Recombinant Rabbit Metallothionein-2D

What is Recombinant Rabbit Metallothionein-2D and how does it differ structurally from other metallothionein variants?

Recombinant Rabbit Metallothionein-2D (MT-2D) belongs to the metallothionein family, which are cysteine-rich, low molecular weight proteins characterized by their ability to bind various metal ions. Metallothioneins are intrinsically disordered proteins with diverse structures, biological functionalities, and metal ion specificity .

While sharing core characteristics with the MT-2 family, MT-2D likely has specific amino acid variations that affect its metal-binding properties and structural dynamics. Based on studies of rabbit metallothioneins, MT-2D would be part of the MT-2 subfamily alongside MT-2A, which has been extensively studied in partially metalated forms . The structural differences from MT-1 would include variations in the amino acid sequence that could modify metal-binding affinities and biological functions.

Rabbit MT variants demonstrate specific expression patterns, with MT-I being predominantly expressed in rabbit blastocysts following zinc induction . The molecular evolution of rabbit metallothioneins includes processed retrogenes like the MT-2 pseudogene, which shows evidence of complex rearrangements involving recombination and deletion events .

What fundamental metal-binding properties characterize Rabbit Metallothionein-2D?

Based on studies of rabbit metallothioneins, MT-2D would likely demonstrate the following metal-binding properties:

• Capacity to bind multiple metal ions including Cd²⁺, Zn²⁺, and Ag⁺ through thiol coordination with cysteine residues

• Formation of metal-thiolate clusters with distinctive spectroscopic properties, including high absorbance at 250 nm and low absorbance at 280 nm when binding cadmium

• Metal-dependent conformational changes reflected in circular dichroism spectral features, particularly an absorption band at 260 nm corresponding to the characteristic metal-thiolate cluster at pH 8.0

• Metal-specific binding strengths characterized by different half-dissociation pH values. For comparison, recombinant mouse MT-I showed half-dissociation pH values of 3.57 for Cd, 1.40 for Cu, and 5.20 for Zn, similar to those from native rabbit MT-I

• Sequential metal binding that progressively stabilizes the protein structure, as observed in studies of partially metalated MT-2A

How is Recombinant Rabbit Metallothionein-2D gene expression regulated in rabbit tissues?

Based on studies of metallothionein expression in rabbit tissues:

Metallothioneins display both constitutive and inducible expression patterns. In rabbit blastocysts, MT is constitutively expressed at low levels, suggesting basal roles in metal homeostasis . Upon exposure to metal ions, expression levels increase significantly through transcriptional regulation.

Zinc exposure demonstrates dose-dependent induction of metallothionein synthesis. In rabbit blastocysts, zinc treatment at 400 μM increased the relative rate of MT synthesis ten-fold . Both MT-I and MT-II showed markedly increased synthesis following zinc treatment, with MT-I being the predominant isometallothionein .

Cadmium exposure produces a different pattern of response compared to zinc. In rabbit blastocysts, exposure to 10 μM Cd²⁺ resulted in a large induction of MT mRNA but only a modest increase in the relative rate of MT synthesis, suggesting post-transcriptional regulation mechanisms . Additionally, cadmium induced an acute stress response, as indicated by dramatic induction of heat-shock protein (HSP-70) gene expression .

The timing of MT expression is developmentally regulated. In rabbits, zinc induction of MT synthesis is detectable on day 4 of gestation, just after the morula-to-blastocyst transition .

What expression systems are most suitable for producing Recombinant Rabbit Metallothionein-2D?

Based on successful protocols for recombinant metallothionein production, researchers should consider the following expression systems:

1. Bacterial expression systems (E. coli):

• Mouse MT-I cDNA has been successfully amplified by PCR, inserted into vector pGEX-4T-1, and expressed in E. coli

• Advantages include high yield, simplicity, and cost-effectiveness

• Using a GST-fusion approach improves solubility and facilitates purification

• Requires optimization of culture conditions, including metal supplementation to stabilize the recombinant MT

2. Mammalian expression systems:

• CHO cells have been used successfully for producing recombinant rabbit proteins

• Advantages include proper folding and post-translational modifications

• More resource-intensive than bacterial systems

Recommended expression protocol for E. coli system:

• Clone the MT-2D cDNA using PCR amplification from rabbit tissue (prostate tissue has been used successfully for other rabbit proteins )

• Insert into an expression vector with an affinity tag (GST tag has proven effective )

• Transform into an appropriate E. coli strain

• Optimize expression conditions (temperature, IPTG concentration, induction time)

• Include reducing agents and appropriate metal ions in the culture medium to promote proper folding

• Consider protease cleavage sites to remove fusion tags after purification

The choice of expression system should be guided by the research requirements and whether post-translational modifications are critical for the intended applications.

