Recombinant Human Uncharacterized protein C10orf105 (C10orf105)

What expression systems are most suitable for producing recombinant C10orf105?

The selection of an appropriate expression system is crucial for successfully producing recombinant C10orf105. While Escherichia coli remains the most commonly used host for recombinant protein production, several challenges must be addressed when expressing human proteins in bacterial systems, including inclusion body formation, metabolic burden, and inefficient protein translocation . For optimal C10orf105 expression, researchers should consider testing multiple systems, including:

• Bacterial systems (E. coli BL21): Suitable for rapid screening and initial expression trials

• Eukaryotic systems: May provide better folding and post-translational modifications

• Specialized E. coli strains: The ackA mutant strain has demonstrated higher recombinant protein production compared to wild-type BL21 due to reduced acetate excretion into the extracellular medium

The choice should be guided by the specific research objectives and protein characteristics, with preliminary expression trials in different systems to determine optimal conditions.

How should promoter strength and plasmid copy number be balanced for C10orf105 expression?

Finding the optimal balance between promoter strength and plasmid copy number is essential for maximizing soluble C10orf105 expression. Studies have demonstrated that metabolic burden associated with transcription and translation of foreign genes can significantly decrease recombinant protein expression . Based on experimental evidence:

Experimental evidence indicates that the metabolic burden associated with strong promoters and high copy number plasmids can paradoxically decrease protein yield . For an uncharacterized protein like C10orf105, starting with a moderate promoter (Plac trc or P tac) combined with a medium-copy number plasmid may provide the most balanced approach.

What strategies can minimize inclusion body formation during C10orf105 expression?

Inclusion body formation represents a major challenge when expressing human proteins in bacterial systems. For C10orf105, several approaches can be implemented to enhance soluble protein production:

• Temperature optimization: Lowering expression temperature to 16-25°C to slow protein synthesis rate

• Induction optimization: Using lower inducer concentrations and gradual induction protocols

• Strain selection: Utilizing the ackA mutant strain which shows higher recombinant protein production with lower acetate accumulation

• Carbon source selection: Using glycerol instead of glucose to reduce acetate production

• Co-expression with chaperones: Including molecular chaperones to assist proper protein folding

• Fusion tags: Adding solubility-enhancing tags such as MBP, SUMO, or thioredoxin

Each strategy should be systematically tested, as the optimal conditions for C10orf105 expression will depend on its specific structural characteristics and folding requirements.

What are the key principles for designing reproducible C10orf105 studies?

Designing reproducible studies for C10orf105 requires adherence to fundamental principles of experimental design. A well-designed experiment should be precise, unbiased, amenable to statistical analysis, and reproducible . Key considerations include:

• Clear objectives: Define specific research questions about C10orf105 function or characteristics

• Focus and simplicity: Design experiments with controlled variables that address one aspect at a time

• Statistical power: Ensure sufficient replicates to detect expected changes in expression or function

• Randomized comparisons: Minimize bias through proper randomization of samples and conditions

• Appropriate controls: Include positive and negative controls relevant to C10orf105 research

As noted by statistician Ronald A. Fisher: "To consult the statistician after an experiment is finished is often merely to ask him to conduct a post mortem examination. He can perhaps say what the experiment died of" . This underscores the importance of consulting statistical expertise during the planning phase rather than after data collection.

What controls are essential when characterizing the function of C10orf105?

Proper controls are critical for functional characterization of an uncharacterized protein like C10orf105. Essential controls include:

• Expression controls:

  • Empty vector expressed under identical conditions

  • Well-characterized protein expressed using the same system

  • Inactive mutant version of C10orf105 (if functional domains can be predicted)

• Experimental controls:

  • Technical replicates to assess method variability

  • Biological replicates to assess biological variability

  • Time-matched controls for time-course studies

  • Vehicle controls for any treatments or stimuli

• Validation controls:

  • Alternative detection methods to confirm key findings

  • Dose-response relationships to verify specificity

  • Complementary approaches to validate interactions

How should researchers approach measuring C10orf105 expression levels?

Accurate quantification of C10orf105 expression requires systematic methodology and appropriate controls. Based on experimental approaches for recombinant proteins:

• Standardized quantification methods:

  • Fluorescent reporter systems (similar to YFP reporter in the provided study)

  • Western blotting with calibrated standards

  • qPCR for transcript-level analysis

• Data normalization strategies:

  • Normalize to total protein content

  • Use multiple housekeeping genes as references

  • Include internal standards for absolute quantification

• Replication requirements:

  • Minimum of three biological replicates

  • Multiple technical measurements per biological replicate

  • Independent experimental repetitions to confirm key findings

• Statistical analysis:

  • Appropriate statistical tests based on data distribution

  • Reporting of all data points, not just means

  • Clear indication of variability (standard deviation or standard error)

This systematic approach ensures that measurements of C10orf105 expression are reliable, reproducible, and amenable to statistical analysis .

