Recombinant Rat UPF0498 protein KIAA1191 homolog (Aa2-141)

What is the molecular structure and function of Rat UPF0498 protein KIAA1191 homolog?

The Rat UPF0498 protein KIAA1191 homolog is a member of the uncharacterized protein family 0498, with structural similarities to the human KIAA1191 protein. While its precise function remains under investigation, it likely participates in cellular nitrogen metabolism pathways similar to other proteins in this family . Structurally, the protein belongs to the aminotransferase family with a pyridoxal-phosphate-dependent domain architecture . Researchers should approach functional studies with multiple methodologies including co-immunoprecipitation and subcellular localization techniques to establish binding partners and cellular distribution patterns.

What expression systems are optimal for producing Recombinant Rat UPF0498 protein KIAA1191 homolog?

For laboratory-scale production of Rat UPF0498 protein KIAA1191 homolog, Escherichia coli expression systems typically yield sufficient protein with ≥80% purity . The truncated construct (Aa2-141) represents a functional fragment that maintains core enzymatic activity while improving expression efficiency. When establishing your expression protocol, consider the following methodology:

• Clone the coding sequence into a vector containing an N-terminal His-tag for purification

• Transform into BL21(DE3) E. coli strain for protein expression

• Induce expression at OD600 of 0.6-0.8 with 0.5mM IPTG

• Harvest after 4-6 hours of induction at 30°C rather than 37°C to improve protein folding

• Verify expression using SDS-PAGE analysis before scaling up production
  For applications requiring higher purity or mammalian post-translational modifications, consider using HEK293 or CHO cell expression systems, though these will require significant protocol optimization.

How can I verify the identity and purity of the expressed protein?

Verification of recombinant Rat UPF0498 protein KIAA1191 homolog requires a multi-method approach:

• SDS-PAGE analysis under reducing conditions should reveal a single band at the expected molecular weight (approximately 16 kDa for the Aa2-141 fragment with His-tag)

• Western blotting using anti-His antibodies confirms the presence of the tagged protein

• Mass spectrometry analysis provides definitive identification through peptide mass fingerprinting

• Size-exclusion chromatography assesses aggregation state and homogeneity

• For functional verification, design activity assays based on predicted aminotransferase activity
  Purity assessment should target ≥80% for most research applications, with higher standards (≥95%) required for structural studies or therapeutic research contexts .

What are the optimal storage conditions for Recombinant Rat UPF0498 protein KIAA1191 homolog?

For maintaining stability and activity of Recombinant Rat UPF0498 protein KIAA1191 homolog, implement the following evidence-based storage protocol:

• Store purified protein at -80°C in small single-use aliquots to avoid freeze-thaw cycles

• Use a stabilizing buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 10% glycerol, and 1mM DTT

• For short-term storage (1-2 weeks), 4°C storage is acceptable if the buffer contains protease inhibitors

• Avoid repeated freeze-thaw cycles as this significantly reduces enzymatic activity

• Monitor protein stability through periodic activity assays and SDS-PAGE analysis
  The addition of stabilizers such as glycerol or trehalose (5-10%) can significantly extend shelf-life by preventing freeze-induced denaturation, particularly important for the Aa2-141 fragment which may have reduced inherent stability compared to the full-length protein.

How should I optimize protein reconstitution after lyophilization?

When reconstituting lyophilized Rat UPF0498 protein KIAA1191 homolog, follow this methodological approach:

• Allow the lyophilized protein to equilibrate to room temperature before opening the container to prevent moisture condensation

• Reconstitute using sterile deionized water or an appropriate buffer (typically PBS or Tris buffer at pH 7.4-8.0)

• Add buffer slowly while gently rotating the vial rather than vortexing to prevent protein denaturation

• Allow 15-30 minutes at room temperature for complete dissolution

• Centrifuge briefly (10,000 × g for 1 minute) to collect all material and remove potential insoluble aggregates

• Verify protein concentration using Bradford or BCA assay considering the amino acid composition of this specific fragment
  For experiments requiring high protein activity, supplement the reconstitution buffer with 1mM pyridoxal phosphate, a common cofactor for aminotransferase family proteins .

How should I design definitive experiments to elucidate the function of Rat UPF0498 protein KIAA1191 homolog?

Designing rigorous experiments to determine the function of this poorly characterized protein requires a systematic approach following established experimental design principles :

• Begin with bioinformatic analysis to identify conserved domains and potential orthologs with known functions

• Design experiments with multiple independent and dependent variables:

  • Independent variables: Protein concentration, substrate concentration, cofactor presence/absence

  • Dependent variables: Substrate conversion, product formation, binding affinity

• Control for extraneous variables by:

  • Using appropriate negative controls (inactive mutant protein)

  • Including positive controls (related proteins with known function)

  • Standardizing environmental conditions (temperature, pH, buffer composition)

• Implement a step-wise investigation methodology:

  • First, determine subcellular localization using immunofluorescence or fractionation

  • Identify binding partners through pull-down assays and mass spectrometry

  • Assess potential enzymatic activities based on aminotransferase family functions

  • Validate findings through gene knockout/knockdown followed by phenotypic analysis
    This multilayered approach controls for confounding variables while systematically building evidence for protein function through hypothesis testing .

What are the key considerations for designing activity assays for this aminotransferase family protein?

