Recombinant Rat WSC domain-containing protein 1 (Wscd1)

What is WSC domain-containing protein 1 (WSCD1) and what are its key characteristics?

WSCD1 (WSC domain containing 1) is a protein characterized by the presence of WSC domains, which are carbohydrate-binding modules approximately 90-100 amino acids in length. In rats, WSCD1 has sulfotransferase activity and is encoded by the WSCD1 gene. The protein has been assigned the UniProt Primary AC: Q505J3 . WSC domains were initially identified in yeast cell wall integrity and stress response proteins, suggesting potential roles in cellular response mechanisms. The rat WSCD1 is one of several proteins containing this conserved domain and may function in protein-protein interactions or carbohydrate binding, similar to other WSC domain-containing proteins .

What is the molecular weight and structure of recombinant rat WSCD1?

While the exact molecular weight of rat WSCD1 is not explicitly stated in the available literature, recombinant proteins typically reflect the native molecular weight with potential modifications based on expression tags and post-translational modifications. Based on similar recombinant proteins, WSCD1 would likely be produced with common purification tags (His, Avi, or Fc) that may affect the final molecular weight. For detection purposes in experimental assays, ELISA kits for rat WSCD1 are calibrated to detect concentrations between 0.156 ng/ml and 10 ng/ml , suggesting standardized parameters for quantification.

What expression systems are commonly used for producing recombinant rat WSCD1?

Based on available information for similar proteins, recombinant rat WSCD1 can be produced in several expression systems including:

• Mammalian cells (particularly HEK293 cells), which provide proper folding and post-translational modifications

• E. coli systems, which offer high yields but may lack proper glycosylation

• Other eukaryotic expression systems that might be suitable for specific research needs

The choice of expression system should be guided by the intended application. For structural studies requiring high purity, bacterial systems might be preferred, while for functional studies requiring native-like activity, mammalian expression systems would be more appropriate.

How can I verify the identity and activity of recombinant rat WSCD1?

Verification of recombinant rat WSCD1 should employ multiple approaches:

• Western blotting: Using validated anti-WSCD1 antibodies to confirm molecular weight and immunoreactivity

• Mass spectrometry: For peptide fingerprinting to confirm protein identity

• Enzymatic activity assays: Since WSCD1 has sulfotransferase activity , specific assays measuring this function can confirm functional integrity

• ELISA: Quantitative detection using calibrated ELISA kits with a validated range of 0.156-10 ng/ml

For activity verification, it's important to note that recombinant proteins may exhibit different activity levels compared to native proteins, and standardization against known controls is recommended.

What are the functional differences between rat WSCD1 and its human ortholog?

• Sequence alignment analysis: Calculating percentage identity and identifying conserved domains and motifs

• Structural prediction: Using computational tools to predict potential functional differences based on structural variations

• Cross-species functional assays: Testing both proteins in parallel sulfotransferase activity assays

Researchers should recognize that while core functions like sulfotransferase activity may be conserved, species-specific differences in regulation, interacting partners, and tissue expression patterns are likely to exist and should be experimentally verified rather than assumed.

How does the sulfotransferase activity of WSCD1 compare to other sulfotransferases?

WSCD1 belongs to the broader family of sulfotransferases but with distinct characteristics. While detailed comparative data for rat WSCD1's enzymatic parameters is limited, researchers investigating this question should:

• Compare kinetic parameters (Km, Vmax) with other sulfotransferases like SULT1A2, SULT4A1, and HS3ST3B1B

• Determine substrate specificity profiles through competitive binding assays

• Evaluate cofactor requirements and pH optima

Based on related sulfotransferases, expected differences may include substrate preferences, catalytic efficiency, and regulatory mechanisms. A systematic biochemical characterization would provide valuable comparative data currently missing from the literature.

What are the known interacting partners of rat WSCD1 and how do they affect its function?

The complete interactome of rat WSCD1 is not fully characterized in the current literature. To investigate this important question, researchers should:

• Employ yeast two-hybrid screening to identify potential protein interactors

• Perform co-immunoprecipitation followed by mass spectrometry to validate interactions

• Use proximity labeling techniques (BioID or APEX) to identify proximal proteins in the cellular context

Based on the sulfotransferase activity of WSCD1 , likely interacting partners may include substrate proteins, regulatory factors that modulate its enzymatic activity, and scaffold proteins that facilitate its subcellular localization. Functional validation of these interactions would provide important insights into WSCD1's biological roles.

What are the optimal conditions for recombinant rat WSCD1 expression and purification?

Optimal expression and purification of recombinant rat WSCD1 would follow these methodological approaches:

Expression system selection:

• For high yield: E. coli systems with appropriate codon optimization

• For proper folding and post-translational modifications: Mammalian expression systems like HEK293

Purification strategy:

• Affinity chromatography using engineered tags (His, Avi, or Fc tags)

• Size exclusion chromatography for higher purity

• Ion exchange chromatography for removing contaminants

Buffer optimization:
Recombinant proteins similar to WSCD1 typically show optimal stability in PBS-based buffers with potential additives to enhance stability:

• 20-50 mM phosphate buffer, pH 7.2-7.4

• 150 mM NaCl

• Potential stabilizers: 5-10% glycerol, 1-2 mM DTT, or 0.1% BSA

For storage, lyophilization and storage at -20°C to -80°C is recommended, similar to other recombinant proteins .

What are the best methods for studying WSCD1 sulfotransferase activity in vitro?