How can researchers resolve contradictory data in C10orf105 functional studies?

When facing contradictory results in C10orf105 functional studies, researchers should implement a systematic troubleshooting approach:

• Verify protein integrity:

  • Confirm protein folding through biophysical methods

  • Assess post-translational modifications

  • Validate activity using complementary functional assays

• Examine experimental variables:

  • Systematically vary buffer conditions, pH, and salt concentrations

  • Test different protein concentrations to identify concentration-dependent effects

  • Evaluate the impact of different tags and their potential interference

• Cross-validate with orthogonal methods:

  • Employ multiple detection technologies for key measurements

  • Use both in vitro and cellular systems to confirm findings

  • Implement genetic approaches to complement biochemical data

• Design controlled comparison studies:

  • Directly compare contradictory conditions in the same experiment

  • Include appropriate positive and negative controls

  • Perform power analysis to ensure sufficient replication

What bioinformatic approaches can guide experimental characterization of C10orf105?

Bioinformatic analysis can provide valuable insights to guide experimental characterization of uncharacterized proteins like C10orf105:

• Sequence-based predictions:

  • Homology detection to identify distant relatives

  • Secondary structure prediction to inform construct design

  • Functional motif identification to predict activity

  • Post-translational modification site prediction

• Structural modeling:

  • Template-based modeling if homologs exist

  • Ab initio modeling using current deep learning approaches

  • Molecular dynamics simulations to predict flexibility

• Interaction predictions:

  • Protein-protein interaction network analysis

  • Ligand binding site prediction

  • Molecular docking to potential binding partners

• Expression optimization tools:

  • Codon optimization for selected expression system

  • mRNA secondary structure prediction at translation initiation sites

  • Signal peptide and transmembrane domain prediction

These computational approaches should be used iteratively with experimental validation, following the principle that experiments should be designed with clear objectives and focus .

How can researchers assess the effect of cellular conditions on C10orf105 function?

Understanding how cellular conditions affect C10orf105 function requires systematic investigation of various parameters:

• Experimental design considerations:

  • Factorial design to efficiently test multiple variables

  • Time-course studies to capture dynamic responses

  • Dose-response relationships to determine sensitivity

• Environmental variables to test:

  • pH variations to identify optimal functional range

  • Ionic strength effects on protein stability and interactions

  • Temperature dependence of activity and folding

  • Redox conditions that might affect disulfide bond formation

• Cellular context variables:

  • Cell type-specific effects through multi-cell line testing

  • Cell cycle dependence through synchronized cultures

  • Stress response effects through controlled cellular stressors

  • Co-factor or binding partner availability

• Analytical approaches:

  • Single-cell analysis to capture heterogeneity

  • Spatial localization studies to determine subcellular distribution

  • Temporal regulation studies to assess dynamic responses

This comprehensive approach allows researchers to define the cellular conditions that influence C10orf105 function, providing context for interpreting experimental results and understanding the protein's physiological role.

What strategies can optimize purification of recombinant C10orf105?

Purifying recombinant C10orf105 requires optimization of multiple parameters to maintain protein stability and activity:

• Expression optimization:

  • Balance promoter strength and plasmid copy number

  • Select appropriate E. coli strain (consider ackA mutant for higher yields)

  • Optimize induction conditions to maximize soluble fraction

• Extraction considerations:

  • Test multiple lysis buffers with varying pH and salt concentrations

  • Evaluate detergent requirements if membrane association is suspected

  • Include protease inhibitors to prevent degradation

• Purification strategy:

  • Select affinity tags based on protein characteristics

  • Design multi-step purification protocol with orthogonal methods

  • Implement on-column refolding if inclusion bodies are unavoidable

• Stability enhancement:

  • Identify stabilizing additives through thermal shift assays

  • Optimize storage conditions to maintain activity

  • Consider engineering stabilizing mutations if native protein is unstable

The purification strategy should be systematically optimized and documented to ensure reproducibility, following the experimental design principles outlined in search result .

How should researchers approach structural characterization of C10orf105?

