Recombinant Mouse UPF0420 protein C16orf58 homolog

What is UPF0420 protein C16orf58 homolog and what are its general characteristics?

The UPF0420 protein C16orf58 homolog is a protein that belongs to the uncharacterized protein family 0420. In mice, this protein consists of 466 amino acids. The designation "UPF" indicates that this is part of an uncharacterized protein family, suggesting that its full functional profile has not yet been completely elucidated. The protein is orthologous to the human C16orf58 protein (encoded on chromosome 16 in humans) . The full amino acid sequence is known and has been documented in protein databases, with mouse UPF0420 protein C16orf58 homolog having the UniProt accession number Q91W34 .

How should recombinant mouse UPF0420 protein C16orf58 homolog be stored for optimal stability?

For optimal stability, recombinant mouse UPF0420 protein C16orf58 homolog should be stored at -20°C. For extended storage, conservation at -20°C or -80°C is recommended. The protein is typically supplied in a Tris-based buffer with 50% glycerol that has been optimized for this specific protein . It's important to note that repeated freezing and thawing is not recommended as this can lead to protein degradation and loss of activity. For ongoing experiments, working aliquots can be stored at 4°C for up to one week .

What expression systems are commonly used to produce recombinant mouse UPF0420 protein C16orf58 homolog?

Based on the available data, E. coli is the primary expression system used for producing recombinant mouse UPF0420 protein C16orf58 homolog. The protein is typically expressed with a His-tag to facilitate purification using affinity chromatography . This bacterial expression system is widely used for recombinant proteins due to its relatively low cost, high yield, and established protocols for optimization. While E. coli has limitations in producing mammalian proteins with complex post-translational modifications, it appears suitable for the expression of this particular protein .

What are the known biological functions and pathways associated with UPF0420 protein C16orf58 homolog?

The UPF0420 protein C16orf58 homolog is still categorized as an uncharacterized protein, indicating that its precise biological functions remain to be fully elucidated. The search results indicate that this protein "involved in several pathways and played different roles in them," but specific pathway details are not provided . This gap in knowledge represents an opportunity for researchers to conduct further investigations to characterize the protein's functions, potentially through techniques such as gene knockout studies, protein-protein interaction analyses, or functional genomics approaches. The lack of detailed pathway information suggests that determining the biological role of this protein remains an active area of research .

What experimental approaches are recommended for studying protein-protein interactions involving UPF0420 protein C16orf58 homolog?

For studying protein-protein interactions involving UPF0420 protein C16orf58 homolog, several established methods would be appropriate. The search results mention that interactions with this protein have been detected using techniques such as "yeast two hybrid, co-IP, pull-down and so on" .

To implement these approaches effectively:

• Yeast Two-Hybrid (Y2H): This system could be used to screen for potential interacting partners by expressing the UPF0420 protein as a bait fusion protein and testing against a library of prey proteins.

• Co-Immunoprecipitation (Co-IP): Using antibodies against the UPF0420 protein or its tagged version to pull down protein complexes from cell lysates, followed by mass spectrometry to identify interacting partners.

• Pull-down Assays: Utilizing the His-tagged recombinant protein as bait to capture interacting proteins from cell lysates, followed by identification of bound proteins.

• Proximity-based Labeling: Methods such as BioID or APEX could be employed to identify proteins in close proximity to UPF0420 protein in living cells.

The choice of method would depend on specific research questions and available resources, but a combination of complementary approaches would provide the most robust results for identifying and validating protein interactions .

How can recombinant UPF0420 protein C16orf58 homolog be used in immunological studies?

While the search results don't provide specific examples of UPF0420 protein C16orf58 homolog being used in immunological studies, insights can be drawn from similar recombinant protein applications. Based on general principles and the information about recombinant proteins like LpxC and GmhA mentioned in the search results, the following approaches would be methodologically sound:

• Antibody Production: The purified recombinant protein could be used as an immunogen to generate specific antibodies for research applications. These antibodies could then be employed in various techniques including Western blotting, immunohistochemistry, or flow cytometry.

• Immunization Studies: Similar to the study on LpxC and GmhA proteins described in the search results, recombinant UPF0420 protein could potentially be used in immunization studies to evaluate its immunogenic properties .

• ELISA Development: As indicated by the product listings, recombinant UPF0420 protein could be used in ELISA assays, either as a standard or for the development of quantitative detection methods .

The appropriate methodology would depend on the specific research objectives and would need to be optimized for this particular protein .

What are the challenges and considerations when using recombinant mouse UPF0420 protein C16orf58 homolog in in vivo studies?

When using recombinant mouse UPF0420 protein C16orf58 homolog in in vivo studies, researchers should consider several important factors:

• Protein Purity and Endotoxin Levels: For in vivo applications, ensuring high purity and low endotoxin levels is crucial to prevent non-specific inflammatory responses that could confound experimental results.

