Recombinant Rat UPF0420 protein C16orf58 homolog

What is Rat UPF0420 protein C16orf58 homolog and what are its key structural characteristics?

Rat UPF0420 protein C16orf58 homolog is an uncharacterized protein belonging to the UPF0420 family. The protein consists of 466 amino acids in its full-length form . The designation "UPF" indicates it belongs to an uncharacterized protein family, while "C16orf58 homolog" denotes its homology to the human chromosome 16 open reading frame 58 protein.

Based on the available recombinant protein products, the rat protein is typically expressed with a His-tag to facilitate purification . Unlike some well-characterized proteins in the search results (such as UBF or Notch-2), there is currently limited published information regarding its three-dimensional structure, functional domains, or cellular role.

What expression systems are commonly used for producing recombinant Rat UPF0420 protein C16orf58 homolog?

The primary expression system used for producing recombinant Rat UPF0420 protein C16orf58 homolog is Escherichia coli (E. coli) . This bacterial expression system offers several advantages for producing this particular protein:

• The available recombinant products are expressed in E. coli with N-terminal His-tags for affinity purification

• The bacterial system appears sufficient for expressing the full-length protein (1-466 amino acids)

By contrast, other complex rat proteins in the literature may require eukaryotic expression systems. For example:

• Rat Agrin is produced in Spodoptera frugiperda (Sf21) cells using baculovirus

• Rat IL-12 is also expressed in Sf21 cells with baculovirus

The successful expression in E. coli suggests that Rat UPF0420 protein C16orf58 homolog may not require extensive post-translational modifications or complex disulfide bonding for its recombinant production.

What are the recommended storage conditions for maintaining recombinant Rat UPF0420 protein C16orf58 homolog stability?

Based on the manufacturer recommendations, recombinant Rat UPF0420 protein C16orf58 homolog requires specific storage protocols to maintain stability:

• Long-term storage: Store at -20°C or -80°C (preferred for extended storage)

• Working aliquots: Store at 4°C for up to one week

• Storage buffer: Typically supplied in Tris-based buffer with 50% glycerol, optimized for this protein

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended

For optimal stability, the lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol (5-50% final concentration) is recommended before aliquoting for long-term storage at -20°C/-80°C .

These storage conditions are comparable to those recommended for other recombinant rat proteins, such as Rat Notch-2 and Rat Fc gamma RIIA/CD32a, which also require storage at -20°C with avoidance of repeated freeze-thaw cycles .

What experimental approaches can be used to characterize the function of UPF0420 protein C16orf58 homolog?

Since UPF0420 protein C16orf58 homolog remains largely uncharacterized, multiple complementary approaches would be required to elucidate its function:

• Bioinformatic analysis:

  • Sequence homology searches across species

  • Structural prediction using AlphaFold or similar tools

  • Domain identification and conserved motif analysis

  • Phylogenetic analysis to identify evolutionary relationships

• Protein interaction studies:

  • Yeast two-hybrid screening to identify binding partners

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling techniques (BioID, APEX)

  • Pull-down assays similar to those mentioned for studying protein interactions

• Subcellular localization:

  • Fluorescent protein tagging and microscopy

  • Subcellular fractionation followed by Western blotting

  • Immunofluorescence with specific antibodies

• Functional genomics:

  • CRISPR-Cas9 knockout/knockdown studies

  • RNA-Seq analysis of differential gene expression in knockout models

  • Phenotypic characterization of knockout models

• Structural studies:

  • X-ray crystallography or cryo-EM

  • NMR spectroscopy for smaller domains

  • Homology modeling based on related proteins with known structures (similar to the approach used for rat UBF )

This multi-faceted approach would provide complementary data to overcome the limitations of any single method.

How can one validate the proper folding and biological activity of recombinant Rat UPF0420 protein C16orf58 homolog?

Validating proper folding and biological activity of an uncharacterized protein presents unique challenges. For recombinant Rat UPF0420 protein C16orf58 homolog, the following approaches would be appropriate:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure content

  • Size exclusion chromatography to confirm monomeric/oligomeric state

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to identify folded domains resistant to digestion

• Functional validation strategies:

  • Development of activity assays based on bioinformatic predictions

  • Cell-based assays examining phenotypic rescue in knockout models

  • Binding assays with predicted interaction partners

  • Comparative activity testing with orthologous proteins from other species

• Post-translational modification analysis:

  • Mass spectrometry to identify modifications present in native vs. recombinant protein

  • Phosphorylation/glycosylation-specific staining

  • Comparison of E. coli-expressed vs. eukaryotic-expressed protein properties

For comparison, other recombinant rat proteins have established activity assays. For example, Recombinant Rat Agrin's activity is measured by its ability to induce acetylcholine receptor (AChR) clustering with an ED50 of 2-6 ng/mL, and by binding to human LRP-4 with Kd<3 nM in functional ELISA . Similarly, Recombinant Rat IL-12's activity is determined by its ability to induce IFN-γ production in activated mouse lymphoblasts with an ED50 of 0.025-0.25 ng/mL .

