Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 (DP2912)

What is Recombinant Desulfotalea psychrophila UPF0316 protein DP2912?

Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 (UniProt ID: Q6AJ39) is a full-length protein consisting of 290 amino acids (positions 1-290) derived from the psychrophilic sulfate-reducing bacterium Desulfotalea psychrophila. The recombinant form is typically produced with an N-terminal His-tag in E. coli expression systems to facilitate purification and downstream applications . The protein belongs to the UPF0316 protein family, whose specific biological functions remain under investigation. Current research suggests potential roles in membrane-associated processes based on its amino acid sequence characteristics.

What are the optimal storage conditions for DP2912?

For optimal stability and activity preservation of Recombinant Desulfotalea psychrophila UPF0316 protein DP2912, the following storage conditions are recommended:

• Upon receipt, store lyophilized protein powder at -20°C or -80°C.

• After reconstitution, store working aliquots at 4°C for up to one week.

• For longer-term storage, add glycerol to a final concentration of 5-50% (50% is recommended) and store at -20°C or -80°C in small aliquots.

• Avoid repeated freeze-thaw cycles as they can significantly decrease protein activity .

These storage recommendations are based on standard practices for maintaining recombinant protein stability while minimizing degradation through molecular aggregation, denaturation, or proteolytic cleavage.

How should I design experiments involving DP2912 protein?

When designing experiments with Recombinant Desulfotalea psychrophila UPF0316 protein DP2912, follow these systematic steps:

• Define your variables clearly:

  • Independent variable: Usually the concentration of DP2912 or experimental conditions affecting its activity

  • Dependent variable: The specific biological or biochemical outcome you're measuring

  • Control for extraneous variables such as temperature, pH, and buffer composition

• Formulate a specific, testable hypothesis about DP2912's function or properties

• Design experimental treatments:

  • Consider a range of protein concentrations (typically 0.1-1.0 mg/mL for initial studies)

  • Include appropriate positive and negative controls

  • Consider including known protein family members for comparative analysis

• Determine group assignments:

  • Between-subjects design: Different samples receive different treatments

  • Within-subjects design: Same samples receive multiple treatments sequentially

• Plan precise measurement methods for your dependent variables

What are the best practices for reconstituting lyophilized DP2912?

For optimal reconstitution of lyophilized Recombinant Desulfotalea psychrophila UPF0316 protein DP2912, follow these methodological steps:

• Bring the vial containing lyophilized protein to room temperature.

• Briefly centrifuge the vial prior to opening to ensure all material is at the bottom and to prevent loss of product.

• Reconstitute the protein in deionized sterile water to a concentration between 0.1-1.0 mg/mL.

• Mix gently by inversion or slow vortexing until completely dissolved. Avoid vigorous shaking that could cause protein denaturation.

• For long-term storage, add glycerol to a final concentration of 5-50% (50% is recommended).

• Aliquot the reconstituted protein into sterile microcentrifuge tubes to avoid repeated freeze-thaw cycles.

• Use freshly reconstituted protein for optimal results in functional assays .

The reconstitution buffer (Tris/PBS-based, pH 8.0) and the presence of 6% trehalose in the storage buffer have been specifically optimized to maintain the structural integrity and functional properties of DP2912 .

How can I validate the functional activity of DP2912 in my experimental system?

Validating the functional activity of Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 requires a multi-faceted approach since its precise biological function remains under investigation:

• Structural Integrity Assessment:

  • SDS-PAGE analysis to confirm proper molecular weight (~32 kDa including His-tag)

  • Western blot using anti-His antibodies to verify tag presence

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

• Binding Assays:

  • Pull-down assays to identify potential binding partners

  • Surface plasmon resonance (SPR) to quantify binding kinetics if potential interactors are identified

  • Membrane association studies using liposome binding assays (recommended given the predicted transmembrane domains)

• Functional Characterization:

  • Design experiments based on predicted properties of UPF0316 family proteins

  • Test for potential enzymatic activities (GTPase, ATPase, etc.)

  • Evaluate temperature-dependent activity profiles (considering its psychrophilic origin)

• Cellular Localization:

  • If using cell-based systems, track protein localization using fluorescently tagged versions

  • Compare localization patterns with known membrane proteins

• Comparative Analysis:

  • Include other UPF0316 family proteins as positive controls

  • Benchmark activity against related proteins with known functions

What approaches should I use to investigate DP2912's potential membrane association?

