Recombinant Mouse UPF0708 protein C6orf162 homolog

What is UPF0708 protein C6orf162 homolog and what are its basic properties?

Recombinant Full Length Mouse UPF0708 protein C6orf162 homolog (UniProt ID: Q9CQQ0) is a small integral membrane protein (Smim8) consisting of 97 amino acids. The full amino acid sequence is: MSSAPDPPTVKKEPLKEKNFENPGLRGAHTTTLFRAVNPELFIKPNKPVMAFGLVTLSLCVAYIGYLHATQENRKDLYEAIDSEGHRYMRRKTSKWD. It is typically produced as a recombinant protein with an N-terminal His tag, expressed in E. coli expression systems .

The protein is classified as part of the UPF (Uncharacterized Protein Family) group, indicating that its specific biological functions have not been fully elucidated. As a membrane protein, it likely plays roles in cellular membrane processes, although the precise function requires further investigation.

How does the mouse UPF0708 protein C6orf162 homolog compare to its human counterpart?

The mouse UPF0708 protein C6orf162 homolog shares sequence homology with the human C6orf162 gene product. The human gene is located on chromosome 6q14.1-q15, a region that has been implicated in various developmental disorders. In particular, microdeletions in this chromosomal region, which include the C6orf162 gene among others, have been associated with neurodevelopmental conditions such as severe autistic disorder, language deficits, and dysmorphic features .

What are the recommended storage and reconstitution protocols for recombinant UPF0708 protein?

For optimal stability and activity of recombinant UPF0708 protein C6orf162 homolog, the following protocols are recommended:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Working aliquots may be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the recommended default)

• Aliquot for long-term storage at -20°C/-80°C

Buffer composition:

• The protein is supplied in Tris/PBS-based buffer containing 6% Trehalose, pH 8.0

What purification approaches yield the highest purity for recombinant UPF0708 protein?

The standard recombinant preparation of UPF0708 protein C6orf162 homolog utilizes His-tag affinity chromatography as the primary purification method. This approach typically yields protein with greater than 90% purity as determined by SDS-PAGE .

For enhanced purification results, a multi-step protocol is recommended:

• Initial purification: Ni-NTA affinity chromatography using the N-terminal His-tag

• Intermediate purification: Size exclusion chromatography to separate the target protein from aggregates and degradation products

• Final polishing: Ion-exchange chromatography to remove contaminants with similar sizes but different charge properties

Researchers should verify protein purity using both SDS-PAGE and Western blot analysis. For studies requiring ultra-high purity (>95%), additional chromatographic steps may be necessary.

How can researchers validate the structural integrity of UPF0708 protein for functional studies?

Multiple complementary approaches can be used to validate the structural integrity of UPF0708 protein:

• Circular Dichroism (CD) spectroscopy: To assess secondary structure elements and confirm proper folding

• Tryptophan fluorescence spectroscopy: To evaluate tertiary structure, as the sequence contains tryptophan residues

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): To determine oligomeric state and detect aggregation

• Thermal shift assays: To evaluate protein stability under various buffer conditions

Similar to approaches used in ubiquitin folding studies, researchers can employ stopped-flow kinetics, NMR spectroscopy, and Förster Resonance Energy Transfer (FRET) to monitor folding dynamics and conformational changes in UPF0708 protein .

What are the implications of UPF0708/C6orf162 in neurodevelopmental disorders?

Genomic studies have revealed that the human C6orf162 gene is located within a chromosomal region (6q14.1-q15) implicated in neurodevelopmental disorders. Microdeletions encompassing this region, which include C6orf162 among 30 other genes, have been associated with severe autistic disorder, absence of oral language, and dysmorphic features .

While the specific contribution of C6orf162 to these phenotypes remains unclear, its presence in this critical genomic region suggests potential roles in neural development or function. Research approaches to investigate this connection might include:

• Targeted gene knockout or knockdown studies in neuronal cell lines

• Expression analysis in neurodevelopmental disease models

• Functional interaction studies with known neurodevelopmental regulators

• Proteomic analyses to identify binding partners in neuronal contexts

It's important to note that the deletion region contains multiple genes, and phenotypic effects likely result from the cumulative impact of several gene disruptions rather than C6orf162 alone.

How can folding dynamics of UPF0708 protein be experimentally characterized?

To characterize the folding dynamics of UPF0708 protein, researchers can adapt methodologies from protein folding studies, such as those used for ubiquitin:

• Stopped-flow kinetics: This approach enables real-time monitoring of folding/unfolding transitions by rapidly mixing the protein with denaturants or refolding buffers and tracking fluorescence changes

• GdnHCl-induced unfolding experiments: By equilibrating the protein at different concentrations of denaturant (e.g., 1M GdnHCl) and then rapidly exposing it to higher concentrations (e.g., 3M GdnHCl), researchers can quantify unfolding rates

• NMR spectroscopy: This provides residue-specific information about structural changes during folding, allowing identification of key stabilizing interactions

• Single-molecule force measurements: These enable direct observation of folding/unfolding transitions at the individual molecule level

• Molecular dynamics simulations: Computational approaches can complement experimental data by providing atomic-level details of folding pathways and energy landscapes

The experimental design should include measurement of both folding and unfolding kinetics to fully characterize the energy landscape of the protein.