What purification strategies yield highest purity Recombinant Rabbit Metallothionein-2D?

Based on protocols for purifying metallothioneins and recombinant proteins, a multi-step purification strategy is recommended:

Step 1: Initial capture

• Affinity chromatography using the fusion tag (GST-tag approach has been successful for recombinant mouse MT-I )

• Thrombin cleavage can be used to remove the GST tag if needed

Step 2: Intermediate purification

• Ion-exchange chromatography is effective for separating metallothioneins from contaminants due to their distinctive charge properties

• For separating different metallothionein isoforms, ion-exchange chromatography has been demonstrated to effectively distinguish MT-I and MT-II

Step 3: Polishing

• Size-exclusion chromatography can provide final purification based on the low molecular weight of metallothioneins

Critical considerations:

• Maintain reducing conditions throughout purification to prevent oxidation of cysteine residues

• Include appropriate metal ions in buffers if metal-loaded metallothionein is desired

• For apo-MT preparation, use chelating agents and acidic conditions carefully

• Flash-freezing in liquid nitrogen, lyophilization, and storage at -20°C has been effective for preserving metallothionein samples

The purification protocol should be validated by confirming the identity and purity of the product using methods such as mass spectrometry, N-terminal sequencing, and spectroscopic characterization .

How can researchers characterize the metal-binding properties of purified Recombinant Rabbit Metallothionein-2D?

Several complementary analytical methods are recommended for comprehensive characterization:

Spectroscopic methods:

• UV-visible spectroscopy: Metal-bound metallothioneins show characteristic absorption profiles. Cadmium-thiolate complexes exhibit high absorbance at 250 nm and low absorbance at 280 nm at pH 8.0, which disappear at pH 2.0 when metals are released

• Circular dichroism (CD) spectroscopy: Provides information on metal-thiolate cluster formation with characteristic bands. For cadmium-thiolate clusters, an absorption band at 260 nm is observed at pH 8.0

Binding strength characterization:
3. Half-dissociation pH determination: Measure the pH at which half of the bound metal dissociates from the protein. This is considered "a criterion to distinguish MT from non-MT metal binding proteins" . For comparison, recombinant mouse MT-I showed half-dissociation pH values of 3.57 for Cd, 1.40 for Cu, and 5.20 for Zn

Structural and stability analysis:
4. Ion mobility mass spectrometry: Used to study conformational preferences of partially metalated metallothioneins

• Collision-induced unfolding: Reveals differences in gas-phase stabilities of metallothioneins with different metal loading

Stoichiometric analysis:
6. Determination of metal:protein and thiol:protein ratios: Confirms proper metal loading and cysteine availability

Recommended experimental design:
Characterize the protein in multiple metalation states, including:

• Apo-form (metal-free)

• Partially metalated forms with different stoichiometries

• Fully metalated form

• Mixed-metal forms to assess competitive binding

This multi-method approach provides comprehensive information about metal-binding properties, structural changes upon metal binding, and relative affinities for different metals.

What analytical methods can confirm the identity and integrity of Recombinant Rabbit Metallothionein-2D?

To ensure the quality and identity of purified recombinant MT-2D, researchers should employ multiple analytical techniques:

Protein identification methods:

• Mass spectrometry: MALDI-TOF mass spectrometry can confirm the amino acid sequence of the recombinant protein

• N-terminal sequencing: Determining the sequence of 10 amino acids at the N-terminus can confirm the identity of the purified protein

• Western blotting: If antibodies are available, Western blotting can confirm the identity of the recombinant protein

Structural integrity assessment:
4. Spectroscopic analysis: Comparing the ultraviolet absorption and CD spectra of the recombinant protein with those of native metallothionein can confirm they have the same metal-thiolate structure

• Functional testing: Measuring metal-binding properties and comparing with expected values for metallothioneins

Purity assessment:
6. SDS-PAGE: To evaluate protein purity and molecular weight

• Determination of metal:protein and thiol:protein ratios: The ratios should match those of wild-type MT to confirm proper folding and metal loading

Data quality criteria:

• Protein sequence should match the expected MT-2D sequence

• Spectroscopic properties should be consistent with metallothionein metal-thiolate structures

• Half-dissociation pH values should be in the typical range for metallothioneins

• Metal:protein and thiol:protein ratios should conform to expected values

If additional amino acids are present (e.g., from fusion tags), researchers should evaluate their impact on protein structure and function, as they may affect properties like immunoreactivity .