What is known about the evolutionary conservation of UPF0420 protein C16orf58 homolog across species?

UPF0420 protein C16orf58 homolog demonstrates evolutionary conservation across multiple mammalian species, suggesting functional importance:

• Cross-species comparison:

  • The mouse ortholog has 466 amino acids, identical in length to the rat protein

  • Commercial recombinant proteins are available for both rat and mouse orthologs, sharing similar structural characteristics

  • The chimpanzee (Pan troglodytes) genome contains a C16H16orf58 gene annotated as "chromosome 16 C16orf58 homolog"

• Sequence identity metrics:

  • While exact sequence identity percentages between rat and other species' C16orf58 homologs are not provided in the search results, the mouse and rat proteins share highly similar physicochemical properties based on their commercial descriptions

  • The UniProt identifier for mouse UPF0420 protein C16orf58 homolog is Q91W34, while the rat ortholog is Q499P8

• Evolutionary significance:

  • The conservation of this protein from rodents to primates suggests it likely serves an important cellular function

  • The preservation of the UPF0420 family across species provides evidence for functional constraints during evolution

Comparative studies with orthologs could provide insights into functionally important domains through identification of highly conserved regions.

What approaches can be used to identify potential interacting partners of UPF0420 protein C16orf58 homolog?

Identifying interaction partners is crucial for understanding the function of uncharacterized proteins like UPF0420 protein C16orf58 homolog. Several complementary approaches can be employed:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged UPF0420 protein C16orf58 homolog in rat cells

  • Perform pull-down with anti-tag antibodies or affinity resins

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by reciprocal pull-downs

• Proximity-dependent labeling:

  • Create fusion proteins with BioID, TurboID, or APEX2

  • Express in cells and activate the enzyme to label proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • This approach captures both stable and transient interactions

• Yeast two-hybrid screening:

  • Screen rat cDNA libraries using UPF0420 protein C16orf58 homolog as bait

  • Validate positive hits by secondary assays

  • Perform domain mapping to identify interaction interfaces

• Co-immunoprecipitation:

  • Generate specific antibodies against UPF0420 protein C16orf58 homolog

  • Perform immunoprecipitation from rat tissue or cell lysates

  • Identify co-precipitating proteins by immunoblotting or mass spectrometry

• Bioinformatic prediction:

  • Use algorithms to predict protein-protein interactions based on:

    • Co-expression patterns

    • Phylogenetic profiles

    • Structural compatibility

    • Domain-domain interaction databases

Similar interaction study methods (yeast two-hybrid, co-IP, pull-down) have been mentioned for studying protein interactions in the search results , indicating these are established approaches in the field.

How might recombinant Rat UPF0420 protein C16orf58 homolog be used in experimental models of disease?

While the specific role of UPF0420 protein C16orf58 homolog in disease models is not directly addressed in the search results, we can propose methodological approaches based on established practices with other recombinant rat proteins:

• Loss-of-function studies:

  • Generate knockout rat models or cell lines using CRISPR-Cas9

  • Employ RNA interference strategies to achieve knockdown

  • Use dominant-negative constructs to disrupt protein function

  • Assess phenotypic changes related to disease progression

• Gain-of-function studies:

  • Administer purified recombinant protein in disease models

  • Overexpress the protein using viral vectors

  • Generate transgenic rat models with enhanced expression

  • Evaluate therapeutic potential in relevant disease contexts

• Biomarker development:

  • Develop antibodies against UPF0420 protein C16orf58 homolog

  • Establish ELISA or other quantitative assays

  • Measure protein levels in various disease states

  • Correlate expression with disease progression or treatment response

• Structure-function analysis:

  • Create domain-specific mutants

  • Test functional consequences in cellular or animal models

  • Identify critical residues for protein activity

  • Design potential inhibitors or activators based on structural insights

For comparison, recombinant activated protein C has been studied in a rat heat stroke model, where it significantly improved survival by ameliorating systemic inflammation, hypercoagulable state, and tissue ischemia . Similarly, approaches used to study recombinant rat Nogo-A in neurite outgrowth models could inform experimental design for UPF0420 protein C16orf58 homolog .

What controls should be included when evaluating the effects of recombinant Rat UPF0420 protein C16orf58 homolog in experimental systems?