Given the amino acid sequence characteristics of DP2912 that suggest membrane association, comprehensive investigation requires multiple complementary approaches:

• Computational Analysis:

  • Apply transmembrane prediction algorithms (TMHMM, Phobius, HMMTOP)

  • Analyze hydrophobicity plots to identify potential membrane-spanning regions

  • Predict secondary structure elements characteristic of membrane proteins

• Biochemical Characterization:

  • Perform membrane fractionation studies in expression systems

  • Conduct detergent solubility screens to identify optimal solubilization conditions

  • Use liposome binding assays with varying lipid compositions to identify preference

• Biophysical Methods:

  • Utilize atomic force microscopy (AFM) to visualize membrane insertion

  • Apply neutron reflectometry to determine orientation in membranes

  • Use fluorescence resonance energy transfer (FRET) to study membrane integration dynamics

• Structural Biology Approaches:

  • Consider crystallization trials with appropriate detergent micelles

  • Apply solution NMR with nanodiscs or bicelles for structure determination

  • Use cryo-electron microscopy if protein forms larger assemblies

• Functional Validation:

  • Design reconstitution experiments in proteoliposomes

  • Measure potential ion conductance or transport activity

  • Assess effects on membrane fluidity and organization

These methodologies provide a comprehensive framework for elucidating the membrane-associated properties of DP2912, which could provide critical insights into its biological function.

How can I apply Quality by Design (QbD) principles when working with DP2912 in formulation studies?

Applying Quality by Design (QbD) principles to formulation studies with Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 requires a systematic approach:

• Define Quality Target Product Profile (QTPP):

  • Identify critical quality attributes of DP2912 (e.g., stability, solubility, activity)

  • Establish acceptance criteria for each attribute

  • Determine analytical methods to measure these attributes

• Design and Implement Design of Experiments (DOE):

  • Select formulation variables to study (pH, buffer composition, excipients)

  • Design factorial or response surface experiments to evaluate multiple variables simultaneously

  • Include center points and replicates for statistical validity

• Characterize the Formulation Design Space:

  • Identify ranges for each formulation component that maintain protein quality

  • Determine interactions between formulation components

  • Establish mathematical models relating formulation variables to quality attributes

• Perform Excipient Robustness Studies:

  • Test the sensitivity of DP2912 to variations in excipient levels

  • Establish acceptable ranges for critical excipients

  • Identify potential stabilizers specific for this psychrophilic protein

• Validate the Design Space:

  • Conduct confirmation runs at the edges of the design space

  • Perform accelerated stability studies to verify predictions

  • Document the relationship between formulation variables and protein quality

The application of QbD principles enables rational formulation development for DP2912, potentially leading to enhanced stability and consistent quality across different research applications.

What techniques are recommended for studying DP2912 protein-protein interactions?

To comprehensively characterize potential protein-protein interactions of Recombinant Desulfotalea psychrophila UPF0316 protein DP2912, employ these methodological approaches:

• In Vitro Binary Interaction Methods:

  • Pull-down assays utilizing the His-tag for affinity purification

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for quantifying interactions in solution

• Structural Characterization of Complexes:

  • X-ray crystallography of co-crystallized complexes

  • Cryo-electron microscopy for larger assemblies

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

  • Cross-linking mass spectrometry (XL-MS) to identify proximity relationships

• Systems-Level Approaches:

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Proximity-dependent biotin identification (BioID)

  • Yeast two-hybrid screening with DP2912 as bait

  • Protein microarray analysis using purified DP2912

• Computational Predictions:

  • Molecular docking simulations with potential partners

  • Coevolution analysis across species

  • Protein-protein interaction network analysis

• Functional Validation:

  • Co-immunoprecipitation from relevant biological systems

  • FRET/BRET assays for detecting interactions in cellular contexts

  • Mutational analysis of predicted interaction interfaces

When implementing these methods, consider the potential membrane association of DP2912, which may necessitate specialized approaches for maintaining its native conformation during interaction studies.

What are common challenges when working with DP2912 and how can they be addressed?

Researchers working with Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 may encounter several challenges, each requiring specific mitigation strategies:

• Solubility Issues:

  • Challenge: Potential aggregation due to hydrophobic regions

  • Solution: Optimize buffer conditions (consider adding mild detergents like 0.01% Triton X-100 or low concentrations of glycerol); test solubility across pH range 6.5-8.5; consider implementing a step-wise dialysis protocol when changing buffer conditions

• Stability Concerns:

  • Challenge: Protein degradation during storage or experiments

  • Solution: Store with 50% glycerol at -80°C; add protease inhibitors to working solutions; minimize freeze-thaw cycles by preparing small aliquots; consider adding reducing agents if cysteine residues are present

• Activity Loss:

  • Challenge: Diminished functional activity after reconstitution

  • Solution: Use freshly reconstituted protein when possible; validate activity with consistent assays; consider temperature effects (given its psychrophilic origin, activity may be optimal at lower temperatures)

• Reproducibility Issues:

  • Challenge: Experimental variability across different protein preparations

  • Solution: Standardize expression and purification protocols; implement quality control measures (SDS-PAGE, western blot) for each batch; maintain detailed records of protein lot characteristics

• Non-specific Interactions:

  • Challenge: His-tag causing artifactual interactions in binding studies

  • Solution: Include appropriate controls; consider tag removal via protease cleavage; validate key findings with differently tagged or untagged protein versions

How can I design and implement controls for experiments involving DP2912?

