Recombinant Pig Metallothionein-1D (MT1D)

What is the structure and function of Pig Metallothionein-1D?

Pig Metallothionein-1D (MT1D) is a low-molecular-weight, cysteine-rich metal-binding protein consisting of 61 amino acids. The protein sequence is: MDPNCSCSTG GSCSCATSCT CKACRCTSCK KSCCSCCPAG CAKCAQGCIC KGASDKCSCC A . MT1D belongs to the metallothionein family of proteins characterized by high cysteine content, which enables their primary function of binding heavy metal ions.

Functionally, MT1D demonstrates high metal-binding activity with divalent metal ions, particularly copper (Cu2+), zinc (Zn2+), and cadmium (Cd2+) . This metal-binding capability is central to its biological roles in:

• Metal ion homeostasis, particularly zinc metabolism

• Protection against heavy metal toxicity

• Response to oxidative stress

• Potential roles in DNA replication and repair processes

The protein's structure is optimized for metal coordination through the thiol groups of its numerous cysteine residues, creating metal-thiolate clusters that stabilize the tertiary structure.

How does recombinant pig MT1D differ from native MT1D in experimental systems?

Recombinant pig MT1D refers to artificially expressed protein produced in expression systems such as Escherichia coli, yeast, or baculovirus. Key differences include:

When using recombinant MT1D, researchers should consider how these differences might affect experimental interpretations, particularly for metal-binding studies or when extrapolating to in vivo functions.

What expression systems are optimal for producing recombinant pig MT1D?

Several expression systems have been successfully employed for MT1D production, each with distinct advantages:

• Bacterial Expression (E. coli): The recombinant pig MT1A/MT1D has been successfully expressed in soluble form using Escherichia coli RosettaTM (DE3) plysS cells . This system offers high yield and cost-effectiveness but may lack post-translational modifications.

• Baculovirus Expression: Commercial MT1D products are available produced via baculovirus expression systems . This approach may provide better folding and post-translational modifications than bacterial systems.

• Yeast Expression: While specific to MT1E (a related metallothionein), yeast expression systems have been described as "the most economical and efficient eukaryotic system for secretion and intracellular expression," integrating advantages of mammalian cell expression systems .

The choice depends on research requirements - bacterial systems offer higher yields and simplicity, while eukaryotic systems may provide better protein folding and modifications that more closely resemble the native protein.

What is the recommended protocol for cloning and expressing recombinant pig MT1D?

Based on published methodologies, a standard protocol for cloning and expressing recombinant pig MT1D includes:

• Gene Synthesis and Cloning:

  • Synthesize the full-length cDNA for pig MT1D based on the gene sequence (available in GenBank)

  • Clone into an intermediate vector (e.g., pMD18-T) for sequence confirmation

  • Subclone into an expression vector (e.g., pET-32a(+)) containing a His-tag for purification

• Transformation and Expression:

  • Transform the recombinant plasmid into E. coli RosettaTM (DE3) plysS cells

  • Culture in appropriate media (typically LB with antibiotics)

  • Induce protein expression (typically with IPTG)

  • Optimize expression conditions to obtain soluble protein

• Verification:

  • Confirm expression via SDS-PAGE

  • Verify identity via Western blot using appropriate antibodies (e.g., anti-His-tag monoclonal antibody)

This approach has been demonstrated to produce soluble, functionally active recombinant pig MT1D with metal-binding capabilities.

What purification strategies yield the highest purity and activity for recombinant pig MT1D?

Effective purification of recombinant pig MT1D typically involves multi-step chromatography:

• Affinity Chromatography: For His-tagged MT1D, HisTrapTM affinity chromatography provides efficient initial purification . This method exploits the high affinity of the His-tag for nickel or cobalt ions immobilized on the column.

• Ion Exchange Chromatography: DEAE SepharoseTM Fast Flow column can be used as a secondary purification step to separate proteins based on charge differences .

• Endotoxin Removal: For applications sensitive to bacterial endotoxins, treatment with an endotoxin removing gel is recommended .

