KRT8 Human, GST
Description
Introduction to KRT8 Human, GST
KRT8 (Keratin 8) Human, GST-tagged is a recombinant protein derived from the human KRT8 gene, fused with a glutathione S-transferase (GST) tag for enhanced solubility and purification efficiency. KRT8 is a type II intermediate filament protein that pairs with keratin 18 (KRT18) to form cytoskeletal networks in epithelial cells, maintaining structural integrity and facilitating cellular signaling . The GST-tagged variant is widely used in biochemical and oncological research due to its stability and utility in protein interaction studies .
Key Attributes:
Functional Domains:
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Rod Domain: Central α-helical region critical for filament assembly.
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Head/Tail Domains: Mediate interactions with cellular organelles and signaling molecules .
Comparative Analysis of Recombinant KRT8 Variants:
| Parameter | KRT8-GST (PRO-298) | KRT8-His (PRO-1567) |
|---|---|---|
| Tag | GST | His-tag |
| Purity | >90% (SDS-PAGE) | >90% (SDS-PAGE) |
| Molecular Mass | 78 kDa | 56 kDa |
| Buffer | Tris-HCl, L-glutathione | Tris-HCl, urea, glycerol |
3.2. Research Applications
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Cancer Biomarker Studies: KRT8 overexpression correlates with tumor aggressiveness in gastric cancer (GC) and clear cell renal cell carcinoma (ccRCC) .
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Metastasis Mechanisms: KRT8 promotes epithelial-mesenchymal transition (EMT) via integrinβ1-FAK and TGFβ/Smad pathways .
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Structural Biology: Used to analyze intermediate filament dynamics and cellular stress responses .
4.2. Mechanistic Insights
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In GC: High KRT8 expression increases p-smad2/3 levels, activating TGFβ-dependent metastasis .
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In ccRCC: KRT8 knockdown reduces cell migration and invasion by suppressing IL-11 autocrine signaling .
Stability and Handling Guidelines
Product Specs
Q&A
What experimental strategies validate KRT8-GST interactions with binding partners in epithelial cells?
Co-immunoprecipitation (co-IP) coupled with GST pull-down assays remains the gold standard for identifying KRT8 interactomes. Researchers should express GST-tagged KRT8 in epithelial cell lines (e.g., HepG2 or MDCK), followed by affinity purification using glutathione-sepharose beads . Western blotting for known partners like KRT18 confirms assay validity . Controls must include empty GST vectors to exclude non-specific binding. For quantitative analysis, surface plasmon resonance (SPR) provides kinetic data (e.g., K<sub>D</sub> values) for KRT8-ligand interactions, as demonstrated in oncofetal chondroitin sulfate studies .
How do researchers address batch-to-batch variability in recombinant KRT8-GST protein production?
Quality control requires three orthogonal assays:
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SDS-PAGE: Verify a single band at ~78 kDa (KRT8 + GST tag) .
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Circular Dichroism: Confirm secondary structure integrity (α-helical content >40%) .
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Functional Assay: Test binding to KRT18 using ELISA or microscale thermophoresis .
Batch variability <15% in these metrics ensures reproducibility. Institutions like the Seattle SNP Project employ similar validation pipelines for protein reagents .
What mechanisms explain contradictory findings about KRT8 mutations in cryptogenic cirrhosis versus cancer progression?
KRT8 variants exhibit context-dependent effects:
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Loss-of-function mutations (e.g., R341W) disrupt filament networks, predisposing hepatocytes to stress-induced apoptosis (familial cirrhosis) .
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Overexpression in carcinomas stabilizes stress granules, promoting chemoresistance .
Resolution requires disease-specific models: CRISPR-Cas9 knock-in for cirrhosis studies vs. xenografts for cancer biology.
How can researchers reconcile discrepancies in KRT8’s utility as a pan-cancer biomarker?
