Recombinant Photobacterium profundum UPF0102 protein PBPRA3228 (PBPRA3228)

What is Photobacterium profundum PBPRA3228 and why is it of interest to researchers?

PBPRA3228 is an uncharacterized protein family (UPF0102) protein from Photobacterium profundum, a deep-sea marine bacterium adapted to high-pressure environments. This protein is of particular interest to researchers studying bacterial adaptation to extreme environments, specifically high hydrostatic pressure conditions. P. profundum is a gram-negative rod with remarkable ability to grow across a wide range of temperatures (0°C to 25°C) and pressures (0.1 MPa to 70 MPa) depending on the strain . The UPF0102 family proteins are generally conserved across bacterial species, suggesting important biological functions despite their currently uncharacterized status.

How does PBPRA3228 compare to other UPF proteins from Photobacterium profundum?

PBPRA3228 belongs to the UPF0102 protein family, while a related protein, PBPRA3258, is classified as a UPF0042 nucleotide-binding protein . Though both are uncharacterized, they likely serve different functions based on their family classifications. PBPRA3258 possesses a nucleotide-binding domain with a sequence suggesting potential involvement in nucleotide metabolism or signaling pathways . Comparatively, the UPF0102 family that includes PBPRA3228 lacks this specific nucleotide-binding motif. Researchers should note these distinctions when designing experiments to elucidate their respective functions in P. profundum physiology.

What are the storage and stability characteristics of recombinant PBPRA3228?

Similar to other recombinant proteins from P. profundum, PBPRA3228 typically demonstrates the following stability characteristics:

• Shelf life in liquid form: approximately 6 months at -20°C/-80°C

• Shelf life in lyophilized form: approximately 12 months at -20°C/-80°C

• Stability is influenced by buffer ingredients, storage temperature, and intrinsic protein properties

For optimal stability, researchers should avoid repeated freeze-thaw cycles, which can lead to protein degradation. Working aliquots may be stored at 4°C for up to one week . For long-term storage, addition of 5-50% glycerol (final concentration) and storage at -20°C/-80°C is recommended.

What expression systems are most effective for producing recombinant PBPRA3228?

Based on protocols established for similar proteins from P. profundum, the most effective expression systems include:

• Mammalian cell systems: Provide proper folding and potential post-translational modifications, though yields may be lower than bacterial systems .

• E. coli-based expression: Can be optimized using vectors like pET29a, which has been successfully used for other P. profundum proteins. The approach typically involves:

  • Gene amplification from chromosomal DNA using specific primers

  • Insertion into expression vectors containing appropriate tags (often hexa-histidine)

  • Transformation into expression strains

  • Induction under controlled conditions

• Native expression: For studying native function, expression in P. profundum itself may be achieved through conjugation methods using helper strains like E. coli with pRK2073 .

Each system offers advantages depending on research objectives, with bacterial systems providing higher yields and mammalian systems potentially offering better protein folding.

What purification strategies are most effective for recombinant PBPRA3228?

Effective purification of PBPRA3228 typically follows this methodological approach:

• Initial clarification: Centrifugation of cell lysate at high speed (typically 15,000-20,000×g) to remove cell debris

• Affinity chromatography: If expressed with a histidine tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices

• Quality assessment: SDS-PAGE analysis, with expected purity of >85% after initial purification

• Further purification (if needed): Ion exchange or size exclusion chromatography

• Buffer exchange and concentration: Using centrifugal concentrators with appropriate molecular weight cutoffs

For researchers studying pressure effects, it's critical to maintain protein samples at conditions that preserve native structure throughout the purification process, as pressure-adapted proteins may show altered stability at atmospheric pressure.

How can I accurately determine the concentration and purity of purified PBPRA3228?

Multiple complementary methods should be employed:

• Spectrophotometric analysis: Using theoretical extinction coefficients based on amino acid composition

• Bradford or BCA assays: For protein quantification relative to standard curves

• SDS-PAGE with densitometric analysis: For purity assessment; expected purity should be >85%

• Western blot: If suitable antibodies are available, for confirmation of protein identity

• Mass spectrometry: For absolute confirmation of molecular weight and potential post-translational modifications

Researchers should note that for maximum accuracy, protein concentrations determined by multiple methods should be compared and reconciled.

