Recombinant Chicken UPF0542 protein C5orf43 homolog (RCJMB04_3o3)

What is the molecular characterization of UPF0542 protein C5orf43 homolog?

The UPF0542 protein C5orf43 homolog (RCJMB04_3o3) from Gallus gallus (Chicken) is a small protein with 74 amino acids in its expression region. Its complete amino acid sequence is: MFDVKAWAVYIVEWAAKDPYGFLTTVILVLTPLFIISAALSWKLAKMIETREREQKKKRKRQENIVKAKRAKKD. The protein has been assigned the UniProt accession number Q5F409 and is classified as part of the UPF0542 protein family .

To characterize this protein thoroughly, researchers should employ multiple complementary approaches:

• Primary structure analysis using mass spectrometry

• Secondary structure prediction using circular dichroism spectroscopy

• Tertiary structure determination using X-ray crystallography or NMR spectroscopy

• Functional domain identification through bioinformatic analysis of conserved regions

How should optimal storage conditions be determined for Recombinant UPF0542 protein stability studies?

• Evaluate protein stability at various temperatures (-80°C, -20°C, 4°C) using activity assays at defined time intervals

• Test buffer composition effects by examining:

  • pH ranges (5.0-9.0)

  • Salt concentrations (0-500 mM NaCl)

  • Additives (glycerol percentages from 10-50%)

• Conduct freeze-thaw cycle testing (1, 3, 5, and 10 cycles)

• Perform aggregation analysis using dynamic light scattering after various storage periods

The results should be presented as a stability matrix showing relative activity retention across all tested conditions.

What expression systems are most appropriate for producing functional Recombinant UPF0542 protein?

Selection of an optimal expression system for Recombinant UPF0542 protein C5orf43 homolog depends on research objectives and required protein characteristics. While bacterial expression in E. coli is commonly employed for recombinant proteins , researchers should consider several factors:

For initial characterization studies, E. coli expression with optimization of induction parameters (IPTG concentration, temperature, and induction time) is often most practical. For the UPF0542 protein specifically, expression optimization should focus on solubility enhancement through fusion tags and modified induction protocols (e.g., using 0.5-1.0 mM IPTG at 25°C for 16 hours) .

How can triple resonance NMR techniques be applied to study UPF0542 protein structure and interactions?

Triple resonance NMR techniques offer powerful approaches for studying protein structure and interactions at atomic resolution. While traditional NMR approaches can be challenging for certain proteins, recent advancements in triple resonance experiments provide enhanced sensitivity and resolution.

To apply triple resonance NMR to UPF0542 protein studies:

• Sample preparation:

  • Express 13C/15N double-labeled protein in minimal media

  • Purify to >95% homogeneity

  • Prepare 0.5-1.0 mM protein in NMR buffer (typically 50 mM phosphate, 50-150 mM NaCl, pH 6.5-7.5)

• Experiment selection:

  • Begin with HSQC to assess sample quality

  • Proceed with triple resonance experiments (HNCA, HN(CO)CA, HNCACB, HN(CO)CACB)

  • For interaction studies, use HMQC-based experiments similar to the H(C)Rh approach described for rhodium complexes

• Analysis workflow:

  • Backbone assignment using established protocols

  • Secondary structure determination from chemical shift indices

  • NOE-based distance restraints for tertiary structure

  • Relaxation measurements for dynamics analysis

Triple resonance approaches can be particularly valuable for detecting subtle conformational changes upon ligand binding or mutation, providing insights into UPF0542 protein function that would be inaccessible through other structural techniques .

What considerations should be addressed when designing chimeric constructs incorporating UPF0542 protein domains?

Designing chimeric constructs with UPF0542 protein domains requires careful consideration of multiple factors to ensure proper expression and functionality:

• Domain boundary identification:

  • Analyze sequence conservation across species

  • Use structure prediction algorithms to identify independently folding units

  • Consider hydrophobic core preservation when selecting fusion points

• Linker design strategies:

  • Flexible linkers (GGGGS)n for independent domain function

  • Rigid linkers (EAAAK)n when domain orientation is critical

  • Specific cleavage sites for post-expression processing

• Expression optimization considerations:

  • Codon optimization for the host system using parameters such as GC content, codon adaptation index (CAI), and codon frequency distribution

  • Signal peptide incorporation if secretion is desired

  • Addition of purification tags (His-tag, GST) with appropriate protease cleavage sites

• Construct validation approaches:

  • In silico structure prediction and validation through Ramachandran plot analysis

  • Experimental validation via circular dichroism spectroscopy

  • Functional assays specific to each domain

For UPF0542 protein specifically, the intact protein is relatively small (74 amino acids), making it potentially suitable as a fusion partner. When designing chimeric constructs, the secondary structure prediction using SOPMA and 3D structure generation using Swiss Model server should be performed to ensure proper domain folding .

