Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A

What is Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A?

Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A is a full-length protein (1-91 amino acids) derived from the African clawed frog (Xenopus laevis). It is a recombinant version expressed in E. coli systems, typically with an affinity tag for purification purposes. The protein is part of the UPF0767 family, and as indicated by its name, it is a homolog of the human C1orf212 protein. The protein is registered in the UniProt database with the identifier A1L2P2 . This protein belongs to a family that has been evolutionarily conserved across vertebrates with homologs present in some invertebrates, plants, and single-celled microorganisms, suggesting important biological functions that have been maintained throughout evolution .

The complete amino acid sequence of the protein is: MWPVLWAAARTYAPYITFPVAFVVGAVGYQLEWFIRGTPEHPVEEQSIQEKREERTLQETMGKDVTQVISLKEKLEFTPKAVLNRNRQEKS . Understanding this protein's structure and function contributes to our broader knowledge of evolutionary biology and comparative protein functionality across species.

How does Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A differ from homolog B?

While both proteins are homologs of the human C1orf212 protein and share significant sequence similarity, there are subtle differences that distinguish homolog A from homolog B. The amino acid sequence of homolog B is MWPVLWAAARTYAPYITFPVAFVVGAVGYQLEWFIRGTPGHPVEEQSILEKREERTLQETMGKDVTQVISLKEKLEFTPKAVLNRNRQEKS , whereas homolog A's sequence is MWPVLWAAARTYAPYITFPVAFVVGAVGYQLEWFIRGTPEHPVEEQSIQEKREERTLQETMGKDVTQVISLKEKLEFTPKAVLNRNRQEKS .

A careful comparison reveals key differences at positions 41 (G vs E) and 47-48 (IL vs IQ) between homologs B and A, respectively. These amino acid substitutions might affect protein-protein interactions or structural conformation, potentially leading to distinct functional roles within the organism. Additionally, homolog B is known by the gene name smim12-b and is also referred to as Small integral membrane protein 12-B . When designing experiments, researchers should carefully consider which homolog is most appropriate for their specific research questions.

What are the recommended storage and handling conditions for this protein?

Proper storage and handling of Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A are critical for maintaining its stability and activity. The protein is typically supplied as a lyophilized powder in a Tris-based buffer with 50% glycerol, optimized for this specific protein . For long-term storage, the protein should be kept at -20°C or -80°C .

To reconstitute the protein, it is recommended to briefly centrifuge the vial prior to opening to ensure the contents are at the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For optimal stability, add glycerol to a final concentration of 5-50% (with 50% being standard) before aliquoting for long-term storage at -20°C/-80°C . Importantly, repeated freeze-thaw cycles should be avoided as they can degrade the protein. Working aliquots can be stored at 4°C for up to one week .

What are the optimal conditions for expressing Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A?

Optimizing expression conditions for Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A requires a systematic approach rather than the traditional one-factor-at-a-time method. Design of Experiments (DoE) methodology is highly recommended for determining optimal expression conditions in E. coli systems .

A comprehensive DoE approach should consider multiple factors simultaneously, including:

• Induction temperature (typically ranging from 16°C to 37°C)

• IPTG concentration (0.1 mM to 1.0 mM)

• Induction duration (4 to 24 hours)

• Media composition (LB, TB, or specialized media)

• E. coli strain selection (BL21(DE3), Rosetta, Arctic Express, etc.)

• Co-expression with chaperones if necessary

Using statistical software packages designed for DoE, researchers can design a minimal set of experiments that efficiently identifies optimal conditions. This approach not only saves time and resources but also accounts for interaction effects between variables that might be missed in traditional approaches. For example, the combined effect of lower temperature and longer induction time might yield better results than optimizing each factor independently .

After expression, purification typically involves affinity chromatography utilizing the His-tag or another fusion tag, followed by size exclusion chromatography if higher purity is required. Protein activity and structural integrity should be verified post-purification through appropriate functional or structural assays.

What structural characteristics have been identified for UPF0767 protein C1orf212 homolog proteins?

The structural characterization of UPF0767 protein C1orf212 homolog proteins remains limited, but computational analyses provide valuable insights. HMM-HMM comparison methods have revealed significant matches to proteins with repetitive alpha helix-rich structures .

