Recombinant Photobacterium profundum UPF0325 protein PBPRA2971 (PBPRA2971)

What is Photobacterium profundum and why is it significant for research?

Photobacterium profundum is a deep-sea piezophilic bacterium that has adapted to function at high pressure environments. It serves as an important model organism for studying adaptation mechanisms to extreme environments, particularly deep-sea conditions. The SS9 strain is well-studied for its pressure-sensitive cellular processes, including motility systems that have been adapted for functionality at depth . Understanding proteins like PBPRA2971 can provide insights into how microorganisms adapt to extreme environmental conditions.

What are the basic characteristics of UPF0325 family proteins?

UPF0325 family proteins belong to the uncharacterized protein families (UPF) group, indicating that their functions are not yet fully understood. Based on structural similarities with other Photobacterium proteins, they likely play roles in cellular processes specific to deep-sea adaptation. These proteins share sequence homology with other bacterial proteins and may be involved in stress responses or specialized metabolic pathways necessary for survival in high-pressure environments .

How should I store and handle recombinant PBPRA2971 protein?

Recombinant proteins from Photobacterium profundum should generally be stored following standard protein storage protocols. Based on similar recombinant proteins like PBPRA2383, it's recommended to store the protein at -80°C for long-term storage, and avoid repeated freeze-thaw cycles . During experimental handling, maintain the protein in appropriate buffer conditions that mimic its native environment, potentially including pressure considerations when relevant to your research questions.

How should I design experiments to investigate the function of PBPRA2971 in pressure adaptation?

Designing experiments to investigate pressure adaptation requires specialized equipment and methodologies. First, define your variables clearly - with pressure serving as your primary independent variable and protein function/activity as your dependent variable . A systematic approach should include:

• Expression and purification of recombinant PBPRA2971 protein

• Baseline functional assays at atmospheric pressure

• Functional assays under incrementally increasing hydrostatic pressure

• Structural stability assessments under various pressure conditions

• Comparative analysis with homologous proteins from non-piezophilic organisms

This experimental design allows you to systematically test how pressure affects protein function while controlling for extraneous variables such as temperature, pH, and salt concentration .

What structural analysis techniques are most effective for characterizing PBPRA2971?

Based on successful structural analyses of other Photobacterium profundum proteins, cryo-electron microscopy has proven effective for high-resolution structural determination . For PBPRA2971, consider the following structural analysis pipeline:

• Initial screening using circular dichroism (CD) to assess secondary structure elements

• X-ray crystallography if the protein readily forms crystals

• Cryo-EM for detailed structural analysis, particularly if the protein is challenging to crystallize

• NMR spectroscopy for analyzing dynamic regions and ligand interactions

• Computational modeling using homology with related proteins

Combining multiple techniques provides complementary structural insights. The 2.97 Å resolution achieved for other Photobacterium profundum proteins by cryo-EM suggests this technique may be particularly valuable .

How do I integrate genetic and structural analyses to elucidate PBPRA2971 function?

A comprehensive approach to understanding PBPRA2971 function should integrate both genetic and structural methodologies:

• Generate gene deletion mutants using suicide vector systems (such as pRL271 with sacB negative selection) as demonstrated with other Photobacterium profundum genes

• Perform complementation studies to verify phenotypes

• Conduct structural analyses on wild-type and mutant proteins

• Map functional domains through site-directed mutagenesis

• Correlate structural features with phenotypic changes

This integrated approach allows for structure-function correlations that can reveal the mechanistic basis of PBPRA2971's role. The established methodology using vectors containing sacB for counterselection has been effectively used in Photobacterium profundum studies and can be adapted for PBPRA2971 .

What experimental design approach is optimal for testing PBPRA2971 interactions with other cellular components?

To investigate protein-protein or protein-substrate interactions involving PBPRA2971, implement a factorial experimental design that systematically examines multiple factors affecting these interactions . The following table outlines a proposed experimental design:

This design enables analysis of not only main effects but also interaction effects between factors, allowing for comprehensive mapping of conditions under which PBPRA2971 forms functional complexes .

How can I optimize PCR-based cloning strategies for PBPRA2971?

Optimizing PCR-based cloning of PBPRA2971 requires attention to the unique characteristics of Photobacterium profundum genes. Based on successful approaches with related genes:

• Design primers with appropriate restriction sites, accounting for the GC content of the target region

• Utilize high-fidelity polymerases capable of amplifying longer fragments, such as the Expand Long-Template PCR system

• Implement a touchdown PCR protocol to enhance specificity

• Consider codon optimization if expressing in heterologous systems

• Verify clone integrity through sequencing before expression studies

Primer design should follow the examples successful with other Photobacterium profundum genes, such as those used for motility genes (e.g., FlaAUPF 5′-AGTCTCGAGTGATCGGCAGTGGGCATACC-3′) .

What are the critical considerations for designing PBPRA2971 deletion mutants?