While KRT8-derived oncofetal chondroitin sulfate (ofCS) shows promise for liquid biopsies , its diagnostic performance varies:
| Cancer Type | Sensitivity (Stage I/II) | AUC | Cohort Size |
|---|---|---|---|
| Colorectal | 62% | 0.81 | 2081 cases |
| Pancreatic | 38% | 0.68 | Same cohort |
Methodological refinements improve consistency:
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Pre-analytical factors: Standardize plasma collection tubes (EDTA vs. heparin alters ofCS recovery) .
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Assay multiplexing: Combine ofCS-CD44 with ctDNA methylation markers (AUC improves from 0.81 to 0.89 in CRC) .
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Machine learning: Integrate KRT8 fragmentomics with proteomic classifiers .
What validation frameworks are recommended for KRT8-GST in mechanotransduction studies?
A tiered approach ensures robustness:
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In vitro: Atomic force microscopy (AFM) measures stiffness changes in KRT8-knockdown epithelia .
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In vivo: Intravital imaging of KRT8-GFP zebrafish models quantifies force propagation during morphogenesis .
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Clinical correlation: Match biophysical data with histopathology (e.g., KRT8<sup>+</sup> ductal carcinomas show 3.2-fold higher compressive stress vs. normal ).
How do post-translational modifications (PTMs) of KRT8-GST influence experimental outcomes?
Common PTMs include:
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Phosphorylation (S73/S431): Regulates filament solubility; use Phos-tag gels to assess .
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O-GlcNAcylation: Impairs stress granule assembly; monitor via click chemistry .
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Proteolytic cleavage: Caspase-6 generates pro-apoptotic KRT8 fragments; block with Z-VEID-FMK inhibitor .
PTM-mimetic mutants (e.g., S73E) are essential for dissecting signaling roles.
Product Science Overview
Cytokeratin 8 is a member of the type II keratin family, which is clustered on the long arm of chromosome 12. These keratins are essential components of the cytoskeleton in epithelial cells, providing structural support and contributing to various cellular functions, including cell signaling, apoptosis, and stress response. CK8 is particularly significant in non-squamous epithelia and is present in the majority of adenocarcinomas and ductal carcinomas .
The recombinant form of Cytokeratin 8, tagged with Glutathione S-transferase (GST), is produced using various expression systems, including E. coli and wheat germ. The GST tag facilitates the purification process through affinity chromatography, ensuring high purity and yield of the recombinant protein.
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Expression in E. coli: The gene encoding CK8 is cloned into an expression vector containing the GST tag. The recombinant plasmid is then transformed into E. coli cells, which are cultured under optimal conditions to induce protein expression. The cells are harvested, lysed, and the recombinant CK8-GST fusion protein is purified using glutathione affinity chromatography .
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Expression in Wheat Germ: An alternative method involves the in vitro wheat germ expression system. This system preserves the correct conformational folding of the protein, which is crucial for its biological function. The recombinant CK8-GST protein is purified similarly using glutathione affinity chromatography .
Cytokeratin 8 undergoes various post-translational modifications, including phosphorylation, glycosylation, and ubiquitination, which regulate its function and interactions with other proteins. These modifications can influence the stability, solubility, and assembly of CK8 filaments.
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Phosphorylation: CK8 is phosphorylated at multiple serine residues, which modulates its interaction with CK18 and other binding partners. Phosphorylation plays a critical role in the dynamic reorganization of the cytoskeleton during cell division, migration, and apoptosis.
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Glycosylation: Glycosylation of CK8 can affect its solubility and stability. This modification is essential for the proper folding and function of the protein within the cellular environment.
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Ubiquitination: Ubiquitination targets CK8 for degradation via the proteasome pathway. This process is crucial for maintaining the balance of cytokeratin levels within the cell and preventing the accumulation of damaged or misfolded proteins.
Recombinant CK8 tagged with GST is widely used in research to study the structure and function of intermediate filaments, as well as their role in various diseases, including cancer. The GST tag allows for easy purification and detection of the protein in various assays, such as Western blotting, ELISA, and immunoprecipitation .
