Recombinant Human UPF0708 protein C6orf162 (C6orf162)

What is UPF0708 protein C6orf162 and what are its key characteristics?

C6orf162 (chromosome 6 open reading frame 162) is a protein encoded by a gene located on human chromosome 6, specifically in the q15-q16.1 region. It belongs to the UPF0708 protein family, which consists of uncharacterized proteins with functions that are not yet fully elucidated in scientific literature. The protein has several synonyms including dJ102H19.2 and DKFZp586E1923 .

The mouse homolog (Smim8; Small integral membrane protein 8) consists of 97 amino acids with the sequence: MSSAPDPPTVKKEPLKEKNFENPGLRGAHTTTLFRAVNPELFIKPNKPVMAFGLVTLSLCVAYIGYLHATQENRKDLYEAIDSEGHRYMRRKTSKWD . The human variant likely shares considerable sequence homology with this mouse variant, though specific differences should be expected as with most orthologous proteins.

What experimental systems have been used to study C6orf162 function?

Several experimental approaches have been employed to investigate C6orf162, including:

• Recombinant protein expression in prokaryotic systems (E. coli)

• Protein tagging strategies (particularly His-tagging) for purification and detection

• Transcriptomic analyses to determine expression patterns in different tissues

• Proteomic approaches to identify interacting partners

The mouse homolog has been successfully expressed as a recombinant protein in E. coli with an N-terminal His tag, which allows for straightforward purification and subsequent functional studies .

What is currently known about C6orf162 tissue distribution and expression patterns?

While the search results don't provide comprehensive information about C6orf162 tissue distribution, researchers typically assess this using:

• Transcriptomic data from RNA-seq experiments similar to those described in other studies

• RT-PCR and qPCR validation of expression across tissue panels

• Immunohistochemistry with validated antibodies

• Public database mining (such as GTEx, Human Protein Atlas)

Detection methodologies often employ statistical significance testing using proportional statistics on exon reads as described in related transcriptomic studies , with stringent criteria including q ≤0.05 according to Benjamini and Hochberg methods and fold-regulation >2 for estimation of up- and downregulated transcripts.

What are the predicted structural features of C6orf162 and their implications for function?

Based on available sequence data, C6orf162 appears to have membrane-associated properties. The mouse homolog (Smim8) is classified as a "small integral membrane protein" , suggesting that the human version may also interact with cellular membranes.

Sequence analysis of the mouse protein reveals hydrophobic regions (e.g., "VAYIGYLHAT") that could form transmembrane domains . Researchers investigating structural features should consider:

• Using bioinformatic tools for predicting transmembrane domains, signal peptides, and post-translational modification sites

• Circular dichroism spectroscopy to assess secondary structure elements

• Limited proteolysis coupled with mass spectrometry to identify stable domains

• Structural studies using X-ray crystallography or NMR for detailed atomic-level information

How might experimental design approaches be optimized for studying C6orf162 interactions?

When investigating protein-protein interactions involving C6orf162, researchers should consider multivariant experimental design approaches similar to those used in other recombinant protein studies . These methods allow for:

• Systematic evaluation of multiple variables simultaneously

• Identification of statistically significant factors affecting protein behavior

• Assessment of interaction effects between experimental variables

• Efficient resource utilization through fractional factorial designs

As noted in relevant protein expression studies: "The multivariant method permits a thoroughly analysis compared to the traditional univariant method, where the response is evaluated changing one variable at a time while fixing the others. Furthermore, the multivariant method enables to characterize the experimental error, to compare the effects of variables between themselves when variables are normalized, and hence, to gather high-quality information with as few experiments as possible" .

What experimental approaches can differentiate between direct and indirect protein interactions with C6orf162?

To establish whether interactions with C6orf162 are direct or indirect, researchers should implement:

• Yeast two-hybrid screening with stringent controls

• Co-immunoprecipitation followed by mass spectrometry

• Proximity labeling approaches (BioID, APEX)

• Surface plasmon resonance or isothermal titration calorimetry for direct binding kinetics

• FRET/BRET assays for interactions in living cells

What are the optimal conditions for soluble expression of recombinant C6orf162?

Based on related recombinant protein expression studies , researchers should consider several factors to optimize soluble expression:

• Expression system selection: While E. coli has been successfully used for the mouse homolog , mammalian or insect cell systems might provide better folding for the human variant.

• Temperature optimization: Lower induction temperatures (15-25°C) often improve solubility by slowing protein synthesis and allowing proper folding.

• Induction parameters: As noted in related studies, "induction times between 4h and 6h presented similar levels of productivity" , suggesting this timeframe might be optimal.

• Media composition: Rich media or defined media supplements may improve folding and solubility.

The statistical experimental design methodology described for other recombinant proteins can be adapted: "This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression... in E. coli, which should contribute to reduce operational costs" .

What purification strategies are most effective for recombinant C6orf162?

For His-tagged recombinant C6orf162 as described for the mouse homolog , the following purification approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins

• Intermediate purification: Ion exchange chromatography based on the protein's predicted isoelectric point

• Polishing step: Size exclusion chromatography to remove aggregates and ensure homogeneity

Specific buffer considerations should include:

• IMAC binding buffer: 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM imidazole

• IMAC elution buffer: 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole

• Final storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0

What storage conditions maximize stability of purified recombinant C6orf162?

Based on recommendations for the mouse homolog, the following storage practices are advised :

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

• Reconstitute 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 optimal) for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• For short-term use, store working aliquots at 4°C for up to one week

• Avoid repeated freezing and thawing, which can lead to protein degradation and loss of activity

What methodologies are most suitable for investigating potential membrane associations of C6orf162?

