Recombinant Cricetulus longicaudatus Glutathione S-transferase P (GSTP1)

What is Cricetulus longicaudatus GSTP1 and what is its function?

Cricetulus longicaudatus (Chinese hamster) Glutathione S-transferase P (GSTP1) is a member of the GST family of enzymes that plays a critical role in cellular detoxification pathways. This enzyme catalyzes the conjugation of reduced glutathione (GSH) to various electrophilic compounds, thereby neutralizing their reactive centers and facilitating their elimination from cells. The scientific description identifies it as a purified recombinant protein commonly used in research settings .

GSTP1 functions primarily in phase II detoxification metabolism, providing protection against xenobiotics and oxidative stress. The enzyme has significant importance in comparative biochemistry studies due to its evolutionary conservation with specific species-dependent variations. When studying Cricetulus longicaudatus GSTP1, researchers should note that while it shares core functional properties with other mammalian GSTs, its specific amino acid composition confers unique catalytic and structural characteristics that make it valuable for comparative analyses.

The protein's role extends beyond simple detoxification, as GSTs have been implicated in cell signaling regulation, stress response pathways, and have been studied extensively in relation to disease models, particularly in the context of drug resistance and antioxidant defense mechanisms.

What are the structural characteristics of Cricetulus longicaudatus GSTP1?

Cricetulus longicaudatus GSTP1 maintains the canonical GST family structure consisting of distinct domains with specialized functions. The protein is identified in SwissProt as Q00285 and possesses specific amino acid characteristics that distinguish it from human GSTP1 (P09211) .

The protein structure includes:

• N-terminal domain (G-site): Contains the highly conserved glutathione binding site

• C-terminal domain (H-site): Contains the hydrophobic substrate binding pocket that determines specificity

• Dimeric quaternary structure: Functional as a homodimer with critical interface interactions

A notable structural feature of Cricetulus longicaudatus GSTP1 is the presence of a Leucine residue at position n+8, which differs from the corresponding position in human GSTP1 . This amino acid difference contributes to the specific binding and catalytic properties of the hamster enzyme.

The protein's three-dimensional structure follows the thioredoxin-like fold characteristic of GST family proteins, with alpha helices and beta sheets arranged to create the active site at the domain interface. This structural arrangement facilitates the activation of the glutathione thiol group, enabling nucleophilic attack on electrophilic substrates during the detoxification process.

What are the optimal storage conditions for Recombinant Cricetulus longicaudatus GSTP1?

The stability and activity of Recombinant Cricetulus longicaudatus GSTP1 are highly dependent on proper storage conditions. According to supplier specifications, the protein should be stored at -20°C for long-term maintenance of activity . This temperature prevents protein degradation and preserves structural integrity essential for enzymatic function.

For optimal preservation, consider these storage recommendations:

• Temperature: Maintain at -20°C for long-term storage as specified by manufacturers

• Buffer composition: Store in a stabilizing buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 150 mM NaCl

  • 10% glycerol as a cryoprotectant

  • 1 mM DTT or other reducing agent

To prevent activity loss from repeated freeze-thaw cycles, it is advisable to prepare small single-use aliquots before freezing. For ongoing experiments, limited storage at 4°C (up to 1 week) may be acceptable, but activity should be verified before critical applications.

Proper storage practices significantly impact experimental reproducibility and validity. Researchers should document storage conditions and protein lot information in their experimental protocols to facilitate troubleshooting and ensure experimental consistency across studies.

What is the typical purity level of commercially available Recombinant Cricetulus longicaudatus GSTP1?

Commercially available Recombinant Cricetulus longicaudatus GSTP1 typically has a purity level exceeding 90% as indicated by supplier specifications . This level of purity is sufficient for most research applications including enzymatic assays, structural studies, and comparative biochemical analyses.

The purity assessment typically involves:

• SDS-PAGE analysis with Coomassie brilliant blue staining to visualize protein bands

• Densitometric analysis to quantify the proportion of the target protein

• Quality control verification through additional methods such as:

  • Western blot analysis using specific antibodies

  • Mass spectrometry for molecular weight confirmation

  • Activity assays to verify functional integrity

For applications requiring exceptionally high purity (e.g., crystallography studies or sensitive interaction analyses), researchers may need to perform additional purification steps. When evaluating commercially available preparations, scientists should review the certificate of analysis to confirm the purity level and the methods used for its determination.

