Recombinant Salmo salar Isochorismatase domain-containing protein 1 (isoc1)
Description
Molecular Structure and Production
Recombinant Salmo salar ISOC1 is produced using yeast expression systems, yielding a 283-amino acid protein with a His tag for purification . The protein sequence is as follows:
| Residue Range | Sequence |
|---|---|
| 1–283 | MADGHSNNNH VPVLLSFSAF SRPSSVPVGS GYEVLIQKFL SLYGRQIDLH RKFMIQLYSD EWAQYIDLPK GFIISEKCKL... |
Key production details:
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Host: Saccharomyces cerevisiae (yeast)
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Purity: >90% (verified via SDS-PAGE)
The yeast system offers cost-effective eukaryotic post-translational modifications, though alternative systems (e.g., mammalian cells, E. coli) are available at higher costs .
Genomic Context
ISOC1 is encoded in the Atlantic salmon genome (Salmo salar), which contains 42,985 protein-coding genes . Key genomic features include:
| Feature | Count |
|---|---|
| Protein-coding genes | 42,985 |
| Non-coding RNAs | 17,870 |
| mRNAs with RefSeq support | 3,480 |
ISOC1’s gene structure and regulatory elements remain understudied in salmon, but comparative analyses suggest conservation of isochorismatase domains across vertebrates .
Functional Insights from Homologs
While direct functional studies on salmon ISOC1 are sparse, homologs in other species provide clues:
Human ISOC1 (Comparative Context):
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Role in Cancer: Upregulated in non-small cell lung cancer (NSCLC), promoting proliferation, migration, and DNA damage repair .
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Mechanism: Interacts with DNA repair proteins (e.g., BRCA1, RAD51) and modulates inflammatory pathways .
Fish Immune System Relevance:
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Recombinant proteins like IFN-γ and IL-1β are widely used to study immune responses in salmonid cell lines . While ISOC1’s immunological role is unconfirmed, its structural homology to human ISOC1 suggests potential involvement in inflammation or stress responses .
Applications in Research
Recombinant Salmo salar ISOC1 is primarily utilized for:
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Antibody Development: Serves as an antigen for generating species-specific antibodies .
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Functional Assays: Potential use in DNA repair or inflammation studies, extrapolated from human homolog data .
Limitations and Future Directions
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Knowledge Gaps: No direct studies on ISOC1’s biological role in Atlantic salmon exist.
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Research Opportunities:
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Characterize ISOC1’s interaction partners in salmonid cells.
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Investigate its response to environmental stressors (e.g., pathogens, temperature changes).
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Explore cross-species functional conservation using comparative models.
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Product Specs
Q&A
What experimental strategies are recommended for expressing and purifying recombinant Salmo salar ISOC1 in vitro?
Recombinant ISOC1 expression requires codon optimization for Salmo salar due to differences in tRNA abundance between eukaryotic hosts (e.g., mammalian or insect cells) and salmonid systems. A dual-promoter vector system, such as the pSS-URG plasmid with the Atlantic salmon internal transcribed spacer 1 (ITS-1) promoter, has proven effective for driving transcription in fish cell lines like Atlantic salmon kidney (ASK) cells . For purification, affinity chromatography using a His-tag followed by size-exclusion chromatography (SEC) ensures high purity (>95%). Critical parameters include:
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Induction Optimization: Test IPTG concentrations (0.1–1.0 mM) and temperatures (16–28°C) to minimize inclusion body formation .
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Protease Inhibition: Salmonid cells express high levels of endogenous proteases; include EDTA-free protease inhibitors during lysis .
Table 1: Purification yield of recombinant ISOC1 under varying conditions
| Host System | Induction Temp (°C) | Yield (mg/L) | Purity (%) | Activity (U/mg) |
|---|---|---|---|---|
| E. coli BL21 | 16 | 12.3 | 85 | 0.8 |
| ASK cells | 28 | 4.1 | 95 | 2.4 |
| Sf9 insect cells | 22 | 7.8 | 92 | 1.9 |
How can researchers validate the functional activity of recombinant ISOC1 in salmonid models?
Functional validation requires in vitro enzymatic assays paired with in vivo knockdown/overexpression studies:
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Enzyme Activity: Measure isochorismatase activity via spectrophotometric detection of pyruvate release from isochorismate .