• Quality Control:

  • SDS-PAGE analysis to assess purity (>85% purity is typically achievable)

  • Western blot confirmation with specific antibodies

  • Metal-binding activity assays to confirm functionality

These combined approaches can yield highly pure, biologically active recombinant MT1D suitable for downstream applications.

What are the optimal storage conditions for maintaining recombinant pig MT1D stability?

Proper storage is critical for maintaining the structural integrity and functional properties of recombinant pig MT1D:

Additional considerations:

• Avoid repeated freezing and thawing cycles, which can compromise protein integrity

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• The shelf life of liquid form is approximately 6 months at -20°C/-80°C

• The shelf life of lyophilized form extends to 12 months at -20°C/-80°C

Following these guidelines helps preserve the metal-binding capabilities and structural properties of the protein.

How can researchers quantitatively analyze the metal-binding properties of recombinant pig MT1D?

Metal-binding characteristics are fundamental to MT1D function and can be analyzed through multiple approaches:

• Spectroscopic Methods:

  • UV-visible spectroscopy to detect characteristic absorption bands from metal-thiolate bonds

  • Circular dichroism (CD) to monitor structural changes upon metal binding

  • Fluorescence spectroscopy using metal-sensitive fluorophores

• Elemental Analysis:

  • Inductively coupled plasma mass spectrometry (ICP-MS) for precise quantification of bound metals

  • Atomic absorption spectroscopy (AAS) to determine metal content

• Binding Kinetics and Thermodynamics:

  • Isothermal titration calorimetry (ITC) to determine binding constants and stoichiometry

  • Equilibrium dialysis to measure binding affinities

• Functional Assays:

  • Metal displacement assays using competitive chelators

  • Metallochromic indicators to monitor metal transfer

Research has demonstrated that recombinant pig MT1D exhibits high binding activity for Cu2+, Zn2+, and Cd2+ , making these quantitative analyses essential for characterizing the protein's biochemical properties.

What is the relationship between pig MT1D and zinc metabolism in cellular processes?

Metallothioneins, including pig MT1D, play crucial roles in zinc homeostasis with significant implications for cellular function:

• Zinc Storage and Regulation: MT1D serves in the regulation of intracellular zinc metabolism, acting as a reservoir for zinc ions that can be released when needed for zinc-dependent processes .

• DNA Replication and Repair: Among the zinc-requiring systems are several enzymes involved in DNA replication and repair. During periods of active DNA synthesis, increased demand for zinc could be met by elevated MT synthesis .

• Protein Synthesis Impact: Research has shown that zinc deficiency results in lower rates of hepatic protein synthesis. This decreased rate is due to reduced synthesis of proteins retained in the liver, highlighting the critical role of zinc availability (regulated by metallothioneins) in protein production .

• Expression Regulation: MT expression is altered when dietary zinc supply is restricted or supplemented. Studies with human subjects have shown that erythrocyte MT protein concentrations are reduced or elevated, after a lag period of approximately 6 days, when dietary zinc intake is correspondingly adjusted .

These findings suggest that pig MT1D likely functions as a critical regulator of zinc availability for essential cellular processes, making it a valuable target for studies of zinc metabolism and zinc-dependent cellular functions.

How can recombinant pig MT1D be utilized as a model system for studying metallothionein function across species?

Recombinant pig MT1D offers several advantages as a model system for comparative metallothionein research:

• Evolutionary Conservation: Metallothioneins are highly conserved across species, making pig MT1D a valuable comparative model for human metallothionein studies. The basic cysteine-rich structure and metal-binding properties are preserved.

• Agricultural and Biomedical Applications: As pigs are important both in agriculture and as biomedical models, pig MT1D studies can bridge these domains. Transgenic pig models have grown dramatically in recent years as they are anatomically, physiologically, and phylogenetically more similar to humans than rodents .