What approaches are recommended for determining the structure of PBPRA3228?

For structural characterization of PBPRA3228, researchers should consider these methodological approaches:

• X-ray crystallography:

  • Requires optimization of crystallization conditions specific to PBPRA3228

  • May require screening hundreds of conditions varying in precipitants, buffers, pH, and additives

  • For pressure-adapted proteins, specialized high-pressure crystallization chambers may provide structures more relevant to native conditions

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Particularly useful for studying protein dynamics

  • Can be performed under variable pressure conditions to assess structural changes

• Cryo-electron microscopy:

  • Emerging technique for membrane-associated proteins

  • May be useful if PBPRA3228 forms larger complexes

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution structural information in solution

  • Useful for studying conformational changes under different pressure conditions

• Computational modeling:

  • Homology modeling based on related UPF0102 proteins

  • Molecular dynamics simulations to predict behavior under varying pressure conditions

What experimental approaches can help determine the biological function of PBPRA3228?

Since PBPRA3228 is an uncharacterized protein, multiple approaches should be employed:

• Genetic manipulation:

  • Gene knockout or knockdown studies in P. profundum

  • Phenotypic analysis under various pressure conditions

  • Complementation studies to confirm phenotype

• Protein interaction studies:

  • Pull-down assays to identify binding partners

  • Bacterial two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

• Comparative genomics:

  • Analysis of gene neighborhood and conservation across strains

  • Presence/absence patterns across P. profundum strains adapted to different depths

• Transcriptomic analysis:

  • RNA-seq under varying pressure conditions to identify co-regulated genes

  • Analysis of expression patterns in different bathytypes

• Biochemical activity assays:

  • Testing for enzymatic activities (hydrolase, transferase, etc.)

  • Ligand binding assays similar to those used for P450 enzymes from P. profundum

How do pressure and temperature affect the structure and function of PBPRA3228?

As a protein from a piezophilic organism, PBPRA3228 likely exhibits specific adaptations to high pressure environments. Research approaches should include:

• High-pressure biophysical techniques:

  • Circular dichroism under varying pressure conditions

  • Fluorescence spectroscopy to monitor conformational changes

  • FTIR spectroscopy for secondary structure analysis

• Activity assays under pressure:

  • Using specialized high-pressure vessels to measure activity

  • Comparing kinetics at different pressures (0.1 MPa to 70 MPa range)

  • Temperature-pressure matrices to assess interaction effects

• Molecular dynamics simulations:

  • Computational prediction of pressure effects on protein dynamics

  • Analysis of water penetration into protein core under different conditions

P. profundum strains show varying adaptations to pressure, with strain SS9 growing optimally at 15°C and 28 MPa, while strain 3TCK grows optimally at 9°C and 0.1 MPa . The protein may therefore exhibit strain-specific structural adaptations that reflect these environmental preferences.

How can PBPRA3228 be used to study bacterial adaptation to extreme environments?

PBPRA3228 represents an excellent model system for studying adaptation to high-pressure environments:

• Comparative analysis across bathytypes:

  • PBPRA3228 can be compared between deep-sea strains (SS9, DSJ4) and shallow-water strains (3TCK, 1230sf1)

  • Sequence variations may provide insights into pressure adaptation mechanisms

• Structure-function relationships:

  • Identification of amino acid substitutions that confer pressure tolerance

  • Engineering these substitutions into proteins from non-piezophilic organisms

• Evolutionary studies:

  • Analysis of horizontal gene transfer patterns

  • Investigation of bathytype conversion mechanisms

• Biophysical investigations:

  • Study of protein flexibility and compressibility as adaptation mechanisms

  • Analysis of hydration patterns under varying pressure conditions

The protein can serve as a model for understanding broader mechanisms of extremophile adaptation, with potential applications extending to astrobiology and origin of life studies.

What are the recommended experimental controls when working with recombinant PBPRA3228?

