Recombinant Chicken UPF0485 protein C1orf144 homolog (RCJMB04_13j19)

What is Recombinant Chicken UPF0485 protein C1orf144 homolog?

Recombinant Chicken UPF0485 protein C1orf144 homolog (RCJMB04_13j19) is a full-length recombinant protein expressed in E. coli with a Uniprot identification number of Q5ZK25. The protein is derived from Gallus gallus (Chicken) and is also known by the target name SZRD1. It consists of 152 amino acids with the complete sequence: MEDEEVAESW EEAADSGEID RRLEKKLKIT QKESRKSKSP PKVPIVIQDD SVPSGPPPQI RILKRPATNG VLSNPNSTTR PAFPVKSLAQ REAEYAEARK RILGSASPEE EQEKPILDRP TRISQPEDIR QPNNVIRQPL GPDGSQGFKQ RR .

What are the optimal storage conditions for Recombinant Chicken UPF0485 protein?

The shelf life of Recombinant Chicken UPF0485 protein C1orf144 homolog is influenced by multiple factors, including storage state, buffer composition, temperature, and the intrinsic stability of the protein. For liquid formulations, the recommended shelf life is approximately 6 months when stored at -20°C to -80°C. Lyophilized formulations exhibit greater stability, with a shelf life of up to 12 months at -20°C to -80°C. To maintain optimal protein integrity, repeated freeze-thaw cycles should be avoided. Working aliquots may be stored at 4°C for up to one week without significant degradation .

How should Recombinant Chicken UPF0485 protein be reconstituted for experimental use?

For optimal reconstitution of Recombinant Chicken UPF0485 protein C1orf144 homolog, the vial should first be briefly centrifuged to ensure all contents settle at the bottom. The protein should be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage stability, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the standard default concentration). Following reconstitution, the solution should be aliquoted to minimize freeze-thaw cycles and stored at -20°C to -80°C for maximum shelf life .

What are the best approaches for designing experiments to study protein-protein interactions involving Recombinant Chicken UPF0485 protein?

When designing experiments to study protein-protein interactions involving Recombinant Chicken UPF0485 protein C1orf144 homolog, researchers should consider implementing multiple complementary techniques. Similar to approaches used for studying ligand-receptor interactions such as those between chicken macrophage stimulating protein and the Sea receptor , affinity chromatography employing the extracellular domain of potential interacting partners can be effective.

For in vitro binding assays, fusion proteins (such as immunoglobulin fusion constructs) can be utilized to identify binding partners. Phosphorylation studies examining downstream signaling events, particularly focusing on common signaling molecules like Shc, Erk1, and Erk2, can provide insight into functional interactions. Additionally, co-immunoprecipitation experiments followed by mass spectrometry analysis can help identify novel binding partners. Western blotting with antibodies against predicted interaction partners can confirm specific binding relationships.

How can researchers address potential inconsistencies in experimental results when working with Recombinant Chicken UPF0485 protein?

When addressing inconsistencies in experimental results with Recombinant Chicken UPF0485 protein C1orf144 homolog, researchers should systematically evaluate multiple factors that could contribute to variability. First, protein quality should be assessed by SDS-PAGE to confirm the expected purity of >85% . Batch-to-batch variations should be monitored through consistent quality control testing.

Storage conditions significantly impact protein stability - researchers should verify proper storage at -20°C/-80°C for long-term storage, with working aliquots maintained at 4°C for no more than one week . The reconstitution protocol should be standardized, ensuring deionized sterile water is used and glycerol is added at consistent concentrations (recommended 50%) .

Researchers should also consider potential post-translational modifications that might affect protein function. Since the protein is expressed in E. coli , it will lack eukaryotic post-translational modifications present in the native chicken protein, which may affect certain experimental outcomes. Conducting parallel experiments with positive and negative controls can help differentiate true biological effects from technical artifacts.

What methodological approaches can be used to study the functional significance of specific domains within Recombinant Chicken UPF0485 protein?

To study the functional significance of specific domains within Recombinant Chicken UPF0485 protein C1orf144 homolog, researchers can employ several strategic methodological approaches:

• Site-directed mutagenesis: Critical amino acid residues can be systematically mutated to assess their contribution to protein function. This approach was successfully used in studies of chicken macrophage stimulating protein, where a specific Cys to Ala substitution (C665A) significantly enhanced protein activity .