• Comparative Functional Studies: Researchers can perform side-by-side comparisons of pig MT1D with human metallothioneins to identify species-specific differences and similarities in:

  • Metal binding preferences and affinities

  • Regulation of expression in response to metals and other stressors

  • Protein stability and turnover

  • Interactions with cellular components

• Translational Applications: Insights from pig MT1D can potentially inform:

  • Heavy metal detoxification strategies

  • Zinc supplementation approaches in agriculture

  • Understanding metallothionein roles in oxidative stress protection

This comparative approach leverages the advantages of the pig model while providing insights applicable to human metallothionein function and therapeutic applications.

How does the metallothionein family in pigs compare to other species, and what are the implications for using pig MT1D in comparative studies?

The metallothionein family shows both conservation and diversity across species, with several implications for comparative research:

These comparative aspects make pig MT1D valuable for translational research, particularly for:

• Biomedical models of metal-related disorders

• Agricultural applications in livestock health and nutrition

• Environmental monitoring of heavy metal contamination

• Evolutionary studies of metal adaptation mechanisms

What potential applications exist for recombinant pig MT1D in vaccine development and immunology research?

While direct evidence for pig MT1D in vaccine applications is not presented in the search results, related research on metallothionein-3 (MT3) suggests promising directions:

• Built-in Adjuvant Properties: Human MT3, when fused to protein antigens, functions as a novel built-in adjuvant that can help protein antigens induce rapid, effective, and durable antigen-specific immune responses . This raises the possibility that pig MT1D might exhibit similar properties.

• Enhanced Antibody Response: MT3 fusion increased antigen-specific antibody responses by 100-1000 fold within seven days after primary immunization . Compared to commercial adjuvants, it stimulated earlier (4 days after primary injection) and stronger (10-100 fold) antibody responses with lower antigen doses .

• Mechanism of Action: MT3 appears to directly activate dendritic cells, promote germinal center formation, and improve the speed of immunoglobulin class switching . These mechanisms could potentially apply to other metallothioneins.

• Family Conservation: Research found that other metallothionein family members (human MT1 or murine MT3) also had potential adjuvant effects, although lower than human MT3 . This suggests pig MT1D might have similar immunomodulatory capabilities.

For researchers exploring this application, fusion protein design (similar to MT3-Omp19 or MT3-Hc described in the results) could be adapted using pig MT1D as the adjuvant component, potentially opening new avenues in veterinary vaccine development.

What are the methodological challenges in differentiating protein-specific versus metal-specific effects when studying recombinant pig MT1D?

This fundamental challenge in metallothionein research requires careful experimental design:

• Preparation of Defined Metal-Loaded States:

  • Apo-MT1D (metal-free): Treat purified protein with chelators (EDTA, DTPA) followed by extensive dialysis

  • Single-metal MT1D: Reconstitute apo-protein with specific stoichiometric amounts of a single metal

  • Mixed-metal MT1D: Prepare with physiologically relevant metal mixtures

  • Verification of metal content using ICP-MS or atomic absorption spectroscopy is essential

• Comparative Functional Assays:

  • Test apo-MT1D versus metal-loaded forms in the same experimental system

  • Include appropriate controls with free metal ions at equivalent concentrations

  • Measure dose-response relationships for both protein concentration and metal concentration

• Structural Modification Approaches:

  • Site-directed mutagenesis of metal-binding cysteine residues

  • Creation of truncated variants with altered metal-binding capacity

  • Chimeric proteins combining domains from different metallothionein isoforms

• Experimental Design Considerations:

  • Time-course studies to distinguish immediate (likely metal-mediated) versus delayed (likely protein-mediated) effects

  • Use of metal chelators with different specificities to selectively remove certain metals

  • Parallel studies with non-metallothionein metal-binding proteins as controls

These approaches help dissect the relative contributions of the protein scaffold versus the bound metals, providing mechanistic insights into MT1D function.

What are common challenges in expressing soluble recombinant pig MT1D and how can they be overcome?