For rigorous research with PBPRA3228, these controls are essential:

• Expression controls:

  • Empty vector transformants

  • Expression of a well-characterized protein using the same system

  • Wild-type P. profundum extracts for comparison

• Purification controls:

  • Mock purifications from non-transformed cells

  • Purification of a standard protein using identical protocols

• Activity assays:

  • Heat-denatured PBPRA3228 as negative control

  • Related proteins from non-piezophilic organisms for comparison

  • Samples exposed to atmospheric pressure for varying times

• Pressure experiments:

  • Multiple pressure levels including 0.1 MPa (atmospheric), optimal (strain-dependent), and inhibitory

  • Gradual versus rapid pressure changes

  • Control proteins with known pressure responses

• Cross-strain validation:

  • Comparison of results between different P. profundum strains (SS9, 3TCK)

  • Heterologous expression in piezosensitive hosts

How can researchers address the challenges of studying pressure-adapted proteins at atmospheric conditions?

Studying pressure-adapted proteins presents unique challenges that can be addressed through:

• Pressure retention strategies:

  • Rapid sample processing after pressure release

  • Addition of stabilizing agents (osmolytes, specific ligands)

  • Use of pressure-resilient buffer systems

• High-pressure experimental setups:

  • Design and utilization of specialized pressure vessels for enzymatic assays

  • Development of high-pressure crystallization chambers

  • Adaptation of spectroscopic techniques for high-pressure measurements

• Computational approaches:

  • Molecular dynamics simulations under varying pressure conditions

  • Prediction of pressure-induced conformational changes

  • Virtual screening for stabilizing compounds

• Methodological considerations:

  • Sample preparation under pressure when possible

  • Recording time after pressure release for all measurements

  • Development of standardized reporting formats for pressure experiments

What bioinformatic approaches are most useful for predicting potential functions of PBPRA3228?

Advanced bioinformatic strategies for functional prediction include:

• Sequence-based approaches:

  • Hidden Markov Model searches against specialized databases

  • Analysis of conserved domains and motifs

  • Detection of subtle sequence patterns using position-specific scoring matrices

• Structural bioinformatics:

  • Threading and fold recognition to identify structural homologues

  • Binding site prediction and comparison

  • Molecular docking with potential ligands

• Systems biology approaches:

  • Gene neighborhood analysis across multiple species

  • Protein-protein interaction network prediction

  • Metabolic pathway gap analysis

• Evolutionary analysis:

  • Phylogenetic profiling to identify co-evolved proteins

  • Detection of positive selection signatures

  • Analysis of substitution patterns in piezophilic versus non-piezophilic organisms

• Integration of multi-omics data:

  • Correlation of expression patterns with metabolomic changes

  • Integration of proteomic and transcriptomic responses to pressure

How can researchers distinguish between pressure-specific effects and general stress responses when studying PBPRA3228?

This methodological challenge requires careful experimental design:

• Multiple stress comparison:

  • Parallel experiments with pressure, temperature, osmotic, and oxidative stresses

  • Development of stress-specific signatures

  • Construction of a stress response matrix distinguishing general versus specific responses

• Time-course experiments:

  • Analysis of immediate versus adaptive responses

  • Identification of temporal patterns specific to pressure adaptation

  • Distinction between primary and secondary effects

• Genetic approaches:

  • Creation of mutant strains with disrupted general stress response pathways

  • Assessment of PBPRA3228 function in these backgrounds

  • Complementation studies with PBPRA3228 variants

• Targeted inhibition studies:

  • Use of specific inhibitors of stress response pathways

  • Combination of inhibitors with pressure challenges

  • Assessment of direct versus indirect effects on PBPRA3228 function

P. profundum SS9 is known to upregulate several stress response genes (htpG, dnaK, dnaJ, groEL) in response to atmospheric pressure . Researchers must distinguish these general stress responses from specific functions of PBPRA3228.

What are the best practices for presenting research data on PBPRA3228?