• Dosage Determination: Based on similar protein studies mentioned in the search results, careful titration of protein dosage would be necessary. For instance, in the immunization study with LpxC and GmhA proteins, 100 μg/100 μL was used .

• Adjuvant Selection: The choice of adjuvant can significantly impact the immune response to the protein. In the referenced study, Freund's complete adjuvant was used .

• Administration Route: The administration route affects biodistribution and immune responses. Subcutaneous injection was used in the referenced immunization study and would be a reasonable starting point .

• Species-Specific Considerations: When using mouse protein in mouse models, potential tolerance issues should be considered, especially if studying immune responses.

• Stability In Vivo: The protein's half-life and stability in physiological conditions need to be assessed to determine appropriate dosing schedules.

A typical experimental design for in vivo studies might follow this format:

This design is based on similar protocols used for recombinant protein studies in mouse models .

What analytical methods are most effective for assessing the quality and activity of recombinant UPF0420 protein C16orf58 homolog preparations?

For comprehensive quality assessment of recombinant UPF0420 protein C16orf58 homolog preparations, a multi-faceted analytical approach is recommended:

• Purity Assessment:

  • SDS-PAGE with Coomassie staining to visualize protein bands and assess purity

  • HPLC or capillary electrophoresis for quantitative purity determination

  • Mass spectrometry for accurate molecular weight confirmation and identification of potential modifications

• Identity Confirmation:

  • Western blot analysis using specific antibodies, as mentioned in the search results for similar proteins

  • Peptide mapping and mass spectrometry for sequence verification

  • N-terminal sequencing to confirm the correct start of the protein

• Structural Integrity:

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Dynamic light scattering for aggregation analysis

• Functional Assessment:

  • Since the specific function of UPF0420 protein is not well-characterized, functional assays would need to be developed based on putative activities

  • If protein-protein interactions are known, binding assays could be employed

  • If enzymatic activity is suspected, appropriate biochemical assays would need to be established

• Endotoxin Testing:

  • Limulus Amebocyte Lysate (LAL) assay for endotoxin quantification, particularly important for in vivo applications

These analytical methods provide complementary information about different aspects of protein quality and should be selected based on the intended application of the recombinant protein .

What strategies can optimize expression and purification of recombinant mouse UPF0420 protein C16orf58 homolog?

To optimize expression and purification of recombinant mouse UPF0420 protein C16orf58 homolog, several strategies can be implemented:

• Expression System Optimization:

  • The search results indicate that E. coli is commonly used for expression

  • Consider testing different E. coli strains (BL21(DE3), Rosetta, Arctic Express) to identify optimal expression

  • Evaluate expression temperature (typically 16-37°C), with lower temperatures often favoring proper folding

  • Test various induction conditions (IPTG concentration, induction time)

• Vector and Tag Selection:

  • His-tags are commonly used for this protein as indicated in the search results

  • Consider testing different tag positions (N-terminal vs. C-terminal) for optimal expression and function

  • Evaluate the impact of tag cleavage on protein stability and function

• Purification Protocol Development:

  • Implement a multi-step purification strategy, starting with affinity chromatography using the His-tag

  • Follow with polishing steps such as ion exchange or size exclusion chromatography

  • Monitor protein purity at each step using SDS-PAGE

• Solubility Enhancement:

  • If facing solubility issues, test different buffer compositions, pH conditions, and additives

  • Consider fusion partners known to enhance solubility (e.g., MBP, SUMO, thioredoxin)

  • Evaluate refolding protocols if the protein forms inclusion bodies

• Stability Considerations:

  • As indicated in the search results, include 50% glycerol in the storage buffer

  • Test various buffer compositions for optimal stability

  • Prepare single-use aliquots to avoid freeze-thaw cycles

A systematic approach to optimization, with careful documentation of conditions and outcomes, will lead to the most efficient production protocol for this specific protein .

How can researchers validate the biological activity of recombinant UPF0420 protein C16orf58 homolog in cellular systems?

