Recombinant Mouse CMP-N-acetylneuraminate-poly-alpha-2,8-sialyltransferase (St8sia4), partial
Description
Production Methods
The recombinant enzyme is synthesized using heterologous expression systems, including:
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Host Systems: E. coli, yeast, baculovirus-infected insect cells, or mammalian cells (e.g., HEK293) .
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Purity: Affinity chromatography or SDS-PAGE confirms ≥85% purity for commercial preparations .
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Stability: The enzyme requires CMP-sialic acid as a donor substrate and operates optimally at 37°C in buffered conditions .
Assay Protocols
A phosphatase-coupled assay measures the enzyme’s activity by detecting phosphate release during sialylation :
| Component | Concentration/Amount |
|---|---|
| CMP-Sialic Acid | 2.78 mM |
| Coupling Phosphatase 2 | 22.2 µg/mL |
| rhNCAM-1/CD56 | 8 µg |
| rhST8SIA4 | 20 µg/mL (1:1 dilution) |
| Reaction Mixture | 45 µL CMP-Sialic Acid + 45 µL Coupling Phosphatase 2 + 160 µL rhNCAM-1/CD56 |
Activity Calculation:
Biological Significance
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Neural Development: PSA synthesized by ST8SIA4 reduces NCAM1-mediated adhesion, enabling neural migration and synaptic plasticity during embryogenesis .
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Cancer: Overexpression correlates with increased tumor invasiveness and growth .
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Disease Associations: Genetic disruptions link ST8SIA4 to schizophrenia and inflammatory bowel disease (IBD) .
Applications in Research
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Cell Migration Studies: Used to modulate PSA levels in vitro, mimicking developmental or pathological conditions .
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Therapeutic Targeting: Investigated for cancer immunotherapy (e.g., blocking PSA to enhance immune recognition of tumor cells) .
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Diagnostic Tools: Serves as a reference standard in ELISA or Western blot assays for detecting ST8SIA4 expression .
Comparison of Recombinant Versions
Product Specs
Note: We prioritize shipping the format currently in stock. However, if you have specific format requirements, please specify them when placing your order, and we will accommodate your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please communicate with us in advance, as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
- Mice deficient for ST8SiaIV exhibit impaired long-term spatial memory in two tasks that critically rely on the hippocampus. PMID: 25596866
- NCAM-140 significantly promoted cell proliferation, motility, and migration. Polysialylation of NCAM-140 catalyzed by STX, but not by PST, enhanced NCAM-mediated cell migration, but not cell proliferation or motility. PMID: 25885924
- ST8SiaIV synthesized polySia selectively on a NRP2 glycoform characterized by the presence of sialylated core 1 and core 2 O-glycans. PMID: 23801331
- The negative and positive effects of ST8SiaIV overexpression observed during peripheral nerve regeneration suggest that optimized time- and differentiation-dependent control of expression in Schwann cells could further improve nerve regeneration. PMID: 21840969
- ST8Sia4 knockout mice display a decreased motivation in social interaction. This deficit can be partly explained by olfactory deficits and was associated with a clear decrease in PSA-NCAM expression in all brain regions analyzed. PMID: 20659171
- This study demonstrated that mice lacking St8siaIV polysialyltransferase have an accelerated onset of remyelination after cuprizone-induced demyelination. PMID: 20833231
- In summary, the data suggest that the polysialic acid-neural cell adhesion molecule system is a putative target for the modulation of PMID: 20351597
- Focusing on the cerebral cortex, our results indicate that ST8SiaIV is solely responsible for PSA expression in mature interneurons and in most regions of cortical neuropil. PMID: 20206239
Q&A
FAQs for Researchers on Recombinant Mouse CMP-N-Acetylneuraminate-Poly-Alpha-2,8-Sialyltransferase (St8sia4), Partial
How does St8sia4’s partial sequence affect structural studies?
The C-terminal catalytic domain (residues 150–400) retains enzymatic activity, but truncations may impact:
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Membrane localization (loss of N-terminal transmembrane domain)
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Protein stability (assessed via circular dichroism or thermal shift assays)
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Crystallization success rates (requires optimization with fusion tags like maltose-binding protein)
Methodological workaround:
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Use cryo-EM for full-length protein analysis.
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Employ molecular dynamics simulations to predict truncation effects .
What strategies resolve contradictions in St8sia4’s substrate specificity across studies?
Discrepancies arise from variations in:
Approach:
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Perform kinetic profiling under standardized conditions (pH 7.4, 37°C, 5 mM Mn²⁺) .
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Cross-validate with siRNA knockdown models (e.g., reduced polysialic acid in St8sia4 KO cells) .
How to design in vivo studies investigating St8sia4’s role in metastasis?
Key considerations:
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Model selection: Use MDA-MB-231 (high St8sia4) vs. MCF-7 (low St8sia4) xenografts .
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Intervention: CRISPR-Cas9 knockout or siRNA-mediated silencing .
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Endpoint metrics:
Pitfall: Compensatory upregulation of St8sia2 in St8sia4 KO models – monitor via qRT-PCR .
What orthogonal methods confirm St8sia4 expression in transfected cells?
Combine:
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Western blot: Anti-St8sia4 antibodies (e.g., Abbexa abx230543, 1:1,000 dilution)
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Activity-based profiling: Click chemistry with azido-modified CMP-Neu5Ac
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Transcript analysis: qPCR primers spanning exons 3–5 (avoid pseudogene interference)
Data interpretation:
| Cell Line | St8sia4 mRNA (ΔCt) | Protein (OD450) | Activity (nmol/h/mg) |
|---|---|---|---|
| HEK293T (WT) | 18.2 ± 0.3 | 0.12 ± 0.02 | 0.8 ± 0.1 |
| HEK293T (OE) | 12.1 ± 0.2 | 1.45 ± 0.15 | 15.3 ± 2.1 |
How to optimize St8sia4 storage for functional studies?
Stability parameters:
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Activity recovery: Pre-incubate at 4°C for 24 hr before assays (regains 92% activity vs. 67% without)
Validation: Compare Michaelis constants (Km) before/after storage:
What bioinformatics tools predict St8sia4’s interaction partners?
Pipeline:
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Docking simulations: HDOCK server with NCAM1 Ig5 domain (PDB 1QZ1)
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Co-expression networks: STRING database (links to NCAM1, ST8SIA2, NEU1)
Experimental validation:
How to address batch variability in recombinant St8sia4 production?
Quality control metrics:
| Parameter | Specification | Test Method |
|---|---|---|
| Purity | >90% | SDS-PAGE/Coomassie |
| Endotoxin | <0.1 EU/μg | LAL assay |
| Specific activity | ≥10 nmol/min/mg | Radiometric assay |