Effective presentation of data follows these principles:

• General guidelines:

  • Keep presentation simple and focused on key findings

  • Present general information before specific details

  • Ensure data directly answers research questions

  • Use past tense when describing results

  • Choose the most appropriate format (text, tables, or graphics) for each data type

• Text presentation:

  • Present data with interpretation, not just raw values

  • Avoid redundant words and qualitative descriptors like "remarkably" or "extremely"

  • Focus on significant findings rather than exhaustive data reporting

• Table design for protein characterization:

  • Include four main components: title, columns, rows, and footnotes

  • Arrange similar data in columns for easier comparison

  • Use consistent units and decimal places

  • Round numbers to meaningful precision

  • Use footnotes for explaining abbreviations and statistical significance

• Graphical representation:

  • Avoid 3D graphs which can make precise value determination difficult

  • Use line graphs for tracking changes over time or pressure conditions

  • Clearly label axes and include appropriate error bars

  • Consider pressure-temperature matrices for visualizing multifactorial effects

How should researchers interpret and analyze contradictory results when studying PBPRA3228?

When facing contradictory results, researchers should:

• Systematic assessment:

  • Evaluate methodological differences between contradictory studies

  • Consider strain-specific variations (SS9 vs. 3TCK)

  • Examine differences in experimental conditions, particularly pressure parameters

• Validation approaches:

  • Replicate experiments using standardized protocols

  • Employ multiple complementary techniques to assess the same parameter

  • Seek independent verification from collaborating laboratories

• Alternative hypotheses formulation:

  • Develop models that could explain seemingly contradictory results

  • Consider pressure-specific context dependencies

  • Evaluate potential conformational equilibria affected by experimental conditions

• Statistical and bioinformatic analysis:

  • Apply robust statistical methods appropriate for small sample sizes

  • Use meta-analysis approaches when multiple datasets are available

  • Employ Bayesian methods to incorporate prior knowledge

Researchers should recognize that proteins from piezophilic organisms often exhibit complex behaviors that vary with environmental conditions, potentially explaining apparently contradictory observations.

What standardized metrics should be used to compare PBPRA3228 from different strains of Photobacterium profundum?

For rigorous cross-strain comparisons, these standardized metrics are recommended:

These metrics should be measured under identical conditions across strains whenever possible, with clear reporting of experimental parameters to enable accurate meta-analysis.

What are the most promising research directions for further characterizing PBPRA3228 function?

Future research on PBPRA3228 should prioritize:

• Comprehensive functional screening:

  • Systematic testing for enzymatic activities

  • Substrate profiling using metabolomic approaches

  • Chemical biology approaches to identify potential ligands

• Integration with high-pressure adaptation mechanisms:

  • Investigation of potential role in membrane homeostasis

  • Analysis of interactions with known pressure-responsive systems

  • Assessment of contribution to pressure sensing or signaling

• Structural biology under native conditions:

  • Development of in situ high-pressure structural analysis techniques

  • Time-resolved structural studies during pressure transitions

  • Comparison of structures across different P. profundum strains

• Evolutionary analysis:

  • Assessment of selection pressures on PBPRA3228 across depth gradients

  • Reconstruction of ancestral sequences and functional testing

  • Analysis of horizontal gene transfer and bathytype conversion mechanisms

• Applications development:

  • Exploration of biotechnological applications of pressure-adapted proteins

  • Development of biosensors or biocatalysts with unique pressure-responsive properties

  • Investigation of potential roles in extremophile synthetic biology

How might PBPRA3228 research contribute to broader understanding of deep-sea microbial adaptation?

Research on PBPRA3228 has significant implications for understanding:

• Evolutionary mechanisms of piezoadaptation:

  • Molecular basis of pressure adaptation at the protein level

  • Role of horizontal gene transfer in bathytype conversion

  • Mechanisms of niche partitioning in the deep sea

• Extremophile physiology:

  • Integration of pressure and cold adaptation mechanisms

  • Cellular stress response networks in extremophiles

  • Energetic constraints of life under extreme conditions

• Marine microbial ecology:

  • Contribution to microbial community function at different ocean depths

  • Role in biogeochemical cycling in deep ocean environments

  • Mechanisms of bacterial adaptation during vertical transport in the water column

• Protein biophysics:

  • Fundamental understanding of pressure effects on protein structure and dynamics

  • Principles of protein adaptation to extreme conditions

  • Role of protein hydration in pressure adaptation