The strongest structural match for the human ortholog is to the beta subunit of human importin, which contains HEAT repeats (with a probability of 87%, though sequence identity is only 8%) . Other significant structural matches include proteins containing HEAT repeats such as:

• Microtubule-binding TOG domains

• PTPA protein phosphatase activator

• Yeast cytoplasmic export protein 1

Sequence conservation analysis using the ConSurf server indicates that the inner side of the curved structure is significantly more conserved than the outer surface. This conservation pattern suggests that the inner surface likely harbors interfaces for binding to interaction partners .

A notable structural feature is the WCF motif, where the tryptophan residues form part of the hydrophobic core, while the phenylalanine residue lies on the surface, potentially contributing to interactions with partner proteins. The model of C1ORF112 reveals two significant conserved surface patches that may function as distinct interfaces for different interactors .

For researchers working with Xenopus laevis UPF0767 protein C1orf212 homolog A, these structural insights can guide the design of experiments to probe protein-protein interactions and functional domains.

What methodologies are recommended for investigating protein-protein interactions of UPF0767 protein C1orf212 homolog A?

Investigating protein-protein interactions of UPF0767 protein C1orf212 homolog A requires a multi-faceted approach. Based on the predicted structural features and potential functional roles, the following methodologies are recommended:

• Pull-down assays: Utilizing the His-tagged recombinant protein as bait to identify interacting partners from Xenopus laevis cell lysates, followed by mass spectrometry analysis.

• Yeast two-hybrid screening: This can identify direct protein-protein interactions, especially considering the predicted binding interfaces on the inner surface of the curved structure.

• Co-immunoprecipitation: If antibodies against UPF0767 protein C1orf212 homolog A are available, this method can identify protein complexes in near-native conditions.

• Proximity-based labeling: BioID or APEX2 fusion proteins can identify proteins in close proximity to UPF0767 protein C1orf212 homolog A in living cells.

• Surface plasmon resonance (SPR) or biolayer interferometry (BLI): These techniques can quantitatively measure binding kinetics with candidate interacting proteins, particularly those involved in DNA repair pathways like BRCA1, BRCA2, or components of the Fanconi anemia pathway.

• Fluorescence resonance energy transfer (FRET): This can detect direct protein-protein interactions in living cells and provide spatial information about where these interactions occur.

When designing these experiments, special attention should be paid to the conserved surface patches, particularly the regions containing the WCF motif, as these are likely involved in protein-protein interactions .

How can Design of Experiments (DoE) be applied to optimize functional studies of Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A?

Design of Experiments (DoE) offers a powerful approach for optimizing functional studies of Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A. Unlike traditional one-factor-at-a-time methods, DoE enables researchers to investigate multiple factors simultaneously, reducing experimental time and resources while accounting for interaction effects .

For investigating the potential role of this protein in DNA repair pathways, a DoE approach might include the following factors:

• Concentration of DNA damaging agents (e.g., UV, hydroxyurea, cisplatin)

• Protein concentration

• Reaction time

• Buffer composition (pH, salt concentration)

• Presence of potential cofactors or interacting proteins

A central composite design or Box-Behnken design would be appropriate for optimizing these factors. The response variables could include DNA binding activity, ATPase activity, or interaction with known DNA repair proteins. Software packages like JMP, Design-Expert, or the R package 'rsm' can facilitate the design and analysis of these experiments .

After completing the experiments according to the DoE matrix, response surface methodology can be applied to identify optimal conditions and understand how different factors interact to affect protein function.

What approaches can be used to investigate the role of UPF0767 protein C1orf212 homolog A in DNA repair pathways?

Given the potential involvement of UPF0767 protein C1orf212 homolog proteins in DNA repair pathways, several experimental approaches can be employed to investigate this function specifically:

• DNA binding assays: Electrophoretic mobility shift assays (EMSA) or fluorescence anisotropy can determine if the protein binds directly to DNA and whether it shows preference for specific structures (e.g., double-strand breaks, replication forks).

• In vitro DNA repair assays: Using damaged plasmid DNA substrates to assess whether the addition of purified UPF0767 protein C1orf212 homolog A affects repair efficiency in cell extracts.

• Immunodepletion and complementation: Depleting the endogenous protein from Xenopus egg extracts using antibodies and then complementing with recombinant wild-type or mutant proteins to assess effects on DNA repair activities.

• Co-localization studies: In Xenopus cell lines, examining whether UPF0767 protein C1orf212 homolog A co-localizes with DNA damage markers (γ-H2AX) or known repair factors (BRCA1, FANCD2) after induction of DNA damage.

