from artifacts related to the recombinant protein preparation or experimental conditions.

How should data tables and figures be properly formatted for publications involving RNF222 research?

Following standard scientific publication practices for data presentation will enhance the clarity and impact of RNF222 research:

• Data table formatting best practices:

  • Clearly label independent variables (e.g., experimental conditions) and dependent variables (measured outcomes)3

  • Include appropriate units of measurement for all numeric data

  • Present standard deviations or standard errors

  • Use consistent significant figures throughout

  • Merge cells for multi-column headers to improve visual organization3

• Figure preparation guidelines:

  • Plot independent variables on the x-axis and dependent variables on the y-axis

  • Include clear labels with units

  • Provide detailed figure legends explaining experimental conditions

  • Use consistent color schemes throughout the manuscript

  • Include statistical significance indicators

What are common challenges in working with recombinant RNF222 and how can they be addressed?

Researchers frequently encounter specific challenges when working with RING finger proteins like RNF222:

• Protein solubility issues:

  • Challenge: RING domain proteins may aggregate due to improper zinc coordination

  • Solution: Include zinc in purification buffers (10-50 μM ZnCl₂), optimize expression temperature (16-18°C), consider fusion tags (MBP, SUMO) to enhance solubility

• E3 ligase activity detection:

  • Challenge: Weak or undetectable activity in vitro

  • Solution: Test multiple E2 enzymes, optimize buffer conditions (pH, salt, reducing agents), consider adding zinc, ensure protein is not oxidized

• Substrate identification difficulties:

  • Challenge: Non-specific binding in pulldown assays

  • Solution: Use stringent washing conditions, include competitors, perform sequential purification steps, validate with multiple approaches

• Antibody specificity:

  • Challenge: Cross-reactivity with other RING proteins

  • Solution: Validate antibodies using knockout controls, use epitope-tagged versions, confirm key findings with multiple antibodies

How can researchers distinguish the specific functions of RNF222 from other RING finger proteins?

Differentiating the unique functions of RNF222 from other RING finger proteins requires targeted experimental approaches:

• Domain-specific analysis:

  • Identify unique domains or motifs outside the RING domain

  • Create chimeric proteins by swapping domains between RNF222 and related proteins

  • Perform structure-function analysis to correlate specific structural elements with functional outcomes

• Tissue/cell-type specificity:

  • Analyze expression patterns across tissues using RT-qPCR

  • Perform immunohistochemistry to determine cellular and subcellular localization

  • Investigate cell-type specific phenotypes using conditional knockout models

• Substrate specificity determination:

  • Compare ubiquitination targets with closely related RING proteins

  • Identify specific recognition motifs in substrates

  • Use competition assays to evaluate substrate preference

When designing CRISPR-based approaches to study RNF222 function, researchers should carefully validate guide RNA specificity to ensure they don't inadvertently target homologous regions in related RING finger proteins .