Creating deletion mutants of PBPRA2971 requires careful genetic manipulation strategies. Based on methodologies established for Photobacterium profundum:

• Construct deletion plasmids using suicide vectors containing counter-selectable markers like sacB

• Amplify upstream and downstream regions (approximately 2.5 kb each) flanking PBPRA2971

• Join these regions through restriction digestion and ligation, creating an in-frame deletion construct

• Introduce antibiotic resistance markers (e.g., kanamycin) for initial selection

• Perform triparental conjugations using helper plasmids like pRK2073

• Select for double recombination events using sucrose counter-selection

• Verify deletions through PCR with control primers spanning the deletion region

This methodology has been successfully applied to create in-frame deletions of multiple genes in Photobacterium profundum SS9, including flagellar and motility genes .

How should I approach the functional characterization of PBPRA2971 using transcriptomics and proteomics?

A comprehensive functional characterization requires integration of multiple omics approaches:

• Transcriptomics Strategy:

  • Compare gene expression profiles between wild-type and PBPRA2971 deletion mutants

  • Analyze expression under varying pressure conditions

  • Identify co-regulated genes that may function in the same pathways

• Proteomics Strategy:

  • Conduct differential protein expression analysis between wild-type and mutant strains

  • Perform protein-protein interaction studies using pull-down assays

  • Map post-translational modifications that may regulate activity

• Integration of Multi-omics Data:

  • Correlate transcriptomic and proteomic datasets to identify consistent patterns

  • Analyze protein complexes containing PBPRA2971 under different conditions

  • Map the regulatory network involving PBPRA2971

This integrated approach enables identification of both direct functions and broader cellular roles of PBPRA2971 .

What are the best practices for assessing pressure effects on PBPRA2971 structure and function?

Assessing pressure effects requires specialized methodologies and equipment:

• Equipment Considerations:

  • Use high-pressure vessels capable of maintaining stable pressure conditions

  • Implement pressure-resistant sampling systems for real-time measurements

  • Consider pressure cycling to assess reversibility of effects

• Experimental Approaches:

  • Conduct enzyme kinetics under varying pressure conditions

  • Perform structural analyses before, during, and after pressure treatment

  • Compare pressure responses of wild-type and mutant proteins

• Data Analysis:

  • Apply appropriate statistical methods for pressure-response curve fitting

  • Calculate activation volumes from pressure-dependent kinetic data

  • Model structural changes using molecular dynamics simulations under pressure

These approaches build on established methodologies for studying pressure-adaptive proteins in deep-sea organisms, with particular attention to the pressure-sensitive cellular processes known in Photobacterium profundum .

How does PBPRA2971 compare structurally and functionally to similar proteins in other Photobacterium species?

A comparative analysis provides evolutionary context for PBPRA2971 function:

• Conduct phylogenetic analysis of UPF0325 family proteins across bacterial species

• Compare sequence conservation in piezophilic versus non-piezophilic organisms

• Analyze structural similarities with characterized proteins like the SiaQM transporter

• Examine functional conservation through complementation studies

This comparative approach can reveal whether PBPRA2971 represents a specialized adaptation to deep-sea environments or serves a more conserved function across bacteria .

How should I design experiments to test hypotheses about PBPRA2971's role in pressure adaptation?

To systematically investigate pressure adaptation roles:

• Formulate specific, testable hypotheses based on preliminary data

• Design control experiments using pressure-sensitive mesophilic homologs

• Implement between-subjects experimental designs comparing wild-type and mutant strains

• Develop assays that can measure protein function across a pressure gradient

• Plan long-term experiments to assess evolutionary adaptation under pressure selection

This hypothesis-driven approach enables rigorous testing of pressure adaptation mechanisms, building on established experimental design principles .

What are the common challenges in expressing and purifying PBPRA2971, and how can they be overcome?

Based on experiences with similar recombinant proteins:

• Challenge: Low expression levels

  • Solution: Optimize codon usage for expression host

  • Solution: Test multiple promoter systems and induction conditions

  • Solution: Consider fusion tags that enhance solubility

• Challenge: Protein instability

  • Solution: Add stabilizing agents to buffer systems

  • Solution: Express truncated domains if full-length protein is unstable

  • Solution: Optimize purification protocols to minimize time and handling

• Challenge: Maintaining native conformation

  • Solution: Purify under conditions mimicking deep-sea environments

  • Solution: Validate structure using multiple analytical techniques

  • Solution: Consider co-expression with natural binding partners

These solutions build on established protein expression and purification methodologies, adapted for the specific challenges of deep-sea bacterial proteins .

How can I validate that recombinant PBPRA2971 maintains its native structure and function?

Validation requires multiple complementary approaches:

• Compare circular dichroism spectra between recombinant protein and native protein (if available)

• Perform functional assays under conditions mimicking the deep-sea environment

• Conduct thermal and pressure stability analyses

• Use antibodies raised against the native protein to confirm structural epitopes

• Verify ligand binding or enzymatic activity comparable to predictions from structural analysis