The estimated >90% purity standard reflects a balance between achieving sufficient purity for research applications while maintaining cost-effectiveness for research laboratories. This level of purity minimizes the influence of contaminants on experimental outcomes while ensuring the protein retains its native structural and functional properties.

What experimental approaches are recommended for studying GSTP1 enzymatic activity?

Several established methodologies can be employed for studying the enzymatic activity of Recombinant Cricetulus longicaudatus GSTP1, each with specific advantages for different research questions:

The spectrophotometric CDNB assay represents the gold standard for GST activity determination. This method utilizes 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate and measures the increase in absorbance at 340 nm as GSH conjugates with CDNB. A typical reaction mixture contains 100 mM potassium phosphate buffer (pH 6.5), 1 mM CDNB, 1 mM GSH, and 1-5 μg purified GSTP1, monitored continuously for 5 minutes at 25°C.

For kinetic analysis, researchers should conduct systematic studies with varying substrate concentrations to determine Km and Vmax values. This approach provides critical insights into the catalytic efficiency of the enzyme. The data can be analyzed using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee plots to extract kinetic parameters.

Inhibition studies represent another important approach, utilizing known GST inhibitors such as ethacrynic acid or S-hexylglutathione. These studies involve measuring enzyme activity in the presence of varying inhibitor concentrations to calculate IC50 and Ki values, providing insights into inhibitor potency and mechanism.

When studying Cricetulus longicaudatus GSTP1 specifically, researchers should consider conducting comparative analyses with human GSTP1 to identify species-specific enzymatic properties that may be relevant to xenobiotic metabolism differences between species. This comparative approach can reveal evolutionary adaptations in detoxification mechanisms.

How can structural analysis techniques be applied to study GSTP1 conformational changes upon substrate binding?

Multiple complementary structural analysis techniques can reveal the conformational dynamics of Recombinant Cricetulus longicaudatus GSTP1 upon interaction with substrates and inhibitors:

X-ray crystallography provides the most definitive method for high-resolution structural determination. Researchers should crystallize the protein in both apo form and with various ligands (GSH, substrate analogs, inhibitors) to capture different conformational states. Data should be collected at high resolution (<2.5 Å) using synchrotron radiation for optimal results. Analysis approaches include difference electron density maps to identify binding sites, structural superposition to identify conformational changes, and B-factor analysis to identify flexible regions. These crystallographic studies can reveal subtle structural rearrangements that occur during the catalytic cycle .

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) provides complementary information on solvent accessibility and protein dynamics in solution. This technique involves diluting the protein into D2O buffer (with and without ligands), quenching at various time points, followed by pepsin digestion and LC-MS analysis. Regions with reduced exchange upon ligand binding indicate protection, providing insights into binding interfaces and allosteric networks.

Molecular Dynamics (MD) simulations offer a computational approach to predict conformational changes that may be difficult to capture experimentally. All-atom simulations in explicit solvent, typically running for 100 ns to 1 μs, can reveal transient states and energetic barriers between conformations. Analysis methods include RMSD/RMSF calculations for structural variations, principal component analysis for major motion modes, and free energy calculations for binding energetics.

For Cricetulus longicaudatus GSTP1, these techniques should focus particularly on the active site, dimer interface, and any regions that differ from human GSTP1, as these may reveal species-specific conformational behaviors relevant to substrate specificity or inhibitor design.

What are the key structural differences between Cricetulus longicaudatus GSTP1 and other mammalian GST isoforms?

Cricetulus longicaudatus GSTP1 exhibits several important structural differences compared to other mammalian GST isoforms that influence its functional properties:

The most notable distinction is in the amino acid composition at specific positions, particularly at position n+8 where Cricetulus longicaudatus GSTP1 has a Leucine residue, which differs from human GSTP1 (SwissProt code P09211) . These amino acid differences contribute to the unique substrate specificity profile of the hamster enzyme and may influence its interaction with various xenobiotics.

The active site architecture of GSTP1 contains specific residues like Tyr7 that are critical for GSH activation. While this catalytic tyrosine is conserved across many GST classes, the surrounding residues that form the G-site (glutathione binding) and H-site (hydrophobic substrate binding) pockets show significant variations between species and isoforms. These differences result in distinct substrate preferences and catalytic efficiencies.

The dimer interface of GSTs is another region of significant structural variation across isoforms. The interface interactions are crucial for maintaining the quaternary structure and can affect the cooperativity between subunits. In Cricetulus longicaudatus GSTP1, the specific interface residues may create a unique communication network between the two active sites in the functional dimer.

