Recombinant Metallothionein-1
Description
Introduction to Recombinant Metallothionein-1
Metallothionein-1 (MT1) belongs to a family of low-molecular-weight proteins (6–7 kDa) characterized by their high cysteine content (30% of residues) and ability to bind heavy metals like zinc (Zn), copper (Cu), cadmium (Cd), and arsenic (As) . Recombinant MT1 is produced in heterologous systems (e.g., E. coli) to study its metal-binding properties, detoxification roles, and therapeutic potential . Unlike native MT1, recombinant forms often include fusion tags (e.g., GST or His-tag) for purification .
Structure and Functional Domains
Recombinant MT1 retains the two-domain structure of its native counterpart:
-
α-domain: Stabilizes Zn and Cd through thiolate clusters, with higher structural stability .
-
β-domain: Less stable, facilitating redox interactions and metal exchange .
The protein’s cysteine residues form metal-thiolate bonds, enabling promiscuous metal binding. Recombinant MT1 produced in E. coli has been shown to bind up to 7 Cd ions, with dissociation pHs consistent with native MT1 (e.g., Cd at pH 3.57) .
Table 1: Metal-Binding Properties of Recombinant MT1
| Metal | Binding Capacity | Dissociation pH |
|---|---|---|
| Cd | 7 ions/protein | 3.57 |
| Cu | 13 ions/protein | 1.40 |
| Zn | 6 ions/protein | 5.20 |
Source: Purified recombinant mouse MT1 expressed in E. coli .
Production Methods
Recombinant MT1 is typically synthesized via bacterial expression systems:
-
Cloning: MT1 cDNA is inserted into vectors like pGEX-4T-1 (GST fusion) or pET-28a (His-tag) .
-
Expression: Induced in E. coli with IPTG, yielding fusion proteins (e.g., GST-MT1) .
-
Purification: Affinity chromatography (e.g., glutathione-Sepharose) followed by thrombin cleavage to release MT1 .
Table 2: Recombinant MT1 Production Parameters
| Parameter | Value |
|---|---|
| Expression Host | E. coli |
| Purification Yield | >50% of total proteins |
| Molecular Weight | 6,600–7,200 Da |
| Purity | >97% |
Source: Mouse and human recombinant MT1 production protocols .
Biological Roles and Applications
Recombinant MT1 is used to study:
-
Heavy Metal Detoxification: Binds toxic metals (Cd, As) and regulates Zn/Cu homeostasis .
-
Inflammation Modulation: Inhibits proinflammatory cytokines (TNF-α, IL-1β) in autoimmune diseases like ankylosing spondylitis .
-
Cancer Therapy: Overexpression suppresses tumor growth and enhances chemosensitivity (e.g., 5-fluorouracil) .
Table 3: MT1’s Role in Disease Models
| Disease | Mechanism |
|---|---|
| Ankylosing Spondylitis | Correlates with disease activity |
| Colorectal Cancer | Enhances cannabidiol’s cytotoxicity |
| Hepatocellular Carcinoma | Inhibits proliferation and invasion |
Research Findings
-
Gene Regulation: MT1 expression is epigenetically controlled via CpG methylation near metal response elements (MREs) .
-
Structural Dynamics: Apo-MT1 adopts a folded globular structure at neutral pH, enabling templated metal binding .
-
Therapeutic Potential: Recombinant MT1 reduces oxidative stress and protects against metal-induced damage in cell models .
Product Specs
Target Background
Q&A
What methodologies are recommended for structural characterization of recombinant MT1?
Structural analysis of recombinant MT1 requires a multi-technique approach:
-
SDS-PAGE confirms purity (>95% as per commercial standards) and approximate molecular mass (36–38 kDa with GST/His tags) .
-
Amino acid sequencing validates N-terminal residues and detects tag-induced modifications .
-
Circular dichroism (CD) spectroscopy assesses metal-thiolate cluster integrity by comparing spectra at pH 8.0 and 2.0 .
Table 1: Structural Parameters of Recombinant MT1
| Parameter | Value/Observation | Source |
|---|---|---|
| Predicted Molecular Mass | 36.0 kDa | |
| Observed Mass (SDS-PAGE) | 38 kDa | |
| Isoelectric Point | 8.4 | |
| Metal Binding Capacity | 6–7 g-atoms per molecule |
How is recombinant MT1 applied in experimental models of immune regulation?
Recombinant MT1 is utilized to:
-
Modulate T-cell differentiation: Incubate dendritic cells (DCs) with 10–50 µg/mL MT1 under ZnCl₂ supplementation to induce FoxP3+ Treg cells via CD86/MHC-II downregulation .
-
Suppress Th17 cells: Use 1–5 µM MT1 in Th17-polarizing conditions (IL-6 + TGF-β) to reduce IL-17A/F expression .