How does partial metalation affect the conformational dynamics of Recombinant Rabbit Metallothionein-2D?

Partial metalation significantly impacts the structural conformations and stability of metallothioneins, with important implications for MT-2D:

Conformational heterogeneity:

• Partially metalated metallothioneins adopt various conformations depending on the number and type of bound metal ions

• For rabbit MT-2A, "the sequential addition of each Cd²⁺ and Zn²⁺ ion results in the incremental stabilization of unique unfolding intermediates"

• This suggests a progressive structural organization as metal ions bind

Metal-specific stability effects:

• Despite similar ion mobility profiles, differently metalated forms of rabbit MT-2A (Cd₄-MT, Zn₄-MT, Ag₄-MT, and Ag₆-MT) show dramatic differences in gas-phase stabilities

• This indicates that the identity of the bound metal, not just the number of bound metals, significantly influences protein stability

Methodological approaches for studying conformational dynamics:

• Ion mobility mass spectrometry: Reveals conformational distributions of differently metalated species

• Collision-induced unfolding: Probes stability differences between metalation states

• Time-resolved spectroscopy: Can capture dynamic conformational changes during metal binding

These findings highlight the complex relationship between metal binding, protein conformation, and stability in metallothioneins. For MT-2D research, characterizing these properties is essential for understanding the protein's biological functions and designing experiments that account for its conformational heterogeneity.

What are the advantages and limitations of using Recombinant Rabbit Metallothionein-2D compared to native rabbit metallothionein?

Advantages of recombinant MT-2D:

• Controlled production: Recombinant expression allows for consistent, scalable production without reliance on animal tissues

• Defined metal content: Metal loading can be precisely controlled during purification

• Protein modifications: Fusion tags can facilitate purification and detection

• Mutational analysis: Recombinant systems enable structure-function studies through site-directed mutagenesis

• Availability: Can be produced in larger quantities than native protein

Limitations and considerations:

• Additional amino acid sequences: Recombinant proteins often contain additional amino acids from fusion tags or cloning artifacts. In recombinant mouse MT-I, six additional amino acids at the N-terminus affected immunoreactivity

• Potential structural differences: While recombinant mouse MT-I showed the same metal-thiolate structure and function as native rabbit MT-I , subtle differences might exist that affect certain properties

• Expression system impacts: The choice of expression system can affect protein folding and post-translational modifications

• Validation requirements: Extensive characterization is needed to confirm that the recombinant protein behaves like the native protein

Experimental evidence of similarity to native protein:

• Recombinant mouse MT-I showed the same metal-binding structure and function as native rabbit MT-I, with similar half-dissociation pH values for metals

• Ultraviolet and CD spectra of recombinant mouse MT-I were very similar to those of native rabbit MT-I, suggesting the same metal-thiolate structure

• The ratios of metal:protein and thiol:protein were the same as those of wild-type MT

These findings suggest that properly produced recombinant MT-2D would likely retain core structural and functional properties of native MT-2D, despite potential differences in specific properties such as immunoreactivity.

How does Recombinant Rabbit Metallothionein-2D respond to various environmental stressors?