• Domain deletion and truncation studies: Creating a series of truncated protein variants that systematically remove specific regions can help identify functional domains. The full sequence (152 amino acids) of Recombinant Chicken UPF0485 protein provides multiple opportunities for strategic truncations.

• Chimeric protein construction: Swapping domains between related proteins can help identify regions responsible for specific functions or interactions.

• Structural analysis: Combining these functional studies with structural analysis techniques such as X-ray crystallography or NMR can provide insights into how specific domains contribute to protein folding and function.

• In silico analysis: Computational approaches like molecular docking and molecular dynamics simulations can predict functional domains and guide experimental design.

What is the optimal protocol for assessing the purity and integrity of Recombinant Chicken UPF0485 protein preparations?

The optimal protocol for assessing purity and integrity of Recombinant Chicken UPF0485 protein C1orf144 homolog involves multiple complementary techniques:

• SDS-PAGE analysis: The protein should be run on a polyacrylamide gel followed by Coomassie blue staining to verify the expected molecular weight and assess purity, which should exceed 85% as specified in the product documentation .

• Western blot analysis: Using antibodies specific to the protein or to any associated tag to confirm identity and detect potential degradation products.

• Mass spectrometry: For precise molecular weight determination and to detect any post-translational modifications or unexpected processing.

• Size exclusion chromatography: To assess aggregation state and homogeneity of the protein preparation.

• Functional assays: Activity-based assays to confirm that the protein maintains its expected biological function, which provides evidence of proper folding and integrity.

The following criteria should be used to evaluate protein quality:

• Single band at expected molecular weight on SDS-PAGE

• Absence of significant degradation products

• Correct amino acid sequence confirmation through mass spectrometry

• Maintenance of biological activity in functional assays

How can researchers optimize expression conditions for Recombinant Chicken UPF0485 protein to maximize yield and activity?

Optimizing expression conditions for Recombinant Chicken UPF0485 protein C1orf144 homolog in E. coli requires systematic evaluation of multiple parameters:

• Expression vector selection: Vectors with strong inducible promoters (T7, tac) provide controlled expression. Consider fusion tags (His, GST, MBP) that can enhance solubility and facilitate purification.

• E. coli strain optimization: BL21(DE3) derivatives are commonly used for recombinant protein expression, but strains like Rosetta or Origami may improve expression of proteins with rare codons or disulfide bonds.

• Growth and induction conditions:

  • Temperature: Lower temperatures (16-25°C) often improve protein folding

  • Induction timing: Typically at mid-log phase (OD600 of 0.6-0.8)

  • Inducer concentration: Optimize IPTG concentration (typically 0.1-1.0 mM)

  • Post-induction time: Usually 3-18 hours depending on protein

• Media composition: Rich media (LB, TB, 2YT) versus defined media with supplements like glucose or glycerol

• Scale-up considerations: Ensure adequate oxygenation and mixing in larger volumes

A systematic optimization matrix should be created to test combinations of these variables, with protein yield and activity assessed for each condition. The optimal conditions should balance maximum protein yield with maintenance of proper folding and biological activity.

What techniques can be used to investigate potential post-translational modifications of Recombinant Chicken UPF0485 protein in different expression systems?

Investigation of potential post-translational modifications (PTMs) of Recombinant Chicken UPF0485 protein C1orf144 homolog across different expression systems requires specialized analytical techniques:

• Mass Spectrometry-Based Approaches:

  • Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Provides comprehensive identification of PTMs

  • MALDI-TOF MS: Useful for detecting mass shifts indicative of modifications

  • Electron Transfer Dissociation (ETD) and Electron Capture Dissociation (ECD): Preserve labile modifications during fragmentation

• Site-Specific Detection Methods:

  • Phosphorylation: Phospho-specific antibodies, Phos-tag gels, or 32P labeling

  • Glycosylation: Periodic acid-Schiff (PAS) staining, lectin blotting, or glycosidase treatments

  • Ubiquitination/SUMOylation: Specific antibodies against ubiquitin or SUMO

• Expression System Comparison:

  • E. coli: As currently used , minimal PTMs (primarily lacks glycosylation)

  • Yeast: Intermediate complexity of PTMs

  • Insect cells: More complex PTMs

  • Mammalian cells: Most physiologically relevant PTMs

• PTM Prediction Tools:

  • NetPhos, PhosphoSite Plus: Phosphorylation prediction

  • NetNGlyc, NetOGlyc: N- and O-glycosylation prediction

  • SUMOplot, UbPred: SUMO and ubiquitination sites prediction

The interpretation of results should consider that E. coli-expressed recombinant proteins typically lack eukaryotic PTMs, which may affect protein function compared to the native form in chicken tissues. Researchers may need to transition to eukaryotic expression systems if specific PTMs are critical for protein function.