-
Replicate inflammatory disease states: Intra-articular injection of 20 µg MT1 in MT1KO mice mitigates osteoarthritis progression by restoring Treg/Th17 balance .
What expression systems are optimal for producing functional recombinant MT1?
-
Prokaryotic systems (E. coli): Yield 50% of total cellular protein as GST-MT1 fusion proteins but may lack post-translational modifications .
-
Eukaryotic systems: Necessary for studies requiring phosphorylation or glycosylation but face challenges with metal ion toxicity during expression .
Critical Consideration: Tag removal via thrombin cleavage alters immunoreactivity due to exposed N-terminal epitopes .
How should researchers address contradictions in reported molecular masses of recombinant MT1?
Discrepancies between predicted (36 kDa) and observed (38 kDa) masses arise from:
-
Tag interference: His/GST tags add ~20 kDa, requiring mass spectrometry for precise validation .
-
Oxidation artifacts: Cysteine residues in MT1 are prone to cysteic acid formation during purification, increasing apparent mass .
Resolution: Compare reduced vs. non-reduced SDS-PAGE and use MALDI-TOF for intact protein analysis .
What experimental designs resolve MT1’s dual role in immune activation/suppression?
MT1 exhibits context-dependent immunomodulation:
-
Pro-tolerogenic effects: In DCs, 10 µM ZnCl₂ + 25 µg/mL MT1 upregulates ILT3 and IL-10, promoting Treg induction .
-
Anti-inflammatory effects: In Th17 cells, MT1 knockout increases IL-17A by 3.2-fold, requiring CRISPR/Cas9-edited T cells for mechanistic studies .
Strategy: Use cell-specific MT1 knockdown models paired with cytokine profiling (ELISA/MSD) to isolate signaling pathways.
What methodologies elucidate MT1’s role in osteoarthritis (OA) pathogenesis?
-
Animal models: Surgical destabilization of medial meniscus in MT1KO mice shows 40% greater cartilage degradation vs. wild-type .
-
Biomarker analysis: Measure synovial IL-1β, IL-6, and TNF-α via multiplex assays; MT1 deficiency elevates these cytokines by 2.5–4x .
-
Treg/Th17 quantification: Flow cytometry (FoxP3+/RORγt+ cells) reveals MT1’s role in balancing effector T-cell subsets .
Table 2: Key Immune Markers in MT1-Mediated OA
| Marker | MT1KO vs. WT (Fold Change) | Significance |
|---|---|---|
| IL-1β | +3.8 | Drives cartilage erosion |
| IL-10 | -2.1 | Reduced anti-inflammatory |
| Treg/Th17 | 0.6 (vs. 1.2 in WT) | Imbalanced immune response |
How can researchers overcome challenges in recombinant MT1 purification?
-
Fusion protein cleavage: Optimize thrombin concentration (2 U/mg protein) and incubation time (16 hr at 22°C) to prevent incomplete tag removal .
-
Metal reconstitution: Dialyze against 10 mM Tris-HCl (pH 8.0) with 0.1 mM ZnSO₄/CdCl₂ to restore metal-binding capacity .
-
Endotoxin removal: Use polymyxin B columns to achieve <1.0 EU/µg for in vivo applications .
What functional assays validate MT1’s antioxidative and metal-binding properties?
-
Metal displacement assays: Measure Cd²+/Zn²+ release at pH 2.0–5.0; MT1 retains 80% metals at pH 3.5 .
-
ROS scavenging: Use DCFH-DA fluorescence in HepG2 cells; 10 µM MT1 reduces H₂O₂-induced ROS by 65% .
-
Electrophoretic mobility shift assays (EMSAs): Confirm MT1’s interaction with NF-κB or AP-1 transcription factors in cytokine regulation .
How do isoform-specific differences impact MT1 functional studies?
-
MT1 vs. MT2: Glucocorticoids upregulate MT2A 4x more than MT1 in humans, necessitating isoform-specific qPCR primers (e.g., MT1A: F-5’-CGCCTCCAAATCGACCTC-3’) .
-
Functional redundancy: Simultaneous knockdown of MT1/MT2 in HepG2 cells reduces metal detoxification by 90%, requiring combinatorial siRNA approaches .
Table 3: Isoform-Specific MT Functions
| Isoform | Key Function | Regulatory Mechanism |
|---|---|---|
| MT1 | Treg induction, Th17 suppression | Zn²+ binding, IL-10 upregulation |
| MT2A | Heavy metal detoxification | Glucocorticoid response |
Methodological Recommendations
-
Dose optimization: For in vitro immune assays, titrate MT1 between 0.1–1.0 mg/mL to avoid cytotoxicity .
-
Metal supplementation: Add 5 µM ZnSO₄ to cell culture media to stabilize MT1’s tertiary structure .
-
Control experiments: Include tag-cleaved MT1 and empty vector lysates to distinguish tag-mediated artifacts .