• Cell-Based Functional Assays:

  • Since specific functions are not well-defined, researchers could perform comparative transcriptomics or proteomics in cells treated with the recombinant protein versus controls

  • Monitor changes in cellular phenotypes (proliferation, migration, morphology) upon protein treatment

  • Assess impact on signaling pathways using phosphorylation-specific antibodies for key signaling molecules

• Localization Studies:

  • Use labeled recombinant protein (fluorescent tag or detectable epitope) to track cellular uptake and localization

  • Perform subcellular fractionation after protein treatment to determine compartmentalization

  • Conduct co-localization studies with known cellular markers

• Protein-Protein Interaction Validation:

  • Implement proximity ligation assays to detect interactions in situ

  • Perform FRET/BRET analyses with fluorescently tagged proteins

  • Use cellular thermal shift assays (CETSA) to assess target engagement

• Loss/Gain of Function Approaches:

  • Compare the effects of recombinant protein addition with those observed in knockdown/knockout systems

  • Complement gene silencing with recombinant protein to assess functional rescue

  • Overexpress the protein and compare effects with exogenous recombinant protein addition

• Competition Assays:

  • Use labeled and unlabeled protein in competition assays to confirm specific binding

  • Develop dose-response curves to characterize activity parameters

  • Use blocking antibodies to confirm specificity of observed effects

These methodological approaches provide a framework for systematically investigating the biological activity of this protein, even in the absence of detailed functional information .

What considerations are important when designing antibodies against mouse UPF0420 protein C16orf58 homolog?

When designing antibodies against mouse UPF0420 protein C16orf58 homolog, several key methodological considerations should be addressed:

• Epitope Selection:

  • Analyze the amino acid sequence provided in the search results for regions with high antigenicity and surface accessibility

  • Consider evolutionary conservation if antibodies need to recognize orthologs in other species

  • Avoid regions with high similarity to other proteins to minimize cross-reactivity

  • Select multiple epitopes from different regions of the protein to increase success probability

• Antibody Format Selection:

  • Determine whether polyclonal or monoclonal antibodies are more appropriate for the intended application

  • For specific epitope recognition, consider monoclonal antibodies

  • For maximum epitope coverage, polyclonal antibodies may be advantageous

  • Evaluate different antibody isotypes based on intended applications

• Immunization Strategy:

  • Use the purified full-length recombinant protein as described in the search results

  • Consider a prime-boost immunization schedule with appropriate adjuvants

  • Monitor antibody titers during immunization to determine optimal harvest timing

  • Include appropriate controls to validate specificity

• Validation Methods:

  • Implement Western blot analysis against both recombinant protein and native protein in tissue lysates

  • Perform immunoprecipitation to confirm antibody-antigen interaction

  • Include knockout/knockdown controls to confirm specificity

  • Test cross-reactivity against related proteins or orthologs

• Application-Specific Optimization:

  • For immunohistochemistry, test different fixation methods and antigen retrieval protocols

  • For flow cytometry, optimize antibody concentration and buffer conditions

  • For ELISA development, determine optimal coating conditions and detection parameters

By addressing these methodological considerations, researchers can develop reliable antibodies against mouse UPF0420 protein C16orf58 homolog that are suitable for their specific research applications .

How might researchers investigate the evolutionary conservation of UPF0420 protein C16orf58 homolog across species?

To investigate the evolutionary conservation of UPF0420 protein C16orf58 homolog across species, researchers could implement the following methodological approach:

• Sequence Retrieval and Multiple Sequence Alignment:

  • Obtain the mouse UPF0420 protein sequence (466 amino acids) from the search results

  • Use bioinformatic tools like BLAST to identify homologs in other species

  • Perform multiple sequence alignments using tools such as MUSCLE, CLUSTAL, or T-Coffee

  • Generate conservation scores for each amino acid position

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood, Bayesian, or distance-based methods

  • Determine the evolutionary relationship between UPF0420 protein C16orf58 homologs

  • Identify key evolutionary events such as gene duplications or losses

  • Compare the evolutionary rate with that of other proteins

• Domain and Motif Analysis:

  • Identify conserved domains and motifs across species

  • Map conservation scores onto predicted structural elements

  • Determine whether functional motifs are more highly conserved than other regions

  • Compare domain architecture across different taxonomic groups

• Synteny Analysis:

  • Examine the genomic context of UPF0420 protein C16orf58 homolog across species

  • Identify conserved gene neighborhoods that might indicate functional relationships

  • Investigate whether genomic rearrangements have occurred around this gene

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to determine whether the protein is under purifying or positive selection

  • Identify specific residues under selection pressure

  • Correlate selection patterns with functional or structural elements

This systematic approach would provide comprehensive insights into the evolutionary history and conservation patterns of UPF0420 protein C16orf58 homolog, potentially revealing functional importance and constraints that have shaped its evolution .

What are the potential applications of recombinant UPF0420 protein C16orf58 homolog in disease research models?