Proper experimental controls are essential when working with recombinant proteins like Rat UPF0420 protein C16orf58 homolog:

• Negative controls:

  • Buffer-only conditions matching the protein storage buffer

  • Heat-denatured protein to control for non-specific effects

  • Unrelated recombinant protein with similar size and tag

  • Mutated versions of the protein lacking predicted functional domains

• Positive controls:

  • Well-characterized proteins with established effects in the experimental system

  • If function becomes known, a protein known to act in the same pathway

• Tag controls:

  • Tag-only protein to control for tag-mediated effects

  • Comparison of differently tagged versions (N-terminal vs. C-terminal)

  • Tag-cleaved protein to confirm activity is independent of the tag

• Dose-response assessment:

  • Serial dilutions to establish dose-dependent effects

  • Determination of EC50/IC50 values

  • Comparison with physiological concentration ranges if known

• Temporal controls:

  • Time-course experiments to determine optimal treatment duration

  • Washout studies to assess reversibility of effects

  • Pre-treatment vs. post-treatment comparisons

This comprehensive control strategy would be similar to approaches used for other recombinant rat proteins, such as the dose-response testing performed with recombinant activated protein C in the rat heat stroke model (testing doses of 0.5-20 mg/kg) .

How should researchers address potential endotoxin contamination in recombinant protein preparations?

Endotoxin contamination is a critical concern when using E. coli-expressed recombinant proteins like Rat UPF0420 protein C16orf58 homolog in biological experiments:

• Endotoxin testing methods:

  • Limulus Amebocyte Lysate (LAL) assay (gel-clot, chromogenic, or turbidimetric)

  • Recombinant Factor C assay (rFC)

  • Endotoxin-specific ELISA

  • Cell-based assays (TLR4 reporter cells)

• Endotoxin removal strategies:

  • Two-phase extraction with Triton X-114

  • Polymyxin B affinity chromatography

  • Anion exchange chromatography

  • Specific endotoxin removal resins

  • Ultrafiltration with specialized membranes

• Experimental controls for endotoxin effects:

  • Inclusion of polymyxin B in experimental systems

  • Testing in TLR4-deficient cells or animals

  • Heat treatment (endotoxin is heat-stable, whereas most proteins are not)

  • Parallel testing with endotoxin standards

• Documentation and reporting:

  • Record endotoxin levels in units/mg protein

  • Calculate maximum endotoxin exposure in experimental systems

  • Include endotoxin levels in materials and methods sections of publications

For recombinant proteins expressed in bacterial systems, endotoxin levels should typically be below 1.0 EU/μg protein for in vitro cell culture applications and below 5.0 EU/kg body weight for in vivo studies.

What methodological approaches can be used to study the effects of post-translational modifications on UPF0420 protein C16orf58 homolog function?

Studying post-translational modifications (PTMs) of UPF0420 protein C16orf58 homolog requires multiple complementary approaches:

• Identification of PTM sites:

  • Mass spectrometry analysis of the native protein from rat tissues

  • Phosphoproteomic, glycoproteomic, or ubiquitinomic analyses

  • Prediction of modification sites using computational tools

  • Site-specific antibodies against common modifications

• Functional analysis of PTMs:

  • Site-directed mutagenesis of modified residues

  • Comparison of E. coli-expressed protein (limited PTMs) with eukaryotic expression systems

  • In vitro enzymatic modification (kinases, glycosyltransferases, etc.)

  • Chemical mimicry of PTMs (phosphomimetic mutations)

• Temporal and spatial regulation of PTMs:

  • Analysis across different tissues, developmental stages, and conditions

  • Identification of enzymes responsible for adding/removing modifications

  • Investigation of PTM crosstalk and combinatorial effects

• Structural impact assessment:

  • Analysis of PTM effects on protein folding and stability

  • Effect on protein-protein interactions

  • Conformational changes induced by modifications

Comparatively, when studying recombinant rat UBF, researchers used electron microscopy and image analysis to examine structural features, then integrated this data with homology modeling to predict atomic structure . Similar approaches could be applied to understand how PTMs affect UPF0420 protein C16orf58 homolog structure and function.

What considerations should be taken into account when designing antibodies against UPF0420 protein C16orf58 homolog?

Developing specific and effective antibodies against UPF0420 protein C16orf58 homolog requires careful planning:

• Epitope selection strategy:

  • Bioinformatic analysis to identify:

    • Surface-exposed regions

    • Non-glycosylated sites

    • Regions with high antigenicity

    • Unique sequences not present in related proteins

  • Consideration of multiple epitopes from different protein regions

  • Avoidance of highly conserved domains if species specificity is required

• Antibody format selection:

  • Polyclonal vs. monoclonal approaches

  • Full-length protein vs. peptide immunization

  • Consideration of antibody applications (Western blot, IP, IHC, IF)

  • Species of origin to avoid cross-reactivity in experimental systems

• Validation requirements:

  • Testing in knockout/knockdown systems

  • Comparison of multiple antibodies against different epitopes

  • Pre-absorption controls with immunizing peptide

  • Cross-reactivity testing against related proteins

• Application-specific considerations:

  • For Western blotting: denaturation-resistant epitopes

  • For immunoprecipitation: epitopes accessible in native state

  • For immunohistochemistry: fixation-resistant epitopes

  • For flow cytometry: epitopes on extracellular domains (if applicable)

This comprehensive approach to antibody development ensures maximum specificity and utility across various experimental platforms.