How does Recombinant Chicken UPF0485 protein C1orf144 homolog relate to protein quality differences between organic and conventional chicken?

While direct research on the relationship between Recombinant Chicken UPF0485 protein C1orf144 homolog and protein quality differences in organic versus conventional chicken is limited, emerging evidence suggests potential connections worth investigating. Recent pilot studies indicate that organic chicken may contain higher density of contractile proteins (actin and myosin) compared to conventional chicken . The UPF0485 protein, as a conserved protein in chicken, could potentially show differential expression or modification patterns between organic and conventional raising methods.

When designing studies to investigate this relationship, researchers should consider:

• Comparative proteomic analysis of UPF0485 protein expression levels in organic versus conventional chicken tissues

• Evaluation of potential post-translational modifications that might differ based on raising methods

• Functional assays to determine if any observed differences translate to altered protein functionality

Such investigations would contribute to the broader understanding of how raising methods impact protein quality and essential amino acid profiles in chicken meat .

What are the challenges in extrapolating in vitro findings with Recombinant Chicken UPF0485 protein to in vivo physiological contexts?

Extrapolating in vitro findings with Recombinant Chicken UPF0485 protein C1orf144 homolog to in vivo physiological contexts presents several significant challenges:

• Expression system limitations: The E. coli-expressed recombinant protein lacks eukaryotic post-translational modifications that may be critical for in vivo function. This limitation is similar to challenges observed with other recombinant proteins, such as chicken macrophage stimulating protein, where specific mutations (e.g., Cys to Ala substitutions) were required to achieve full activity in recombinant systems .

• Structural considerations: The in vitro preparation may not perfectly replicate the three-dimensional configuration of the protein in its native cellular environment, where interactions with other cellular components may influence folding and function.

• Concentration and localization discrepancies: The physiological concentration and subcellular localization of the protein in vivo may differ significantly from experimental conditions used in vitro.

• Absence of interacting partners: In vivo, the protein likely functions within complex protein networks and signaling pathways that are difficult to replicate in simplified in vitro systems.

• Species differences: When using the chicken protein for comparative studies with other species, researchers must consider evolutionary differences in protein function and regulation.

To address these challenges, researchers should implement validation strategies that bridge in vitro and in vivo contexts, such as developing cell-based assays that more closely mimic physiological conditions, and ultimately confirming key findings in appropriate animal models.

What future research directions might elucidate the functional role of Recombinant Chicken UPF0485 protein in cellular signaling pathways?

Future research to elucidate the functional role of Recombinant Chicken UPF0485 protein C1orf144 homolog in cellular signaling pathways should pursue several promising directions:

• Interactome mapping: Employing techniques such as affinity purification followed by mass spectrometry to identify binding partners, similar to approaches used for identifying ligand-receptor interactions . This would help position the protein within cellular signaling networks.

• Phosphoproteomic analysis: Investigating whether the protein undergoes phosphorylation in response to specific stimuli and identifying responsible kinases. Drawing inspiration from studies on other signaling proteins that demonstrated phosphorylation of molecules like Shc, Erk1, and Erk2 , researchers could examine similar pathways for UPF0485 protein.

• CRISPR-Cas9 knockout studies: Generating knockout cell lines or animal models to observe phenotypic consequences of protein absence, providing insights into physiological functions.

• Domain-specific functional analysis: Building on the methodological approaches discussed earlier, systematic investigation of how specific protein domains contribute to signaling functions.

• Comparative evolutionary analysis: Examining how the protein's function has evolved across species by comparing the chicken variant with homologs from other organisms.

• Integration with systems biology: Placing findings within broader cellular signaling networks using computational modeling to predict pathway interactions and functional roles.

These research directions would significantly advance understanding of this protein's role in cellular signaling and potentially identify novel therapeutic targets or biological markers for future applications.