Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog B

What is the basic structure of UPF0767 protein C1orf212 homolog B?

UPF0767 protein C1orf212 homolog B is a 91-amino acid protein from Xenopus laevis with UniProt accession number Q68EV8. The amino acid sequence (MWPVLWAAARTYAPYITFPVAFVVGAVGYQLEWFIRGTPGHPVEEQSILEKREERTLQETMGKDVTQVISLKEKLEFTPKAVLNRNRQEKS) shows a full-length protein with expression region 1-91 . The sequence contains hydrophobic regions, particularly at the N-terminus, suggesting potential membrane association characteristics, although detailed structural studies have not been extensively reported in the literature.

What is known about the functional role of UPF0767 protein C1orf212 homolog B?

While specific functional roles of UPF0767 protein C1orf212 homolog B in Xenopus laevis remain largely uncharacterized, its classification as an UPF (Uncharacterized Protein Family) member indicates it belongs to a group of proteins with conserved sequences but undefined functions. Other Xenopus laevis proteins, such as the cortical granule lectin-1 (XCGL-1), have well-established roles in fertilization membrane development . Research using recombinant UPF0767 protein C1orf212 homolog B could potentially elucidate its biological function, possibly related to developmental processes or cellular signaling pathways in Xenopus, similar to how XCGL-1's role was identified.

How does UPF0767 protein C1orf212 homolog B compare to its homologs in other species?

As a homolog to human C1orf212 protein, UPF0767 protein C1orf212 homolog B likely shares conserved structural features and possibly functional similarities. Comparative sequence analysis between Xenopus laevis UPF0767 protein C1orf212 homolog B and human small integral membrane proteins (such as SMIM12 ) may reveal evolutionary relationships and conserved domains. Researchers should conduct bioinformatic analyses using tools like BLAST, multiple sequence alignments, and phylogenetic tree construction to identify conserved motifs that could suggest functional roles.

What are the optimal storage conditions for maintaining protein stability?

The recombinant protein should be stored in a Tris-based buffer with 50% glycerol, which has been optimized for this specific protein . For short-term storage (up to one week), working aliquots can be maintained at 4°C. For medium-term storage, -20°C is recommended, while long-term storage requires -80°C . To prevent degradation, avoid repeated freeze-thaw cycles as this can significantly reduce protein activity and structural integrity. Dividing the stock solution into single-use aliquots immediately upon receipt is strongly recommended.

What expression systems are most suitable for producing functional UPF0767 protein C1orf212 homolog B?

While the search results don't specifically address expression systems for UPF0767 protein C1orf212 homolog B, insights can be drawn from successful approaches with other Xenopus proteins. For instance, XCGL-1 has been successfully expressed in bacterial (E. coli) and eukaryotic systems (HEK293T mammalian cells and Trichoplusia ni insect cells) . For UPF0767 protein C1orf212 homolog B, researchers should consider:

• Bacterial expression: Cost-effective but may require optimization of solubility and refolding protocols

• Mammalian expression: Potentially better for proper folding and post-translational modifications

• Insect cell expression: Often provides a good balance between yield and proper protein processing

The choice should be guided by the experimental requirements, especially if post-translational modifications are critical for the protein's function.

What purification strategies yield the highest purity and activity?

Effective purification strategies would likely include:

• Affinity chromatography: Using nickel or cobalt resins if the protein includes a histidine tag

• Size exclusion chromatography: For separating the protein from aggregates and other contaminants

• Ion exchange chromatography: Based on the protein's isoelectric point

For quality control, researchers should verify:

• Purity by SDS-PAGE (>95% recommended for most applications)

• Identity by Western blotting and/or mass spectrometry

• Activity through appropriate functional assays (once established)

How can researchers effectively characterize the oligomeric state of UPF0767 protein C1orf212 homolog B?

Based on characterization methods used for similar Xenopus proteins like XCGL-1, researchers should employ multiple complementary techniques:

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): To determine absolute molecular weight and oligomeric state in solution

• Analytical Ultracentrifugation: For detailed analysis of sedimentation properties and molecular weight determination

• Non-reducing and reducing SDS-PAGE: To identify potential disulfide-linked oligomers, as demonstrated with XCGL-1

• Cross-linking studies: To capture transient protein-protein interactions

• Native PAGE: To analyze native oligomeric states under non-denaturing conditions

What techniques are most informative for structural characterization?

For comprehensive structural characterization, researchers should consider:

• X-ray crystallography: For high-resolution structural determination if the protein can be crystallized

• Cryo-electron microscopy: Particularly useful if the protein forms larger complexes

• Nuclear Magnetic Resonance (NMR): For solution structure determination and dynamics studies if the protein is of suitable size

• Circular Dichroism (CD): For secondary structure composition assessment

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): To probe conformational dynamics and solvent accessibility

For initial characterization, CD spectroscopy provides a relatively quick assessment of proper folding and secondary structure content.

How should researchers approach potential post-translational modifications analysis?

Drawing from studies on other Xenopus proteins, researchers should investigate:

• N-linked glycosylation: Using PNGase F and Endo H digestion followed by SDS-PAGE analysis, similar to methods used for XCGL-1

• Disulfide bond mapping: Using mass spectrometry after non-reducing proteolytic digestion

• Phosphorylation sites: Through phospho-specific antibodies or mass spectrometry approaches

• Other modifications: Using comprehensive LC-MS/MS analysis with appropriate database searching

A systematic approach comparing experimentally determined mass with theoretical mass can reveal the presence and nature of modifications.

What binding assays are recommended for identifying interaction partners?