Electrostatic surface properties also differ between GST isoforms, with each class exhibiting a characteristic charge distribution pattern. These electrostatic differences influence substrate approach and binding, particularly for charged or polar substrates. The specific charge distribution in Cricetulus longicaudatus GSTP1 contributes to its distinct substrate specificity compared to other mammalian GSTs.

How can site-directed mutagenesis be utilized to study the functional domains of GSTP1?

Site-directed mutagenesis provides powerful insights into structure-function relationships in GSTP1 by allowing researchers to systematically alter specific amino acids and observe the resulting functional changes.

Key residues for targeted mutagenesis include:

• G-site residues involved in GSH binding (e.g., Tyr7, which activates the GSH thiol group)

• H-site residues that determine substrate specificity and influence binding of various xenobiotics

• Dimer interface residues that affect quaternary structure and potential cooperativity

• Residues that differ between Cricetulus longicaudatus GSTP1 and human GSTP1, particularly the Leucine at position n+8

For mutagenesis strategy, researchers should consider:

• Conservative substitutions to assess the importance of specific chemical properties

• Non-conservative substitutions to probe dramatic functional changes

• Alanine scanning to systematically neutralize side chain contributions

• Substitutions that mimic the corresponding residues in human GSTP1 to investigate species-specific differences

Functional analysis of mutants should include:

• Enzymatic activity assays comparing wild-type and mutant proteins using multiple substrates

• Thermal stability assessments to evaluate effects on protein folding and stability

• Binding affinity measurements for GSH and various substrates/inhibitors

• Structural analysis when possible to correlate functional changes with structural alterations

This systematic mutational approach provides valuable mechanistic insights that cannot be obtained through other methods. By correlating the effects of specific amino acid changes with functional outcomes, researchers can develop detailed models of how GSTP1 recognizes and processes diverse substrates, informing both basic enzymology and applications in xenobiotic metabolism research.

What methodologies are recommended for crystallization of Recombinant Cricetulus longicaudatus GSTP1?

Crystallization of Recombinant Cricetulus longicaudatus GSTP1 requires careful attention to protein preparation and crystallization conditions to obtain diffraction-quality crystals suitable for structural studies.

For protein preparation:

• Purify to >95% homogeneity using sequential chromatography steps beyond the standard >90% purity

• Verify monodispersity by dynamic light scattering to ensure sample uniformity

• Concentrate to 10-15 mg/mL in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT

• Remove any aggregates by centrifugation at 20,000 × g for 10 minutes before setting up crystallization trials

Initial screening should employ commercial sparse matrix screens using sitting or hanging drop vapor diffusion methods. Typical drop composition includes 1 μL protein solution mixed with 1 μL reservoir solution, equilibrated against 500 μL reservoir solution at 18-20°C. Based on previous studies with GST proteins, promising conditions often contain PEG 3350 (15-25%) or ammonium sulfate (1.6-2.2 M) at pH 6.5-8.5.

For co-crystallization with ligands:

• Pre-incubate protein with 5-10 mM GSH for GSH-bound structures

• For substrate or inhibitor complexes, include 1-5 mM of the compound of interest

• Ensure the ligand is soluble in the crystallization buffer and does not precipitate

Optimization strategies should fine-tune promising conditions by varying pH (±0.5 units), precipitant concentration (±2%), protein concentration, temperature, and including additives like divalent cations (Mg²⁺, Ca²⁺ at 10-20 mM) which often improve crystal quality.

Successful crystallization can be assessed by crystal morphology (single, well-formed crystals are preferred) and, ultimately, by diffraction quality. Crystals should be carefully cryoprotected using reservoir solution supplemented with 20-25% glycerol before flash-freezing in liquid nitrogen for data collection at synchrotron facilities.

What are the recommended protocols for expression and purification of Recombinant Cricetulus longicaudatus GSTP1?

The efficient expression and purification of Recombinant Cricetulus longicaudatus GSTP1 involves several critical steps that must be carefully optimized to obtain high-quality protein for research applications.

For expression system selection, Escherichia coli remains the most common and cost-effective platform. BL21(DE3) or Rosetta strains are recommended hosts when using pET series vectors (e.g., pET-28a with His-tag) or pGEX series (with GST-tag) for expression. The choice between these systems depends on downstream applications – His-tagged constructs offer smaller tags but may have lower solubility, while GST-fusion proteins typically show enhanced solubility but require tag removal for certain applications.