• Protein complex analysis: Mass spectrometry analysis of immunoprecipitated protein complexes in untreated versus DNA-damaged conditions to identify damage-specific interactions.

• Functional genomics approaches: CRISPR-Cas9 knockout or knockdown of the gene in Xenopus cell lines followed by sensitivity testing to various DNA damaging agents.

These approaches should be designed considering the predicted structural features of the protein, particularly the conserved surface patches that may mediate interactions with DNA repair proteins .

What considerations are important when using Xenopus laevis as a model organism for studying UPF0767 protein C1orf212 homolog A?

Xenopus laevis offers several advantages as a model organism for studying UPF0767 protein C1orf212 homolog A, but specific considerations must be addressed when designing experiments:

• Tetraploidy consideration: Xenopus laevis is allotetraploid, meaning it has four copies of most genes. Researchers should verify whether both homologs (A and B) are expressed and have redundant or distinct functions .

• Developmental stage selection: Different developmental stages may exhibit varying expression levels of UPF0767 protein C1orf212 homolog A. RT-PCR or Western blot analysis across developmental stages can guide appropriate timepoint selection for experiments .

• Appropriate controls for in vivo studies: When conducting in vivo studies, proper controls are essential. For example, when assessing protein function through morpholino knockdown, include control morpholinos and validate knockdown efficiency .

• Xenopus egg extract systems: The Xenopus egg extract system provides a powerful biochemical tool for studying DNA replication and repair. These cell-free extracts can be used to assess the role of UPF0767 protein C1orf212 homolog A in various DNA transactions .

• Transgenic approaches: Consider using CRISPR-Cas9 genome editing to create knockout lines or introduce tagged versions of the protein for visualization and biochemical studies.

• Comparative analysis with human ortholog: Given the evolutionary conservation, comparative functional studies between Xenopus UPF0767 protein C1orf212 homolog A and human C1ORF212 can provide insights into conserved mechanisms .

As an established model organism with a large volume of background information in the literature, Xenopus laevis provides an excellent system for investigating both the in vitro biochemical functions and the in vivo physiological roles of UPF0767 protein C1orf212 homolog A .

What are common challenges in working with Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A and how can they be addressed?

Working with Recombinant Xenopus laevis UPF0767 protein C1orf212 homolog A presents several technical challenges that researchers should anticipate and address:

• Protein solubility issues: If the protein forms inclusion bodies during expression, consider:

  • Lowering the induction temperature to 18-20°C

  • Reducing IPTG concentration to 0.1-0.2 mM

  • Co-expressing with chaperones like GroEL/GroES

  • Using solubility-enhancing fusion tags like SUMO or MBP

• Protein stability concerns: The protein may show limited stability after purification. To address this:

  • Add glycerol (20-50%) to storage buffer

  • Include reducing agents (1-5 mM DTT or 0.5-2 mM TCEP)

  • Consider adding specific stabilizing agents based on protein characteristics

  • Always store in small aliquots to avoid freeze-thaw cycles

• Low expression yield: If expression yields are suboptimal:

  • Optimize codon usage for E. coli

  • Try different expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Implement Design of Experiments approach to systematically optimize expression conditions

• Protein function verification: As a protein with incompletely characterized function, verifying activity can be challenging:

  • Develop activity assays based on predicted functions in DNA repair

  • Use thermal shift assays to verify proper folding

  • Verify structural integrity through circular dichroism or limited proteolysis

• Interaction studies challenges: When investigating protein-protein interactions:

  • Ensure protein is not aggregated before use

  • Consider native conditions versus denaturing/renaturing approaches

  • Use multiple complementary methods to confirm interactions

By anticipating these challenges and implementing appropriate strategies, researchers can enhance their success in working with this protein.

How can site-directed mutagenesis be used to investigate functional domains in UPF0767 protein C1orf212 homolog A?