Given that the mouse homolog is classified as a small integral membrane protein , researchers should consider these approaches for investigating membrane associations:

• Subcellular fractionation: Differential centrifugation to separate membrane fractions, followed by Western blotting to detect C6orf162

• Detergent extraction profiles: Testing solubility in different detergents to characterize membrane association strength

• Protease protection assays: To determine topology of membrane-inserted regions

• Fluorescence microscopy: Using tagged versions to visualize subcellular localization

• Liposome reconstitution: For in vitro assessment of membrane insertion capabilities

These approaches should be complemented with appropriate controls and statistical analysis as described in related literature: "Significance was tested using proportional statistics on total exon reads using method by Kal et al. (1999)... with stringent criteria being applied, involving q ≤0.05 according to Benjamini and Hochberg, fold-regulation >2 for estimation of up- and downregulated transcripts" .

How can researchers address potential artifacts in C6orf162 functional studies?

When conducting functional studies with recombinant C6orf162, researchers should implement these controls to minimize artifacts:

• Expression tag influence: Compare N-terminal vs. C-terminal tagged versions, and include tag-only controls

• Overexpression artifacts: Use inducible expression systems with titrated expression levels

• Cross-species validation: Compare results between human C6orf162 and mouse Smim8 to confirm conserved functions

• Multiple detection methods: Employ orthogonal approaches to validate observed phenotypes

• CRISPR/Cas9 knockout controls: Generate complete knockouts to compare with knockdown and overexpression phenotypes

What are the recommended approaches for identifying potential interacting partners of C6orf162?

To identify proteins that interact with C6orf162, researchers should consider:

• Affinity purification-mass spectrometry (AP-MS): Using tagged C6orf162 as bait to capture interacting proteins

• Proximity-dependent biotin identification (BioID): For detecting transient or weak interactions in living cells

• Co-immunoprecipitation with specific antibodies: For endogenous protein interactions

• Protein microarrays: To screen for interactions with predefined protein sets

• In silico prediction followed by experimental validation: Using tools that predict protein-protein interactions based on sequence and structural features

For validation and characterization of interactions, researchers should implement stringent statistical criteria similar to those used in other studies: "q ≤0.05 according to Benjamini and Hochberg, fold-regulation >2 for estimation of up- and downregulated transcripts, and also q ≤0.05 for estimation of significant GO categories" .

How can researchers address common expression issues with recombinant C6orf162?

When encountering difficulties with C6orf162 expression, consider the following approaches:

• Insoluble expression: Modify induction conditions (temperature, IPTG concentration, duration) or use solubility-enhancing fusion tags (SUMO, MBP, TrxA)

• Low expression yield: Optimize codon usage for the host organism or use stronger promoters

• Toxicity to host cells: Use tightly regulated expression systems or lower-copy-number plasmids

• Protein degradation: Add protease inhibitors during purification or use protease-deficient host strains

• Improper folding: Co-express with molecular chaperones or use eukaryotic expression systems

Statistical experimental design approaches can systematically address these issues, as they "allow the rapid and economical determination of optimal culture conditions with fewer experiments and minimal resources" .

What quality control measures should be implemented for recombinant C6orf162 preparations?

Researchers should implement these quality control measures:

• Purity assessment: SDS-PAGE analysis with a minimum target of 90% purity

• Identity confirmation: Western blotting and/or mass spectrometry

• Homogeneity evaluation: Size exclusion chromatography and dynamic light scattering

• Functional testing: Activity assays specific to predicted functions

• Endotoxin testing: For preparations intended for cell culture or in vivo experiments

• Stability assessment: Accelerated stability studies under different storage conditions

For the mouse homolog, greater than 90% purity as determined by SDS-PAGE has been reported as a quality standard .

What considerations are important when designing antibodies against C6orf162?

When developing or selecting antibodies against C6orf162, researchers should consider:

• Epitope selection: Choose unique, solvent-accessible regions that don't include predicted transmembrane domains

• Cross-reactivity: Test against related proteins, particularly when studying across species

• Validation methods: Use knockout/knockdown controls, recombinant protein standards, and orthogonal detection methods

• Application specificity: Validate antibodies separately for Western blotting, immunoprecipitation, and immunofluorescence

• Lot-to-lot variation: Establish quality control measures to ensure consistent performance across antibody batches

How might C6orf162 be relevant to disease processes based on predicted functions?

While specific disease associations for C6orf162 are not detailed in the search results, researchers investigating potential roles in disease should consider:

• Expression correlation: Analyze transcriptomic datasets for differential expression in disease states

• Genetic association studies: Evaluate SNPs or mutations in C6orf162 for disease associations

• Pathway involvement: Investigate whether C6orf162 participates in pathways implicated in disease processes

• Membrane protein relevance: Given its potential membrane association, consider roles in membrane integrity, transport, or signaling

What experimental models would be most suitable for studying C6orf162 in disease contexts?

Researchers investigating C6orf162 in disease contexts should consider these model systems:

• Cell line models: Select cell lines that endogenously express C6orf162 at detectable levels

• CRISPR/Cas9 knockout models: Generate cell lines with C6orf162 deletion

• Mouse models: Consider studies with the mouse homolog (Smim8)

• Patient-derived samples: Compare expression levels in relevant disease tissues

• In silico approaches: Use structural prediction and virtual screening to identify potential modulators

Appropriate statistical analyses should be implemented, similar to those described for other molecular studies: "Significance was tested using proportional statistics on total exon reads... with stringent criteria being applied, involving q ≤0.05 according to Benjamini and Hochberg, fold-regulation >2 for estimation of up- and downregulated transcripts" .