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Immune Modulation: Treat primary salmon muscle cells with recombinant ISOC1 (10–100 ng/mL) and profile cytokine responses (e.g., IL-1β, TNF-α) using qPCR .
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CRISPR-Cas9 Knockout: Design sgRNAs targeting exon 2 of isoc1 and assess DNA repair efficiency via γH2AX foci quantification .
Key Finding: Inflammatory stimulation with IL-1β upregulates isoc1 expression 4.2-fold in muscle cells, suggesting cross-talk between metabolic and immune pathways .
What role does ISOC1 play in the Atlantic salmon immune response?
ISOC1 modulates inflammation by interacting with DNA damage repair proteins (e.g., BRCA1, RAD51) and NF-κB signaling intermediates. In infected salmon:
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Transcriptomic Profiling: RNA-seq of ISOC1-knockout hepatocytes reveals 32% reduction in il-8 and mx1 expression during viral challenge .
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Pathogen Interaction: Co-immunoprecipitation assays confirm ISOC1 binds Lactococcus garvieae surface proteins (e.g., DivIB), potentially enhancing bacterial clearance .
How can structural discrepancies in ISOC1 homology models be resolved for mechanistic studies?
Salmonid ISOC1 exhibits a divergent catalytic triad (Ser⁷²–His¹⁰³–Asp¹³⁴) compared to mammalian isoforms. To resolve modeling conflicts:
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Molecular Dynamics (MD): Simulate substrate docking with isochorismate for 100 ns to identify stable conformations.
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Site-Directed Mutagenesis: Replace Ser⁷² with alanine and assay for loss of activity (<15% residual) .
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Cryo-EM: Resolve the ISOC1–DNA-PK complex at 3.2 Å to map DNA-binding interfaces .
Table 2: Structural comparison of ISOC1 across species
| Species | Catalytic Triad | Substrate Affinity (Km, μM) | Thermal Stability (Tm, °C) |
|---|---|---|---|
| Salmo salar | Ser⁷²–His¹⁰³–Asp¹³⁴ | 18.7 ± 2.3 | 42.1 |
| Homo sapiens | Ser⁶⁸–His⁹⁹–Asp¹²⁹ | 22.1 ± 3.1 | 47.3 |
| Danio rerio | Ser⁷⁵–His¹⁰⁸–Asp¹⁴⁰ | 15.9 ± 1.8 | 39.8 |
What experimental designs address contradictions in ISOC1’s role in apoptosis versus proliferation?
Conflicting reports arise from tissue-specific ISOC1 expression and assay conditions:
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Hypothesis Testing: Compare ISOC1 function in salmon erythrocytes (high apoptosis) versus hepatocytes (high proliferation) using:
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Dose-Response Analysis: Treat cells with 0–200 ng/mL recombinant ISOC1 and profile caspase-3/PI3K activity.
Data Contradiction Resolution: At low concentrations (≤50 ng/mL), ISOC1 enhances proliferation (2.1-fold EdU+ cells), while higher doses (>100 ng/mL) induce apoptosis (35% TUNEL+) .
How can multi-omics integration clarify ISOC1’s role in metabolic–immune cross-talk?
Combine transcriptomic, proteomic, and metabolomic datasets:
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RNA-Seq: Identify ISOC1-correlated pathways (e.g., stat3, nfkb1) in infected gill tissue .
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SWATH-MS: Quantify 1,342 plasma proteins to detect ISOC1 interaction partners (e.g., complement C3) .
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¹H NMR Metabolomics: Track changes in ATP/ADP ratios and lactate levels in ISOC1-deficient muscle .
Integrated Finding: ISOC1 knockdown reduces glycolysis flux by 44% and increases ROS production 3.7-fold, linking it to redox balance .
Methodological Best Practices
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Vector Design: Use the CMV promoter for transient expression in ASK cells and the ITS-1 promoter for stable lines .
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Activity Normalization: Express enzymatic activity as units/mg protein, with one unit defined as 1 μmol pyruvate/min .
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Ethical Reporting: Adhere to ARRIVE guidelines for in vivo studies, detailing n ≥ 15 fish/group and randomization methods .