How can contradictory results in UPF0542 protein localization studies be resolved?

Resolving contradictory results in protein localization studies requires systematic investigation using complementary approaches:

• Methodological reconciliation strategy:

  • Compare fixation protocols (paraformaldehyde vs. methanol)

  • Assess antibody specificity through Western blot analysis

  • Evaluate expression level effects (physiological vs. overexpression)

  • Consider cell type-specific differences in localization machinery

• Multi-technique verification approach:

  Technique	Resolution	Live/Fixed	Advantages	Limitations
  Immunofluorescence	~200 nm	Fixed	Multiple protein detection	Fixation artifacts
  Fluorescent protein fusion	~200 nm	Live	Dynamic studies	Tag interference
  Biochemical fractionation	N/A	Fixed	Quantitative	Poor spatial resolution
  Proximity labeling	~10 nm	Live	In situ neighbors	Non-specific labeling
  Super-resolution microscopy	10-50 nm	Both	High resolution	Complex analysis

• Computational analysis of protein sequence:

  • Predict localization signals (NLS, NES, signal peptides)

  • Analyze post-translational modification sites affecting localization

  • Examine interaction partners with known localizations

• Design of definitive experiments:

  • CRISPR/Cas9 endogenous tagging to maintain physiological expression

  • Split fluorescent protein complementation to confirm interaction-dependent localization

  • Inducible expression systems to monitor localization kinetics

When addressing UPF0542 protein localization specifically, sequence analysis suggests potential membrane association based on the presence of hydrophobic regions within its amino acid sequence (MFDVKAWAVYIVEWAAKDPYGFLTTVILVLTPLFIISAALSWKLAKMIETREREQKKKRKRQENIVKAKRAKKD) , which should be experimentally verified.

What purification strategy yields the highest purity and recovery of Recombinant UPF0542 protein?

Developing an optimal purification strategy for Recombinant UPF0542 protein requires a multi-step approach that balances purity, yield, and functional integrity:

• Initial capture phase:

  • Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA agarose is recommended for His-tagged UPF0542 protein

  • Optimize binding conditions: test various imidazole concentrations (5-20 mM) in binding buffer to reduce non-specific binding

  • For denaturing conditions, use 8M urea or 6M guanidine-HCl buffer systems

  • For native conditions, include mild detergents (0.1% Triton X-100) if membrane association is suspected

• Intermediate purification:

  • Ion exchange chromatography (IEX) based on theoretical pI calculation from amino acid sequence

  • Size exclusion chromatography (SEC) to remove aggregates and separate oligomeric states

• Polishing and validation:

  • Reverse-phase HPLC for highest purity requirements

  • Verify purity by SDS-PAGE (12% gel concentration) and Western blotting using anti-His antibodies

  • Confirm identity by mass spectrometry (expected MW based on sequence: ~8.5 kDa)

• Scale-up considerations:

  • Implement tangential flow filtration for concentration rather than centrifugal concentrators

  • Monitor and minimize endotoxin levels for downstream biological applications

  • Validate batch consistency through activity assays and analytical SEC

The purification protocol should be optimized iteratively, with full documentation of recovery and purity at each step to identify bottlenecks in the process.

How should researchers design controlled experiments to determine UPF0542 protein function?