Based on studies of metallothioneins in stress responses, MT-2D would likely demonstrate the following responses to environmental stressors:

Metal-induced responses:

• Zinc exposure: In rabbit blastocysts, zinc treatment induced metallothionein synthesis in a dose-dependent manner, with a ten-fold increase at 400 μM-Zn²⁺

• Cadmium exposure: Cadmium (10 μM-Cd²⁺) induced a large increase in MT mRNA but only a modest increase in MT synthesis rate in rabbit blastocysts

• Metal-specific effects: Different metals induce different expression patterns and have different toxicity profiles

Stress pathway interactions:

• Heat shock response: Cadmium exposure in rabbit blastocysts induced not only metallothionein expression but also "a dramatic induction of heat-shock protein (HSP-70) gene expression" , indicating cross-talk between metal stress and other stress response pathways

• Developmental timing: Zinc induction of MT synthesis in rabbit embryos was detected on day 4 of gestation, just after the morula-to-blastocyst transition , suggesting developmental regulation of stress responses

Cellular protection mechanisms:

• Toxicity mitigation: Metallothioneins likely protect against metal toxicity by sequestering toxic metals. Cadmium was "found to be toxic to the day-6 blastocyst" , and metallothionein induction represents a protective response

• Post-transcriptional regulation: The discrepancy between MT mRNA induction and protein synthesis rates following cadmium exposure suggests complex post-transcriptional regulation mechanisms

Experimental approaches for studying stress responses:

• Exposure of cells expressing recombinant MT-2D to various stressors (metals, oxidative agents, heat)

• Analysis of gene expression changes, protein stability, and cellular protection

• Comparative studies with MT-deficient cells to assess protective functions

• Location studies to determine subcellular redistribution during stress

What role does Recombinant Rabbit Metallothionein-2D play in metal detoxification pathways?

Based on the general functions of metallothioneins described in the research, MT-2D likely contributes to metal detoxification through several mechanisms:

Constitutive protective functions:

• Metallothionein is "constitutively expressed at low levels in the blastocyst" , suggesting a basal role in metal homeostasis that MT-2D would likely share

• This constitutive expression provides a first line of defense against sudden metal exposure

Inducible protection mechanisms:

• Expression levels increase in response to metal exposure, enhancing cellular capacity for metal sequestration

• The significant induction by zinc (ten-fold increase in synthesis rate at 400 μM-Zn²⁺ ) demonstrates a dynamic response to changing metal concentrations

Metal-specific binding properties:

• Different metals bind to metallothioneins with different affinities, as indicated by their distinct half-dissociation pH values

• For example, based on half-dissociation pH values from recombinant mouse MT-I (Cd: 3.57, Cu: 1.40, Zn: 5.20 ), cadmium and copper are bound more tightly than zinc, suggesting preferential sequestration of toxic metals

Isoform-specific roles:

• Different metallothionein variants may have specialized roles in metal detoxification

• In rabbit blastocysts, "both MT-I and MT-II were markedly increased following zinc treatment, with MT-I being the predominant isometallothionein"

Experimental approaches to study detoxification function:

• Metal competition assays to determine preferential binding of different metals

• Cell viability studies with different metal challenges in the presence/absence of MT-2D

• Subcellular localization studies to track metal sequestration

• Analysis of metal transfer to other metalloproteins

These functions position MT-2D as an important component of cellular metal detoxification systems, with particular relevance in tissues exposed to fluctuating metal concentrations or environmental toxicants.

What are common obstacles in expressing and purifying Recombinant Rabbit Metallothionein-2D?

Researchers should anticipate and prepare for these common challenges:

Expression challenges:

• Protein stability issues: As a metal-binding protein with high cysteine content, MT-2D may be unstable without appropriate metal ions or reducing conditions

  • Solution: Include metal ions in culture media and reducing agents such as TCEP (0.6-1 mM has been used for apo-MT )

• Toxicity to expression host: High levels of metallothionein expression or added metals might stress the host cells

  • Solution: Optimize induction conditions and metal concentrations; consider using metal-resistant host strains

• Low expression levels: Small proteins sometimes express poorly in heterologous systems

  • Solution: Use strong promoters, optimize codon usage, or use fusion tags that enhance expression

Purification challenges:

• Oxidation and aggregation: The high cysteine content (20% of amino acids) makes metallothioneins prone to oxidation and aggregation

  • Solution: Maintain reducing conditions throughout purification; use TCEP rather than DTT or β-mercaptoethanol for better stability

• Metal loss during purification: Metallothioneins can lose bound metals during purification, especially at low pH

  • Solution: Control pH carefully and consider adding metals to purification buffers if metalated forms are desired

• Additional amino acid effects: Recombinant proteins often contain extra amino acids that may affect function. Six additional amino acid residues in recombinant mouse MT-I affected its immunoreactivity

  • Solution: Design constructs to minimize extra sequences or include protease cleavage sites for tag removal

• Heterogeneous metalation: Different molecules in the preparation may bind different numbers or types of metal ions

  • Solution: Use defined metal reconstitution protocols after purification of apo-protein

Validation challenges:

• Confirming correct folding: Without a rigid tertiary structure, confirming proper folding of metallothioneins is challenging

  • Solution: Use spectroscopic methods and metal-binding assays to confirm appropriate structure

What storage and handling protocols maintain stability of Recombinant Rabbit Metallothionein-2D?