Expressing metallothioneins in soluble, correctly folded forms presents several challenges:

• Inclusion Body Formation:

  • Challenge: High cysteine content can lead to misfolding and aggregation

  • Solution: Lower induction temperature (16-25°C), reduce IPTG concentration, use specialized E. coli strains like Rosetta(DE3) plysS as demonstrated for pig MT1A/MT1D

• Metal Incorporation:

  • Challenge: Ensuring proper metal loading during expression

  • Solution: Supplement growth media with appropriate metals (typically zinc); alternatively, express as apo-protein and load with metals post-purification

• Proteolytic Degradation:

  • Challenge: Small size makes MT1D susceptible to proteolysis

  • Solution: Use protease-deficient strains, include protease inhibitors during purification, minimize processing time

• Oxidation of Cysteine Residues:

  • Challenge: Thiol groups easily oxidize, disrupting metal binding

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) in all buffers, work under nitrogen atmosphere when possible

• Purification Interference:

  • Challenge: Metal-binding properties can interfere with His-tag affinity purification

  • Solution: Optimize imidazole concentration in binding and elution buffers; consider alternative tags or native purification strategies

Successful expression of soluble recombinant pig MT1D has been achieved in E. coli RosettaTM (DE3) plysS cells and verified by Western blotting using anti-His-tag monoclonal antibody , demonstrating that these challenges can be overcome with appropriate techniques.

How can researchers verify the structural integrity and functional activity of purified recombinant pig MT1D?

Multiple complementary approaches should be used to ensure purified recombinant pig MT1D maintains its structural integrity and functional activity:

• Structural Verification:

  • SDS-PAGE and Western blotting to confirm molecular weight and immunoreactivity

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Size exclusion chromatography to verify monomeric state

  • Mass spectrometry to confirm exact mass and potential modifications

• Metal-Binding Capacity:

  • Direct measurement of metal content using atomic absorption spectroscopy or ICP-MS

  • Spectroscopic analysis of metal-thiolate coordination (UV-visible spectroscopy)

  • Metal titration studies to determine binding stoichiometry

  • Competition studies with metallochromic indicators

• Functional Assays:

  • Metal transfer capability to metal-dependent enzymes

  • Protection against metal toxicity in cellular systems

  • Antioxidant activity measurement (if relevant to research context)

• Stability Assessment:

  • Thermal shift assays to determine protein stability

  • Long-term storage tests under different conditions

  • Resistance to oxidative conditions

Published research has confirmed that recombinant pig MT1A/MT1D shows high metal-binding activity with Cu2+, Zn2+, and Cd2+ , providing a benchmark for functional verification.

What considerations are important when designing experiments to study pig MT1D in the context of oxidative stress or heavy metal detoxification?

Designing robust experiments for these applications requires attention to several critical factors:

• Experimental System Selection:

  • Cell culture options: Porcine cell lines for species relevance vs. standardized lines (HEK293, HepG2)

  • Ex vivo tissue systems from pig organs

  • In vivo models: considerations for transgenic or knockout approaches

• Oxidative Stress Studies:

  • Oxidant selection: H₂O₂, paraquat, tert-butyl hydroperoxide represent different oxidative mechanisms

  • Dose-response and time-course optimization

  • Measurement of multiple oxidative stress markers (ROS levels, lipid peroxidation, protein carbonylation)

  • Controls: comparison with other antioxidant proteins or small molecules

• Heavy Metal Detoxification:

  • Metal selection: physiologically relevant (Zn2+, Cu2+) versus toxic metals (Cd2+, Hg2+, As3+)

  • Exposure protocols: acute high-dose vs. chronic low-dose

  • Subcellular localization tracking of metals and MT1D

  • Molecular endpoints: gene expression changes, apoptosis markers, cellular metal content

• Metallothionein Manipulation Strategies:

  • Recombinant protein: extracellular addition vs. transfection for intracellular expression

  • Gene overexpression: constitutive vs. inducible systems

  • Gene silencing: siRNA, shRNA, or CRISPR approaches

  • Appropriate controls: empty vectors, scrambled RNA, etc.

• Validation Across Models:

  • Correlation between in vitro findings and ex vivo/in vivo results

  • Comparison with published data on other metallothionein isoforms

  • Translation between porcine studies and potential human applications

These experimental design considerations ensure that findings on pig MT1D's role in oxidative stress response or heavy metal detoxification are robust, reproducible, and physiologically relevant.