Designing controlled experiments to elucidate UPF0542 protein function requires a comprehensive approach:

• Comparative genomics foundation:

  • Analyze conservation across species

  • Identify co-evolved gene clusters

  • Examine expression patterns in different tissues/conditions

• Loss-of-function experimental design:

  • CRISPR/Cas9 knockout with phenotypic characterization

  • RNAi-mediated knockdown with dose-response assessment

  • Dominant-negative mutant expression

• Gain-of-function experimental design:

  • Controlled overexpression using inducible promoters

  • Rescue experiments in knockout backgrounds

  • Ectopic expression in heterologous systems

• Interaction mapping strategy:

  • Yeast two-hybrid screening with appropriate controls

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling (BioID or APEX) in relevant cellular compartments

• Essential controls for rigorous interpretation:

  • Empty vector controls for all expression experiments

  • Scrambled siRNA/sgRNA controls for knockdown/knockout

  • Inactive mutant controls (e.g., predicted active site mutations)

  • Cell viability assessments to rule out non-specific toxicity

  • Time-course experiments to distinguish primary from secondary effects

For the UPF0542 protein specifically, its small size (74 amino acids) and sequence characteristics suggest potential roles in protein-protein interactions or membrane-associated functions that should be systematically tested through these controlled experimental designs.

What are the most effective methods for analyzing post-translational modifications of UPF0542 protein?

Analysis of post-translational modifications (PTMs) on UPF0542 protein requires an integrated workflow combining enrichment, detection, and quantification strategies:

• Comprehensive PTM mapping strategy:

  PTM Type	Enrichment Method	Detection Technique	Quantification Approach
  Phosphorylation	TiO2/IMAC	LC-MS/MS, Phos-tag gels	SILAC, TMT labeling
  Glycosylation	Lectin affinity	PNGase F/mass shift	Glycopeptide MS/MS
  Ubiquitination	K-ε-GG antibodies	Ubiquitin remnant MS	Spectral counting
  Acetylation	Anti-acetyl lysine	MS/MS, Western blot	Label-free quantification
  Methylation	Anti-methyl antibodies	High-resolution MS	Heavy methyl SILAC

• Site-specific analysis workflow:

  • In silico prediction of modification sites using algorithms specific to each PTM

  • Site-directed mutagenesis of predicted sites to confirm functional relevance

  • Generation of site-specific antibodies for high-throughput monitoring

  • Parallel reaction monitoring (PRM) for targeted MS analysis of modified peptides

• Temporal dynamics assessment:

  • Pulse-chase experiments with metabolic labeling

  • Time-resolved proteomics following stimulation

  • In vitro enzyme assays with purified modification enzymes

• Functional impact determination:

  • Correlation of modification status with protein activity

  • Structural analysis of modified vs. unmodified protein

  • Interactome analysis conditioned on modification state

Given the small size of UPF0542 protein and its sequence characteristics, particular attention should be paid to potential phosphorylation sites within the C-terminal region containing multiple lysine residues (KLAKMIETREREQKKKRKRQENIVKAKRAKKD) , which may regulate its interactions or localization.

How can CRISPR/Cas9 technology be optimized for studying UPF0542 protein function in avian systems?

Optimizing CRISPR/Cas9 technology for UPF0542 protein function studies in avian systems requires addressing several unique challenges:

• Delivery optimization strategy:

  • Viral vectors: Lentivirus for cell lines, adeno-associated virus for in vivo applications

  • Non-viral approaches: Lipofection for cell lines, electroporation for primary cells

  • In ovo injection techniques for developmental studies

• Guide RNA design considerations:

  • Target sequence specificity verification against chicken genome

  • Evaluation of guide efficiency using prediction algorithms

  • Design of homology-directed repair templates for knock-in experiments

  • Off-target analysis specific to Gallus gallus genome

• Validation protocol development:

  • T7 Endonuclease I assay for initial editing efficiency assessment

  • Deep sequencing to quantify editing precision and off-target effects

  • RT-qPCR and Western blot to confirm knockdown at RNA and protein levels

  • Phenotypic characterization using standardized assays

• Experimental design for functional studies:

  • Generation of cell line panels (knockout, knockdown, domain deletions)

  • Complementation studies with wild-type and mutant constructs

  • Integration with transcriptomic and proteomic profiling

  • Developmental timing considerations for in vivo studies

Given the limited information available specifically for UPF0542 protein C5orf43 homolog, comparative analysis with mammalian orthologs can guide hypothesis generation for functional studies in avian systems.

What computational approaches can predict interaction partners of UPF0542 protein?