Based on protocols for metallothionein handling in the research literature, the following recommendations are provided:

Short-term storage (days to weeks):

• Buffer composition:

  • Include reducing agents: TCEP at 0.6-1 mM has been used successfully

  • pH control: Maintain pH above the half-dissociation pH of bound metals (e.g., above pH 5.5 for zinc-containing MT)

  • Consider including stabilizing metal ions

• Temperature:

  • Store at 4°C for short periods (days)

  • Avoid repeated freeze-thaw cycles

Long-term storage (weeks to months):

• Lyophilization protocol:

  • Flash-freeze small aliquots in liquid nitrogen

  • Lyophilize the flash-frozen samples

  • Store lyophilized powder at -20°C

• Reconstitution:

  • Reconstitute in appropriate buffer with reducing agents

  • For metal-free studies, add chelating agents during reconstitution

  • For metalation studies, add specific metal ions during reconstitution

Handling considerations:

• Oxidation prevention:

  • Work under nitrogen atmosphere when possible

  • Prepare fresh reducing agents regularly

  • Consider using oxygen-scavenging systems for sensitive experiments

• Metal contamination:

  • Use metal-free reagents and acid-washed glassware for apo-MT work

  • Be aware that even high-purity reagents can contain trace metals

• Concentration effects:

  • Higher concentrations may increase aggregation risk

  • Consider stabilizing additives for concentrated samples

Stability monitoring:

• Regular quality checks:

  • UV-visible spectroscopy to confirm metal-thiolate integrity

  • Mass spectrometry to detect oxidation or degradation

  • Functional assays to confirm metal-binding capacity

These handling protocols are essential for maintaining the integrity of recombinant MT-2D during storage and experimental procedures.

What experimental design considerations are critical when studying metal-binding kinetics of Recombinant Rabbit Metallothionein-2D?

When designing metal-binding kinetics experiments with recombinant MT-2D, researchers should consider:

Pre-experimental considerations:

• Protein preparation:

  • Start with fully characterized apo-MT-2D (metal-free)

  • Confirm protein concentration using amino acid analysis rather than less reliable spectrophotometric methods

  • Verify the reduced state of all cysteine residues

• Metal solution preparation:

  • Use high-purity metal salts with known concentration

  • Prepare metal solutions in the same buffer as the protein

  • Control for potential metal contamination in buffers

Experimental design:

• Reaction conditions:

  • Control temperature precisely (typically 25°C or 37°C)

  • Maintain constant pH using appropriate buffers

  • Consider ionic strength effects on binding

  • Use anaerobic conditions to prevent thiol oxidation

• Time scales:

  • Design experiments to capture both fast initial binding events (milliseconds to seconds) and slower reorganization events (minutes to hours)

  • Use rapid mixing techniques (stopped-flow) for fast reactions

• Monitoring methods:

  • Select appropriate spectroscopic techniques:

    • UV-visible spectroscopy for metal-thiolate bonds (e.g., Cd-S absorption at 250 nm)

    • Circular dichroism for conformational changes and cluster formation

    • Fluorescence for protein conformational changes or metal-specific signals

  • Consider real-time mass spectrometry for metalation state distribution

Data analysis:

• Kinetic models:

  • Apply appropriate models for sequential binding of multiple metals

  • Consider cooperative binding effects

  • Distinguish between different binding sites (α and β domains)

• Competition experiments:

  • Design metal competition studies to determine relative binding preferences

  • Use metallochromic indicators for real-time monitoring

Controls and validation:

• Essential controls:

  • Metal-only and protein-only controls

  • Comparison with known metallothioneins (e.g., rabbit MT-I)

  • Validation with multiple complementary techniques

By addressing these considerations, researchers can obtain reliable kinetic data on the metal-binding properties of recombinant rabbit MT-2D, which is essential for understanding its biological functions and potential applications.