Structural characterization of an uncharacterized protein like C10orf105 requires a multi-technique approach:

• Primary structure confirmation:

  • Mass spectrometry to verify sequence and modifications

  • N-terminal sequencing to confirm proper processing

  • Peptide mapping to ensure complete coverage

• Secondary structure analysis:

  • Circular dichroism to determine α-helix and β-sheet content

  • FTIR spectroscopy as complementary secondary structure method

  • Thermal denaturation to assess structural stability

• Tertiary structure determination:

  • X-ray crystallography if protein can be crystallized

  • NMR spectroscopy for smaller domains or full protein

  • Cryo-electron microscopy for larger complexes

  • Small-angle X-ray scattering for solution structure

• Dynamic properties:

  • Hydrogen-deuterium exchange to map flexible regions

  • Limited proteolysis to identify domain boundaries

  • Molecular dynamics simulations to predict motion

This comprehensive structural characterization provides essential information about C10orf105's potential function and mechanism, guiding subsequent functional studies.

What approaches can identify potential interaction partners of C10orf105?

Identifying interaction partners is crucial for understanding the function of uncharacterized proteins like C10orf105:

• In vitro approaches:

  • Pull-down assays using purified C10orf105 as bait

  • Surface plasmon resonance to measure binding kinetics

  • Protein microarrays to screen for interactions systematically

• Cellular approaches:

  • Co-immunoprecipitation from cells expressing C10orf105

  • Proximity labeling methods (BioID, APEX) to identify proximal proteins

  • Fluorescence resonance energy transfer to detect direct interactions

  • Yeast two-hybrid screening for binary interactions

• Systems biology approaches:

  • Affinity purification-mass spectrometry to identify complexes

  • Correlation analysis of expression data across tissues/conditions

  • Network analysis to predict functional associations

• Validation strategies:

  • Reciprocal pull-downs to confirm interactions

  • Domain mapping to identify interaction surfaces

  • Functional assays to assess biological significance

This multi-layered approach provides complementary evidence for interaction partners, helping to place C10orf105 in its biological context and suggest potential functions.

How can CRISPR/Cas9 technology be applied to study C10orf105 function?

CRISPR/Cas9 technology offers powerful approaches for investigating C10orf105 function:

• Gene knockout strategies:

  • Complete knockout to assess loss-of-function phenotype

  • Conditional knockout to study tissue-specific effects

  • Double knockouts with potential interacting partners

• Gene modification approaches:

  • Knock-in of reporter tags for localization studies

  • Introduction of point mutations to assess functional residues

  • Domain deletions to map functional regions

• Transcriptional regulation:

  • CRISPRi for reversible gene silencing

  • CRISPRa for upregulation of endogenous C10orf105

  • Epigenetic modifiers to study chromatin-level regulation

• High-throughput screening:

  • CRISPR screens to identify genetic interactions

  • Synthetic lethality screens to find dependencies

  • Pooled screens with selective pressures relevant to hypothesized function

When designing CRISPR experiments for C10orf105, researchers should adhere to the experimental design principles outlined previously, ensuring clear objectives, sufficient power, and appropriate controls .

What transcriptomic approaches are valuable for understanding C10orf105 function?

Transcriptomic analyses provide insights into C10orf105's function by examining its expression patterns and effects on gene expression:

• Expression profiling:

  • RNA-seq across tissues and developmental stages

  • Single-cell RNA-seq to identify cell type-specific expression

  • Analysis of transcriptional regulation under various conditions

• Differential expression analysis:

  • Compare gene expression changes after C10orf105 overexpression

  • Analyze transcriptome after C10orf105 knockdown/knockout

  • Identify correlating genes across large datasets

• Alternative splicing analysis:

  • Examine if C10orf105 undergoes alternative splicing

  • Investigate if C10orf105 affects splicing of other transcripts

  • Map important isoforms and their tissue distribution

• Network analysis:

  • Construct co-expression networks

  • Identify transcription factors regulating C10orf105

  • Map pathway enrichment after C10orf105 perturbation

These approaches should be designed following proper experimental design principles, with appropriate replication and controls to ensure reproducible results .

How can researchers design experiments to determine C10orf105 subcellular localization?

Determining the subcellular localization of C10orf105 requires a systematic experimental approach:

• Fluorescent protein fusion strategies:

  • C-terminal and N-terminal tagging to identify optimal configuration

  • Split fluorescent protein complementation to verify interactions

  • Photoactivatable fluorescent proteins for dynamic studies

• Immunofluorescence approaches:

  • Generate and validate specific antibodies

  • Co-staining with organelle markers

  • Super-resolution microscopy for detailed localization

• Biochemical fractionation:

  • Systematic subcellular fractionation

  • Western blotting of fractions

  • Mass spectrometry analysis of organelle proteomes

• Live-cell imaging:

  • Real-time tracking during cellular processes

  • Response to cellular stimuli or stress

  • Correlation with functional assays