Predicting UPF0542 protein interaction partners requires integration of multiple computational approaches:

• Sequence-based prediction methods:

  • Motif identification for known interaction domains

  • Conservation analysis to identify co-evolved residues

  • Machine learning approaches trained on known protein-protein interactions

• Structure-based prediction techniques:

  • Homology modeling followed by molecular docking

  • Binding site analysis using fpocket or SiteMap

  • Molecular dynamics simulations to assess interaction stability

  • Interface residue prediction using PIER or ProMate

• Network-based approaches:

  • Gene co-expression analysis across tissues and conditions

  • Phylogenetic profiling to identify functionally related proteins

  • Literature-based relationship extraction using NLP

  • Integration of high-throughput interaction data from related species

• Validation strategy development:

  • Design of targeted experiments to confirm high-confidence predictions

  • Cross-validation using orthogonal computational methods

  • Sensitivity analysis to assess prediction robustness

For UPF0542 protein specifically, analysis of its amino acid sequence (MFDVKAWAVYIVEWAAKDPYGFLTTVILVLTPLFIISAALSWKLAKMIETREREQKKKRKRQENIVKAKRAKKD) suggests potential for membrane association and protein-protein interactions through its lysine-rich C-terminal region, which should guide computational prediction parameters.

How can researchers address solubility issues with Recombinant UPF0542 protein expression?

Addressing solubility challenges in Recombinant UPF0542 protein expression requires a systematic troubleshooting approach:

• Expression condition optimization:

  • Temperature reduction during induction (37°C → 25°C or 16°C)

  • IPTG concentration titration (0.1 mM to 1.0 mM)

  • Media supplementation with osmolytes (sorbitol, betaine)

  • Co-expression with molecular chaperones (GroEL/ES, DnaK/J)

• Construct modification strategies:

  • Fusion tags selection matrix:

    Fusion Tag	Size	Effect on Solubility	Purification Method
    MBP	42 kDa	High enhancement	Amylose resin
    SUMO	11 kDa	Moderate enhancement	IMAC
    Thioredoxin	12 kDa	Moderate enhancement	IMAC
    GST	26 kDa	Variable	Glutathione resin
    NusA	55 kDa	High enhancement	IMAC

  • Codon optimization for Gallus gallus-derived protein

  • Truncation constructs to identify soluble domains

  • Site-directed mutagenesis of hydrophobic residues

• Extraction condition optimization:

  • Detergent screening (non-ionic: Triton X-100, NP-40; zwitterionic: CHAPS)

  • Ionic strength variation (100-500 mM NaCl)

  • pH optimization (6.0-9.0) based on theoretical pI

  • Addition of stabilizing co-factors or ligands

• Refolding strategy development (if inclusion bodies are unavoidable):

  • On-column refolding during IMAC purification

  • Dilution refolding with redox pair (GSH/GSSG)

  • Dialysis-based refolding with decreasing denaturant gradient

  • Chaperone-assisted refolding

Given the small size of UPF0542 protein (74 amino acids) and its sequence characteristics, particular attention should be paid to the hydrophobic regions that may contribute to aggregation during expression .

What strategies can overcome detection limitations in UPF0542 protein interaction studies?

Overcoming detection limitations in protein interaction studies requires implementation of sensitive techniques and proper experimental design:

• Enhanced detection methodology selection:

  • Proximity ligation assay (PLA) for in situ interaction detection

  • FRET/BRET for real-time interaction monitoring

  • Single-molecule pull-down for detection of low-abundance complexes

  • Crosslinking mass spectrometry for transient interaction capture

• Signal amplification approaches:

  • Tyramide signal amplification for immunodetection

  • Rolling circle amplification for proximity assays

  • Multi-epitope tagging for enhanced antibody recognition

  • Click chemistry-based labeling for improved signal-to-noise ratio

• Enrichment strategy optimization:

  • Tandem affinity purification for complex stability

  • Size exclusion chromatography for native complex isolation

  • Density gradient fractionation for compartment-specific interactions

  • Immunoprecipitation under optimized buffer conditions

• Control implementation for result validation:

  • Interaction-deficient mutants as negative controls

  • Known interaction partners as positive controls

  • Competition assays with excess unlabeled protein

  • Reciprocal tagging to confirm bidirectional pull-down

For UPF0542 protein specifically, its small size may present detection challenges that can be addressed through specialized approaches such as chemical crosslinking prior to purification or the use of split reporter systems (luciferase complementation assay, split-GFP) to enhance signal detection.