What approaches can be used to identify interaction partners of UPF0708 protein?

Identifying interaction partners is crucial for understanding the biological function of UPF0708 protein. Multiple complementary approaches can be employed:

• Affinity purification coupled with mass spectrometry (AP-MS): Using the His-tagged recombinant protein as bait to capture protein complexes from cellular lysates

• Yeast two-hybrid (Y2H) screening: For detecting binary protein-protein interactions

• Proximity labeling approaches: Such as BioID or APEX, which can identify proteins in the vicinity of UPF0708 in living cells

• Co-immunoprecipitation followed by Western blotting: To validate specific interactions identified by high-throughput methods

• Crosslinking mass spectrometry: To capture transient interactions and provide information about interaction interfaces

Given the membrane localization of UPF0708, particular attention should be paid to detergent conditions that preserve membrane protein interactions while allowing sufficient solubilization.

How can researchers design experiments to elucidate the function of UPF0708 in cellular contexts?

A systematic approach to functional characterization of UPF0708 protein would include:

Table 1: Experimental Design Framework for UPF0708 Functional Characterization

What are the key considerations for developing antibodies against UPF0708 protein?

Developing specific antibodies against UPF0708 protein requires careful planning:

• Epitope selection:

  • Analyze the protein sequence for immunogenic regions

  • Avoid transmembrane domains, which may not be accessible in native protein

  • Consider using multiple epitopes from different regions of the protein

  • The N-terminal region (MSSAPDPPTVKKEPLK) contains hydrophilic residues that may serve as good epitopes

• Antibody validation strategies:

  • Western blot against recombinant protein and endogenous protein

  • Immunoprecipitation followed by mass spectrometry

  • Immunofluorescence with knockout/knockdown controls

  • Peptide competition assays to confirm specificity

• Cross-reactivity considerations:

  • Test antibodies against human C6orf162 to determine species cross-reactivity

  • Evaluate potential cross-reactivity with related small membrane proteins

• Application-specific optimization:

  • Different applications (Western blot, immunoprecipitation, immunofluorescence) may require different antibody properties

  • Validate antibodies specifically for each intended application

How can xenonucleus-based approaches be applied to study UPF0708 protein folding?

The xenonucleus approach described for ubiquitin offers an innovative strategy for studying UPF0708 protein folding. This methodology involves:

• Identification of the folding nucleus: Using molecular dynamics simulations or experimental approaches to determine which segment of UPF0708 folds first

• Construction of a xenonucleus: Synthesizing a peptide corresponding to the identified folding nucleus region and conformationally constraining it through disulfide bonds or other modifications

• Folding kinetics analysis: Using stopped-flow fluorescence measurements to assess how the xenonucleus affects folding and unfolding rates of the full-length protein

• Interaction characterization: Employing NMR spectroscopy or FRET to confirm direct interaction between the xenonucleus and the protein

• Functional consequences: Evaluating whether the xenonucleus affects not only folding but also functional properties of the protein

This approach could provide valuable insights into the folding mechanism of UPF0708 and potentially identify strategies to modulate its folding pathway for experimental or therapeutic purposes.

What genetic models would be most appropriate for studying UPF0708/C6orf162 function in vivo?

For comprehensive in vivo characterization of UPF0708/C6orf162 function, multiple genetic models should be considered:

Table 2: Genetic Models for UPF0708/C6orf162 Functional Studies

Given the potential neurodevelopmental relevance, particular attention should be paid to neuronal differentiation and function in these models.

How might structural biology approaches advance our understanding of UPF0708 protein?

Structural characterization of UPF0708 protein would significantly advance functional understanding. Key approaches include:

• X-ray crystallography: For high-resolution structure determination, though membrane proteins present challenges for crystallization

• Cryo-electron microscopy (cryo-EM): Particularly valuable if UPF0708 forms part of larger complexes

• Nuclear Magnetic Resonance (NMR) spectroscopy: Useful for characterizing dynamic regions and solution behavior

• Computational structure prediction: Leveraging recent advances in AI-based prediction methods like AlphaFold2

Structural data would inform hypotheses about:

• Transmembrane topology and membrane insertion mechanism

• Potential binding pockets for small molecules or interaction interfaces for protein partners

• Conserved structural features shared with proteins of known function

• Mechanisms of potential involvement in neurodevelopmental processes

What is the potential relationship between UPF0708/C6orf162 and other genes in the 6q14.1-q15 region?

The chromosomal region 6q14.1-q15 contains multiple genes that, when collectively deleted, have been associated with neurodevelopmental disorders . Understanding the potential functional relationships between UPF0708/C6orf162 and other genes in this region could provide insights into disease mechanisms:

• Co-expression analysis: Identifying tissues or developmental stages where C6orf162 is co-expressed with other genes in the region

• Protein-protein interaction studies: Determining whether C6orf162 directly interacts with proteins encoded by neighboring genes

• Pathway analysis: Identifying shared signaling pathways or biological processes among the proteins encoded in this region

• Combinatorial genetic models: Creating combinatorial knockouts or knockdowns to identify synergistic effects

• Comparative phenotyping: Contrasting the phenotypes of individual gene disruptions with multi-gene deletions

Key genes in this region that may functionally interact with C6orf162 include SYNCRIP, SNX14, ME1, and HTR1E, among others .