Site-directed mutagenesis provides a powerful approach for investigating functional domains in UPF0767 protein C1orf212 homolog A. Based on structural predictions and sequence conservation analysis, several strategic approaches can be implemented:

• Targeting the WCF motif: As this motif appears functionally important, with tryptophan residues forming part of the hydrophobic core and phenylalanine potentially involved in protein interactions, systematic mutation of these residues can reveal their contribution to protein structure and function :

  • Conservative substitutions (W→F, F→Y) to maintain aromatic character

  • Non-conservative substitutions (W→A, F→A) to disrupt function while maintaining structure

  • Charged substitutions (W→E, F→K) to dramatically alter local properties

• Mutating conserved surface patches: The two conserved surface patches identified as potential interaction interfaces can be systematically mutated to assess their role in protein-protein interactions :

  • Alanine scanning mutagenesis across conserved surface residues

  • Charge reversal mutations (positive to negative or vice versa) to disrupt electrostatic interactions

  • Introduction of bulky residues to sterically hinder potential interactions

• Truncation mutants: Creating systematic truncations can identify minimal functional domains:

  • N-terminal truncations to assess the role of the membrane-association region

  • C-terminal truncations to investigate regulatory domains

  • Internal deletions targeting predicted structural elements

After generating these mutants, their functional impact should be assessed through:

• Protein folding and stability assays (circular dichroism, thermal shift)

• Protein-protein interaction assays compared to wild-type

• Functional complementation in knockout/knockdown systems

• Cellular localization studies if specific localization is identified

This systematic mutagenesis approach can provide detailed insights into structure-function relationships of this poorly characterized protein.

How should researchers interpret evolutionary conservation data for UPF0767 protein C1orf212 homolog proteins?

Interpreting evolutionary conservation data for UPF0767 protein C1orf212 homolog proteins requires a nuanced approach that considers both sequence and structural conservation across species:

• Sequence conservation analysis: Sequence alignment of C1orf212 homologs across species reveals that this protein is evolutionarily well-conserved across vertebrates, with homologs also present in some invertebrates, plants, and single-celled microorganisms . This high degree of conservation suggests fundamental biological importance. When analyzing sequence conservation:

  • Focus on residues conserved across all species as these likely serve critical structural or functional roles

  • Identify lineage-specific conservation patterns that might indicate specialized functions

  • Pay special attention to the WCF motif and other highly conserved motifs as potential functional elements

• Structural conservation interpretation: The predicted structural features, particularly the HEAT repeat-like structure with conserved inner surface, provide important functional clues :

  • The inner surface of the curved structure shows significantly higher conservation than the outer surface, suggesting this region harbors interfaces for binding to interaction partners

  • The conserved WCF motif, where tryptophan residues contribute to the hydrophobic core and phenylalanine is surface-exposed, likely plays a role in protein-protein interactions

  • The presence of two distinct conserved surface patches suggests potential for different binding partners

• Comparative genomics approach: Beyond simple conservation, researchers should consider:

  • Gene synteny analysis (conserved gene neighborhoods across species)

  • Co-evolution with potential interaction partners

  • Correlation between evolutionary rates and functional importance of different protein regions

By integrating these multiple perspectives on evolutionary conservation, researchers can generate testable hypotheses about functionally important regions and guide experimental design to elucidate the role of UPF0767 protein C1orf212 homolog A.

What statistical approaches are recommended for analyzing data from functional studies of UPF0767 protein C1orf212 homolog A?

When conducting functional studies of UPF0767 protein C1orf212 homolog A, appropriate statistical approaches are essential for robust data analysis and interpretation:

• Design of Experiments (DoE) analysis: For optimization studies using DoE approaches, statistical analysis should include :

  • Analysis of variance (ANOVA) to determine significant factors

  • Response surface methodology to model the relationship between factors and responses

  • Residual analysis to validate model assumptions

  • Contour or 3D surface plots to visualize optimal conditions

• Protein-protein interaction studies:

  • For quantitative binding assays (SPR, BLI), fit binding curves to appropriate kinetic models (1:1 binding, two-state binding)

  • For co-immunoprecipitation studies, use appropriate controls and quantify band intensities using densitometry with statistical comparison between conditions

  • For proximity labeling studies, implement statistical enrichment analysis to identify significantly enriched proteins

• DNA repair functional assays:

  • Implement dose-response modeling for sensitivity to DNA damaging agents

  • Use linear mixed effects models for time-course experiments

  • Kaplan-Meier survival analysis for clonogenic survival assays

• Comparative studies between wild-type and mutant proteins:

  • Paired statistical tests when comparing the same sample under different conditions

  • Multiple comparison corrections (Bonferroni, Benjamini-Hochberg) when testing multiple mutants

  • Consider using bootstrapping or permutation tests for small sample sizes

• Integration of multiple data types:

  • Principal component analysis to identify patterns across multiple experimental outcomes

  • Hierarchical clustering to group similar experimental conditions or protein variants

  • Bayesian network analysis to infer causal relationships between variables