When developing activity assays for Rat UPF0498 protein KIAA1191 homolog, consider these methodology-focused guidelines:

• Based on aminotransferase family characteristics, design assays that measure:

  • Transamination activity (similar to ALT) using alanine and 2-oxoglutarate as initial substrates

  • Pyruvate and glutamate formation as reaction products

  • Cofactor requirements (typically pyridoxal phosphate for this enzyme family)

• Implement multiple detection methods:

  • Spectrophotometric coupled enzyme assays for real-time monitoring

  • HPLC analysis for precise quantification of reaction products

  • Mass spectrometry for definitive identification of novel substrates or products

• Establish assay conditions through systematic optimization:

  • pH range evaluation (typically 7.0-8.5 for aminotransferases)

  • Temperature dependence (25-37°C)

  • Metal ion requirements or inhibition

  • Substrate specificity testing with multiple potential amino acid substrates

• Validate assay reliability through:

  • Determining linear range, detection limits, and reproducibility

  • Testing with known inhibitors of aminotransferases (e.g., aminooxyacetic acid)

  • Comparing with related enzymes like ALT as positive controls
    This comprehensive assay development approach enables accurate characterization of enzymatic parameters (Km, Vmax, kcat) essential for understanding physiological function.

How can I address contradictory findings when characterizing Rat UPF0498 protein KIAA1191 homolog?

When faced with contradictory experimental results in protein characterization studies, implement this systematic resolution framework:

• Identify potential sources of experimental variation:

  • Protein batch-to-batch inconsistencies (verify with quality control testing)

  • Post-translational modification differences between expression systems

  • Assay-specific artifacts (validate using orthogonal methods)

  • Sample contamination (confirm with mass spectrometry analysis)

• Apply statistical approaches to quantify variation:

  • Perform replicate experiments (minimum n=3) for robust statistical analysis

  • Calculate confidence intervals to determine significance of contradictory findings

  • Use ANOVA or appropriate statistical tests to identify significant variables

• Resolve contradictions methodologically:

  • Design experiments that directly test competing hypotheses

  • Systematically modify one variable at a time to identify critical factors

  • Consider protein conformation changes under different experimental conditions

• Document all experimental conditions meticulously, including:

  • Exact buffer compositions and pH

  • Lot numbers of key reagents

  • Precise temperature and incubation times

  • Instrument calibration status
    This approach transforms contradictory results into valuable insights about conditional protein behavior or previously unrecognized experimental variables .

What computational tools are most effective for predicting substrate specificity of this uncharacterized protein?

To predict substrate specificity of Rat UPF0498 protein KIAA1191 homolog, employ this computational analysis workflow:

• Sequence-based approaches:

  • Perform multiple sequence alignment with characterized aminotransferase family members

  • Identify conserved active site residues through homology modeling

  • Use BLAST and HHpred to identify distant homologs with known substrates

• Structure-based predictions:

  • Generate homology models using AlphaFold or RoseTTAFold

  • Perform molecular docking with potential substrates

  • Calculate binding energies for candidate molecules

  • Analyze active site architecture for substrate constraints

• Machine learning integration:

  • Train prediction algorithms on known aminotransferase-substrate pairs

  • Implement fingerprint-based similarity searching for substrate prediction

  • Validate computational predictions with focused biochemical assays

• Data integration methodology:

  • Create a consensus prediction by weighting results from multiple approaches

  • Prioritize candidates for experimental validation

  • Design targeted assays for top-predicted substrates
    This computational-experimental feedback loop accelerates functional characterization while minimizing resource-intensive broad-spectrum substrate screening approaches.

How can I establish a reliable cell-based system to study the physiological role of UPF0498 protein KIAA1191 homolog?

Developing cell-based systems for physiological studies requires careful consideration of cellular contexts and detection methods:

• Cell line selection strategy:

  • Primary rat hepatocytes provide physiologically relevant context for liver-expressed proteins

  • Established rat cell lines (e.g., H4IIE, PC12) offer experimental consistency

  • Consider tissue-specific expression patterns when selecting model systems

• Genetic manipulation approach:

  • CRISPR/Cas9 knockout to eliminate endogenous expression

  • RNAi for dose-dependent knockdown studies

  • Overexpression systems with inducible promoters for temporal control

  • Tagged constructs (GFP, FLAG) for localization and interaction studies

• Phenotypic analysis methodology:

  • Measure cellular metabolism parameters (glucose uptake, ATP production)

  • Assess growth rates and cell cycle progression

  • Examine stress responses and adaptive pathways

  • Monitor transcriptional changes using RNA-seq

• Pathway integration analysis:

  • Perform phosphoproteomics to identify signaling pathways affected

  • Use metabolomics to detect changes in aminotransferase-related metabolites

  • Implement interactome mapping to position the protein within cellular networks
    This systematic approach enables correlation of molecular function with cellular phenotypes, providing physiological context for biochemical findings.

What are the most effective approaches for studying protein-protein interactions involving Rat UPF0498 protein KIAA1191 homolog?

To comprehensively map protein-protein interactions, implement this multi-method strategy:

• In vitro interaction analysis:

  • Pull-down assays using recombinant His-tagged protein as bait

  • Surface Plasmon Resonance (SPR) for quantitative binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Cross-linking mass spectrometry to capture transient interactions

• Cell-based interaction methods:

  • Co-immunoprecipitation using antibodies against the tagged protein

  • Proximity labeling approaches (BioID, APEX) to capture contextual interactions

  • Fluorescence resonance energy transfer (FRET) for direct interaction visualization

  • Mammalian two-hybrid assays for validation of specific interactions

• Bioinformatic prediction integration:

  • Use existing interactome databases to identify potential binding partners

  • Apply structural docking simulations to predict interaction interfaces

  • Analyze co-expression patterns across tissues and conditions

• Functional validation methodology:

  • Mutagenesis of predicted interaction interfaces

  • Competition assays with peptide mimetics

  • Phenotypic rescue experiments in knockout systems This comprehensive approach provides both high-confidence direct interactions and broader network context, essential for understanding multifunctional proteins like KIAA1191 homolog.