For studying the sulfotransferase activity of WSCD1, researchers should consider these methodological approaches:

Standard sulfotransferase assay:

• Incubate purified recombinant WSCD1 with potential substrates and 35S-labeled PAPS (3'-phosphoadenosine 5'-phosphosulfate)

• Measure transfer of radioactive sulfate to substrate molecules

• Quantify activity through scintillation counting or autoradiography

Non-radioactive alternatives:

• HPLC-based detection of sulfated products

• Colorimetric assays measuring phosphate release

• Coupled enzyme assays that produce detectable products

Reaction conditions optimization:

• Buffer composition: Typically Tris or phosphate buffers (pH 7.0-7.5)

• Cofactor requirements: PAPS concentration optimization

• Divalent cation requirements: Test effects of Mg2+, Mn2+, Ca2+

• Temperature and time course determination

These approaches would provide quantitative data on WSCD1's enzymatic parameters and substrate preferences.

How can I design experiments to study the tissue-specific expression of rat WSCD1?

To investigate tissue-specific expression patterns of rat WSCD1, a multi-faceted approach is recommended:

mRNA expression analysis:

• qRT-PCR across a panel of rat tissues using validated WSCD1-specific primers

• RNA-seq analysis for comprehensive transcriptomic profiling

• In situ hybridization for spatial localization within tissue sections

Protein expression analysis:

• Western blotting of tissue lysates using validated anti-WSCD1 antibodies

• Immunohistochemistry on tissue sections

• ELISA-based quantification (using kits with detection range of 0.156-10 ng/ml)

Sample preparation:
For tissue homogenates, optimization of extraction buffers is crucial:

• RIPA buffer for general protein extraction

• Specialized buffers containing protease inhibitors when analyzing enzymatic activity

• Sample dilution to ensure measurements fall within the linear range of detection (0.156-10 ng/ml for ELISA)

Comparing expression data across developmental stages and in response to physiological stimuli would provide comprehensive understanding of WSCD1's expression regulation.

How can I troubleshoot low expression yields of recombinant rat WSCD1?

When encountering low expression yields of recombinant rat WSCD1, consider this systematic troubleshooting approach:

Expression system issues:

• Codon optimization for the expression host

• Testing alternative promoters (T7, CMV, EF1α)

• Optimizing induction conditions (temperature, inducer concentration, timing)

Protein stability considerations:

• Co-expression with chaperones to improve folding

• Addition of protease inhibitors during extraction

• Testing fusion partners that enhance solubility (MBP, SUMO, GST)

Purification optimization:

• Screening different affinity tags (His, Avi, Fc)

• Testing various elution conditions

• Adding stabilizing agents to buffers (glycerol, reducing agents)

What controls should I include when measuring WSCD1 sulfotransferase activity?

Proper experimental controls are essential for reliable measurement of WSCD1 sulfotransferase activity:

Positive controls:

• Well-characterized sulfotransferases with known activity (e.g., SULT1A2, SULT4A1)

• Commercially available active sulfotransferase preparations

Negative controls:

• Heat-inactivated WSCD1 (95°C for 10 minutes)

• Reaction mixture lacking PAPS cofactor

• Enzymatically inactive WSCD1 mutant (active site mutation)

Validation controls:

• Dose-response curve with varying enzyme concentrations

• Time course measurements to ensure linear reaction range

• Known inhibitors of sulfotransferases to confirm specificity

Including these controls allows proper normalization of data and identification of potential confounding factors in the experimental system.

How do I interpret contradictory results between WSCD1 ELISA and Western blot analyses?

When facing discrepancies between ELISA and Western blot results for WSCD1, consider this analytical framework:

Methodological differences:

• ELISA measures soluble protein in native conformation

• Western blot detects denatured protein separated by size

Potential causes of discrepancies:

• Antibody recognition issues:

  • Different epitopes recognized in each method

  • Epitope masking in native versus denatured states

• Sample preparation effects:

  • Denaturation might affect antibody recognition

  • Buffer components interfering with one assay but not the other

• Assay sensitivity differences:

  • ELISA detection range (0.156-10 ng/ml) versus Western blot sensitivity

  • Quantitative (ELISA) versus semi-quantitative (Western) nature of the assays

Resolution approaches:

• Use multiple antibodies targeting different epitopes

• Validate results with a third method (e.g., mass spectrometry)

• Standardize sample preparation across methods

• Ensure proper standard curves and controls in ELISA

When reporting contradictory results, transparent documentation of methodological details is essential for proper scientific interpretation.

What are the current knowledge gaps and future research directions for rat WSCD1?

Current knowledge about rat WSCD1 remains limited, with several important areas requiring further investigation:

Structural characterization:

• Complete 3D structure determination by X-ray crystallography or cryo-EM

• Structure-function relationships of the WSC domain in WSCD1

Functional aspects:

• Comprehensive substrate specificity profiling

• Regulatory mechanisms controlling WSCD1 activity

• Physiological roles in different tissues

Cellular biology:

• Subcellular localization and trafficking

• Integration in signaling networks

• Roles in normal physiology versus disease states

Future research should address these knowledge gaps through interdisciplinary approaches combining molecular biology, biochemistry, structural biology, and systems biology. Development of specific tools such as conditional knockout models and specific inhibitors would significantly advance understanding of WSCD1 biology and potential therapeutic applications.

How can I effectively compare my WSCD1 research findings with existing literature?

For effective comparison of WSCD1 research findings with existing literature, follow these methodological guidelines:

• Standardized reporting:

  • Use consistent nomenclature (UniProt accession Q505J3)

  • Report complete methodological details enabling reproducibility

  • Present quantitative data with appropriate statistical analysis

• Comparative analysis framework:

  • Create tables comparing your key parameters with published values

  • Highlight methodological differences that might explain discrepancies

  • Consider species differences when comparing with human or mouse orthologs

• Meta-analysis approach:

  • Systematically evaluate all available data on WSCD1

  • Weight evidence based on methodological rigor

  • Identify consensus findings versus conflicting results