A typical expression protocol for E. coli systems involves:

• Transformation of expression plasmid into competent cells

• Culture in LB or 2xYT medium with appropriate antibiotics

• Growth at 37°C to OD600 of 0.6-0.8

• Induction with 0.5-1.0 mM IPTG

• Continued expression at 18-25°C for 16-20 hours (overnight)

For purification, the strategy depends on the affinity tag used:

• For His-tagged protein: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, with washing using 20-40 mM imidazole and elution with 250-300 mM imidazole

• For GST-fusion protein: Glutathione Sepharose affinity chromatography with elution using 10-20 mM reduced glutathione

Additional purification steps may include size exclusion chromatography (Superdex 75 or 200) to remove aggregates and ensure homogeneity. Quality control should verify >90% purity by SDS-PAGE , confirm identity by Western blot or mass spectrometry, and assess enzymatic activity using standard GST assays.

Finally, the purified protein should be concentrated to 1-5 mg/mL, flash-frozen in liquid nitrogen as small aliquots, and stored at -20°C as recommended by suppliers to maintain stability and activity for subsequent experiments.

What are the considerations for using Recombinant Cricetulus longicaudatus GSTP1 in protein-protein interaction studies?

Investigating protein-protein interactions (PPIs) involving Recombinant Cricetulus longicaudatus GSTP1 requires careful experimental design to generate reliable and physiologically relevant data.

Potential interaction partners to consider include:

• Other detoxification enzymes in metabolic pathways

• Stress-response proteins

• Signaling molecules (particularly those involved in cellular stress responses)

• Transcription factors that regulate detoxification pathways

• Regulatory proteins that might modulate GSTP1 activity

For in vitro binding assays, several complementary approaches should be considered:

• Pull-down assays using immobilized tagged GSTP1 to capture interaction partners from cell lysates or purified candidate proteins

• Surface Plasmon Resonance (SPR) to measure binding kinetics and determine association/dissociation rate constants

• Isothermal Titration Calorimetry (ITC) for direct measurement of binding thermodynamics

When designing these experiments, researchers must include appropriate controls:

• Negative controls: non-interacting protein pairs, binding site mutants

• Positive controls: known GSTP1 interaction partners or GSTP1 homodimer formation

• Buffer controls: optimize conditions to minimize non-specific binding while maintaining protein stability

A common challenge in PPI studies is distinguishing specific interactions from non-specific binding. This can be addressed by:

• Optimizing buffer conditions (salt concentration, detergents, reducing agents)

• Using competition assays with excess unlabeled protein

• Validating interactions through multiple independent methods

• Confirming the biological relevance of interactions through functional assays

For Cricetulus longicaudatus GSTP1 specifically, researchers should consider species-specific interaction patterns and how they might differ from human GSTP1 interactions. This comparative approach can provide valuable insights into evolutionarily conserved versus species-specific protein-protein interaction networks involving GST enzymes.

What strategies can address low enzymatic activity of Recombinant Cricetulus longicaudatus GSTP1?

When encountering low enzymatic activity with Recombinant Cricetulus longicaudatus GSTP1, a systematic troubleshooting approach can identify and resolve the underlying issues.

First, protein quality assessment is essential:

• Verify purity by SDS-PAGE (should be >90% as specified)

• Check for degradation by western blot with anti-GSTP1 antibodies

• Assess aggregation state by size exclusion chromatography or dynamic light scattering

• Confirm protein concentration using BCA or Bradford assay with appropriate standards

Buffer optimization can significantly impact enzymatic activity:

• Test different pH values (pH 6.0-8.5) as the optimal pH may differ from human GSTP1

• Evaluate the effect of ionic strength (50-300 mM NaCl)

• Add reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain reduced cysteine residues

• Screen different buffer systems (phosphate, Tris, HEPES) as buffer components can influence activity

Substrate and co-substrate considerations include:

• Verify GSH quality and prepare fresh solutions (GSH can oxidize over time)

• Test different GSH concentrations (1-10 mM)

• Screen alternative substrates beyond CDNB (e.g., DCNB, ethacrynic acid)

• Ensure proper substrate solubility in the reaction mixture

For assay conditions, optimize:

• Enzyme concentration (0.1-5 μg per reaction)

• Reaction temperature (25-37°C)

• Incubation time for slow reactions

• Addition of stabilizing agents like BSA (0.1-1 mg/mL) for dilute enzyme solutions

If these approaches fail to restore activity, consider protein refolding or obtaining a new protein preparation. Including a positive control (such as commercial human GSTP1) in parallel assays can help distinguish between protein-specific issues and problems with the assay system itself.