How can researchers troubleshoot solubility issues with recombinant Rat UPF0420 protein C16orf58 homolog?

Solubility challenges are common with recombinant proteins and may require systematic troubleshooting:

• Buffer optimization strategies:

  • pH screening (typically pH 5.5-8.5 in 0.5 unit increments)

  • Salt concentration variations (50-500 mM NaCl)

  • Addition of stabilizing agents:

    • Glycerol (5-20%)

    • Reducing agents (DTT, β-mercaptoethanol)

    • Non-ionic detergents (0.01-0.1% Triton X-100, NP-40)

    • Amino acids (arginine, glutamate)

    • Osmolytes (trehalose, sucrose)

• Expression and purification modifications:

  • Lower induction temperature (16-20°C)

  • Co-expression with chaperones

  • Use of solubility-enhancing fusion tags (MBP, SUMO, TRX)

  • Refolding from inclusion bodies if necessary

  • Alternative expression systems (yeast, insect cells)

• Analytical approaches:

  • Dynamic light scattering to assess aggregation

  • Differential scanning fluorimetry for stability profiling

  • Size exclusion chromatography to monitor oligomeric state

  • Visual inspection for precipitation at various concentrations

• Storage considerations:

  • Aliquoting to avoid freeze-thaw cycles

  • Flash-freezing in liquid nitrogen

  • Lyophilization with appropriate excipients

  • Storage temperature optimization

For recombinant Rat UPF0420 protein C16orf58 homolog specifically, the recommended storage buffer contains Tris-based buffer with 50% glycerol , which suggests that glycerol is an important stabilizing agent for this protein.

What approaches can be used to verify the identity and purity of recombinant Rat UPF0420 protein C16orf58 homolog preparations?

Verifying identity and purity is critical for ensuring reliable experimental results:

• Purity assessment methods:

  • SDS-PAGE with Coomassie or silver staining (>90% purity is typical for research-grade proteins)

  • High-performance liquid chromatography (HPLC)

  • Capillary electrophoresis

  • Size exclusion chromatography

  • Western blotting with tag-specific and protein-specific antibodies

• Identity confirmation approaches:

  • Mass spectrometry:

    • Peptide mass fingerprinting

    • Tandem MS for sequence confirmation

  • N-terminal sequencing (Edman degradation)

  • Immunological detection with specific antibodies

  • Functional assays (once established)

• Contaminant testing:

  • Host cell protein (HCP) ELISA

  • DNA contamination assays

  • Endotoxin testing (as discussed previously)

  • Microbial contamination testing

• Quantification methods:

  • UV spectroscopy (A280)

  • Bradford or BCA protein assays

  • Amino acid analysis for absolute quantification

  • Comparison with known standards

Commercial recombinant proteins typically undergo rigorous quality control testing, with purity levels >90% as determined by SDS-PAGE being standard for research applications .

What strategies can be employed to improve the yield of functional recombinant Rat UPF0420 protein C16orf58 homolog?

Maximizing yield of functional protein requires optimization at multiple levels:

• Expression optimization:

  • Codon optimization for the expression host

  • Promoter selection for appropriate expression level

  • Induction conditions (temperature, inducer concentration, duration)

  • Media formulation and feeding strategies

  • Scale-up considerations for larger batches

• Purification enhancement:

  • Optimization of cell lysis conditions

  • Selection of appropriate chromatography methods:

    • IMAC for His-tagged proteins

    • Ion exchange chromatography

    • Hydrophobic interaction chromatography

  • Minimizing unnecessary purification steps to reduce losses

  • Buffer optimization during each purification stage

• Protein stability improvement:

  • Addition of protease inhibitors during purification

  • Identification and elimination of proteolytic cleavage sites

  • Use of stabilizing buffers and additives

  • Minimizing time at room temperature during processing

• Refolding strategies (if required):

  • Dilution-based refolding

  • On-column refolding

  • Gradient-based refolding methods

  • Pulse renaturation

  • Use of folding enhancers (arginine, small molecule chaperones)

From the search results, we know that recombinant Rat UPF0420 protein C16orf58 homolog has been successfully expressed as a full-length (1-466 aa) protein with an N-terminal His-tag in E. coli , suggesting that bacterial expression can yield functional protein without the need for complex eukaryotic expression systems.