To identify potential interaction partners, researchers should consider:

• Pull-down assays: Using tagged UPF0767 protein C1orf212 homolog B as bait

• Yeast two-hybrid screening: For identifying protein-protein interactions

• Biolayer Interferometry (BLI): For quantitative binding kinetics, similar to methods used for XCGL-1

• Surface Plasmon Resonance (SPR): For detailed binding kinetics and affinity measurements

• Isothermal Titration Calorimetry (ITC): For thermodynamic characterization of binding interactions

How can researchers determine if UPF0767 protein C1orf212 homolog B binds specific ligands?

Based on approaches used with other Xenopus proteins like XCGL-1, researchers should:

• Perform glycan array screening: To identify potential carbohydrate ligands, if the protein has lectin-like properties

• Conduct small molecule screening: Using thermal shift assays (differential scanning fluorimetry) to identify stabilizing ligands

• Employ fluorescence-based binding assays: Such as fluorescence polarization or FRET

• Utilize molecular docking and virtual screening: For in silico prediction of potential binding partners

• Conduct biolayer interferometry (BLI) studies: Similar to those used for XCGL-1, which demonstrated binding to various galactose-containing carbohydrates

An integrated approach combining computational predictions with experimental validation would be most effective.

What cellular assays can help elucidate the protein's function in Xenopus development?

To investigate the role of UPF0767 protein C1orf212 homolog B in Xenopus development, researchers should consider:

• Morpholino knockdown studies: To assess loss-of-function phenotypes in developing embryos

• CRISPR/Cas9-mediated gene editing: For creating knockout or knock-in models

• mRNA overexpression studies: To assess gain-of-function effects

• Spatiotemporal expression analysis: Using in situ hybridization and immunohistochemistry

• Rescue experiments: To confirm specificity of observed phenotypes

• Ex vivo tissue culture assays: To assess effects on specific developmental processes

These approaches would help place the protein in relevant developmental pathways, similar to how XCGL-1 was identified as critical in fertilization membrane development .

How does UPF0767 protein C1orf212 homolog B compare structurally and functionally to other Xenopus proteins?

While direct comparisons are not available in the provided search results, researchers should consider:

• Sequence homology analysis: Comparing with well-characterized Xenopus proteins like XCGL-1

• Structural modeling: Using homology modeling based on proteins with solved structures

• Domain architecture comparison: Identifying conserved functional domains

• Expression pattern analysis: Comparing tissue distribution and developmental timing

• Functional assay comparisons: Testing if the protein shares functional properties with better-characterized proteins

For instance, XCGL-1 has been shown to bind galactose-containing carbohydrates and play a role in fertilization . Similar binding studies and developmental analyses would be valuable for UPF0767 protein C1orf212 homolog B.

What evolutionary insights can be gained from studying this protein across species?

Evolutionary analysis should include:

• Phylogenetic analysis: Constructing trees to understand evolutionary relationships

• Synteny analysis: Examining conservation of genomic context across species

• Selection pressure analysis: Calculating dN/dS ratios to identify conserved functional regions

• Ancestral sequence reconstruction: To understand evolutionary trajectories

• Comparison with mammalian homologs: Particularly human C1orf212 and related proteins

Such analyses could reveal whether the protein has undergone adaptive evolution or maintained conserved functions across vertebrates, providing insights into its biological significance.

What are common challenges in working with recombinant UPF0767 protein C1orf212 homolog B and how can they be addressed?

Based on experiences with similar proteins:

• Solubility issues: Consider optimizing buffer conditions (pH, salt concentration, additives) or using solubility tags (MBP, SUMO)

• Protein aggregation: Implement size exclusion chromatography quality control steps and optimize storage conditions

• Low activity: Ensure proper folding through CD spectroscopy and consider refolding protocols if necessary

• Degradation: Add protease inhibitors during purification and assess stability under various conditions

• Batch-to-batch variability: Implement standardized quality control metrics for each preparation

For glycosylated proteins, expression in eukaryotic systems may be necessary for proper folding and function, as demonstrated with XCGL-1 .

How can researchers validate that their recombinant protein preparation retains native structure and function?

Validation should include:

• Secondary structure analysis: Using circular dichroism to compare with predicted structures

• Thermal stability assessment: Using differential scanning fluorimetry

• Limited proteolysis: To assess proper folding through proteolytic susceptibility patterns

• Activity assays: Once established, specific functional assays should be used to confirm activity

• Binding studies: Verification of interaction with known partners, once identified

Researchers should establish multiple quality control criteria that must be met before using the protein in downstream applications.

What are promising research applications for UPF0767 protein C1orf212 homolog B in developmental biology?

Potential research directions include:

• Developmental role investigation: Using knockdown/knockout approaches in Xenopus embryos

• Protein interaction network mapping: Identifying binding partners during different developmental stages

• Structural biology studies: Determining high-resolution structure to inform function

• Comparative studies with mammalian homologs: To understand evolutionary conservation of function

• Integration with systems biology approaches: To place the protein in relevant developmental pathways

Like XCGL-1, which was found to be critical in fertilization membrane development , UPF0767 protein C1orf212 homolog B may have specific roles in Xenopus development that could inform broader understanding of vertebrate development.

How might advanced computational approaches enhance our understanding of this protein?

Advanced computational methods could include:

• Molecular dynamics simulations: To understand conformational dynamics and potential binding sites

• Machine learning approaches: For prediction of function based on sequence and structural features

• Network analysis: To predict functional associations based on co-expression data

• Integrative modeling: Combining experimental data with computational predictions

• Prediction of intrinsically disordered regions: Which may be important for function or regulation

These approaches could generate testable hypotheses about the protein's function when experimental data is limited.