While the specific role of UPF0420 protein C16orf58 homolog in disease contexts is not explicitly detailed in the search results, several methodological approaches could be employed to investigate its potential applications in disease research:

• Expression Analysis in Disease Models:

  • Compare expression levels of UPF0420 protein C16orf58 homolog in healthy versus diseased tissues

  • Conduct temporal expression studies during disease progression

  • Perform single-cell analyses to identify cell type-specific expression patterns in disease contexts

• Functional Studies in Disease Models:

  • Generate knockout or knockdown models to assess the impact on disease phenotypes

  • Administer recombinant protein therapeutically to determine potential beneficial effects

  • Investigate protein-protein interactions specifically altered in disease states

• Biomarker Development:

  • Evaluate UPF0420 protein C16orf58 homolog as a potential biomarker for specific diseases

  • Develop detection methods using the recombinant protein as a standard

  • Assess correlation between protein levels and disease severity or progression

• Therapeutic Target Validation:

  • Use the recombinant protein in high-throughput screening assays to identify potential modulators

  • Develop protein-based therapeutics if functional studies indicate beneficial effects

  • Generate antibodies against specific epitopes for targeted interventions

• Mouse Model Development:

  • Create transgenic mouse models with altered expression of UPF0420 protein

  • Challenge these models with disease-inducing conditions

  • Administer recombinant protein in established disease models similar to the approach used with LpxC and GmhA proteins described in the search results

These approaches provide a methodological framework for investigating the potential roles and applications of UPF0420 protein C16orf58 homolog in various disease contexts, which could lead to novel diagnostic or therapeutic strategies .

How can researchers address challenges in determining the three-dimensional structure of UPF0420 protein C16orf58 homolog?

Determining the three-dimensional structure of UPF0420 protein C16orf58 homolog presents several challenges, particularly given its uncharacterized nature. Researchers can employ the following methodological approaches to address these challenges:

This comprehensive approach addresses various challenges in structural biology and provides multiple avenues to determine the three-dimensional structure of this uncharacterized protein .

What are the most promising research directions for further characterizing UPF0420 protein C16orf58 homolog?

Based on the current state of knowledge reflected in the search results, several promising research directions emerge for further characterizing UPF0420 protein C16orf58 homolog:

• Comprehensive Functional Characterization:

  • Systematic knockout/knockdown studies in cellular and animal models

  • High-throughput interactome analysis to identify binding partners

  • Transcriptomic and proteomic profiling following perturbation of protein expression

  • Development of specific functional assays based on preliminary findings

• Structural Biology Investigations:

  • Determination of three-dimensional structure through X-ray crystallography or cryo-EM

  • Structure-function relationship studies through site-directed mutagenesis

  • Identification of functional domains and critical residues

  • Computational modeling and simulation to predict dynamic properties

• Physiological Role Elucidation:

  • Generation and characterization of transgenic mouse models

  • Tissue-specific and developmental expression profiling

  • Phenotypic analysis of gene-modified animals under various conditions

  • Investigation of potential roles in specific physiological processes

• Disease Association Studies:

  • Analysis of expression in various disease models

  • Investigation of genetic variations and their impact on protein function

  • Evaluation as a potential biomarker or therapeutic target

  • Development of modulators (activators or inhibitors) based on functional insights

• Evolutionary and Comparative Studies:

  • Detailed phylogenetic analysis across species

  • Comparative functional studies of orthologs

  • Investigation of selection pressures that have shaped protein evolution

  • Identification of conserved features that may indicate functional importance

These research directions provide a roadmap for systematically unraveling the biological significance of UPF0420 protein C16orf58 homolog, potentially leading to novel insights into cellular processes and disease mechanisms .

How can researchers contribute to standardizing methods for working with poorly characterized proteins like UPF0420 protein C16orf58 homolog?

Researchers can contribute to standardizing methods for working with poorly characterized proteins like UPF0420 protein C16orf58 homolog by implementing several methodological approaches:

• Protocol Optimization and Validation:

  • Develop detailed, reproducible protocols for expression and purification as mentioned in the search results

  • Systematically test and document buffer conditions, storage parameters, and stability factors

  • Establish quality control criteria specific to this protein class

  • Create reference standards for assessing batch-to-batch consistency

• Assay Development and Standardization:

  • Design and validate functional assays based on predicted activities or structural features

  • Establish positive and negative controls for each assay

  • Determine assay sensitivity, specificity, and reproducibility metrics

  • Share assay protocols through protocol repositories or method-focused publications

• Data Sharing and Integration:

  • Deposit all sequence, structural, and functional data in appropriate public databases

  • Contribute to specialized databases for uncharacterized protein families

  • Implement FAIR (Findable, Accessible, Interoperable, Reusable) data principles

  • Create or contribute to protein-specific knowledge bases

• Collaborative Research Networks:

  • Establish research consortia focused on UPF0420 protein family characterization

  • Implement round-robin testing of protocols across multiple laboratories

  • Organize workshops or conferences specifically addressing methodological challenges

  • Develop shared resources and reagents available to the broader research community

• Reporting Standards Development:

  • Create comprehensive reporting templates for experimental work with this protein

  • Establish minimum information requirements for publications

  • Develop standardized nomenclature and terminology

  • Promote the use of structured formats for data presentation