Designing appropriate controls for experiments with Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 is essential for result validation and interpretation:

• Negative Controls:

  • Buffer-only conditions lacking DP2912 to establish baseline measurements

  • Heat-denatured DP2912 to distinguish between specific activity and non-specific effects

  • Irrelevant proteins of similar size and tag configuration to control for tag-related effects

• Positive Controls:

  • Well-characterized proteins from the same UPF0316 family if available

  • Known interaction partners or substrates if established

  • Activity standards relevant to the hypothesized function

• Internal Controls:

  • Concentration gradients of DP2912 to establish dose-dependency

  • Time-course experiments to determine kinetic parameters

  • Multiple methodological approaches to confirm the same finding

• Validation Controls:

  • Site-directed mutants of key residues to confirm functional importance

  • Tag-free versions of the protein to eliminate tag interference

  • Competitive inhibition assays to establish specificity

• Experimental Design Controls:

  • Technical replicates (minimum triplicate) for statistical validity

  • Biological replicates using independent protein preparations

  • Randomization of sample processing to minimize systematic errors

Implementation of these control strategies within a systematic experimental design framework enhances data reliability and facilitates accurate interpretation of results relating to DP2912's properties and functions.

What data analysis approaches are recommended for DP2912 protein characterization studies?

Comprehensive data analysis for Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 characterization requires multiple analytical approaches:

• Biophysical Characterization Data:

  • Apply non-linear regression for binding isotherms (SPR, ITC)

  • Implement deconvolution algorithms for circular dichroism spectra

  • Use peak fitting for size-exclusion chromatography profiles

  • Apply thermal shift analysis models for stability assessments

• Functional Assay Analysis:

  • Calculate enzyme kinetic parameters if enzymatic activity is identified

  • Apply appropriate statistical tests comparing experimental conditions

  • Use time-course modeling for dynamic processes

  • Implement dose-response curve fitting for concentration-dependent effects

• Structural Data Analysis:

  • Employ molecular dynamics simulation analysis for conformational studies

  • Use comparative modeling techniques referencing related protein structures

  • Apply clustering algorithms for conformational ensemble analysis

  • Implement secondary structure prediction validation

• Omics Data Integration:

  • Perform enrichment analysis for potential interaction partners

  • Apply network analysis for placing DP2912 in biological pathways

  • Use machine learning approaches for functional prediction

  • Implement phylogenetic analysis for evolutionary context

• Statistical Considerations:

  • Apply appropriate parametric or non-parametric tests based on data distribution

  • Use multivariate analysis for complex datasets (principal component analysis, factor analysis)

  • Implement power analysis to determine appropriate sample sizes

  • Apply multiple testing corrections for high-throughput data

How might psychrophilic properties of DP2912 be leveraged in research applications?

The psychrophilic origin of Recombinant Desulfotalea psychrophila UPF0316 protein DP2912 offers unique research opportunities:

• Comparative Structural Biology:

  • Investigate cold-adaptation mechanisms by comparing DP2912's structure with mesophilic homologs

  • Analyze flexibility-rigidity balances characteristic of cold-adapted proteins

  • Examine potential reduced hydrophobic core packing or increased surface hydrophilicity

• Enzymatic Activity at Low Temperatures:

  • If enzymatic function is identified, characterize activity profiles across temperature ranges (0-37°C)

  • Determine activation energy and thermodynamic parameters

  • Investigate potential applications in low-temperature biochemical processes

• Membrane Dynamics in Cold Environments:

  • Study membrane association properties at reduced temperatures

  • Investigate interactions with lipids having different phase transition temperatures

  • Examine potential roles in maintaining membrane fluidity in cold conditions

• Protein Engineering Applications:

  • Identify structural elements responsible for cold adaptation

  • Transfer these elements to mesophilic proteins to enhance low-temperature activity

  • Develop biocatalysts with improved cold-temperature performance

• Ecological and Evolutionary Studies:

  • Investigate the role of DP2912 in psychrophilic adaptation

  • Perform comparative genomics with related proteins from different temperature niches

  • Study potential horizontal gene transfer patterns of UPF0316 family genes

The unique psychrophilic properties of DP2912 represent a valuable model system for understanding protein adaptation to extreme environments and may yield insights applicable to biotechnology, bioremediation, and fundamental understanding of protein evolution.