Recombinant Photobacterium profundum UPF0178 protein PBPRA1738 (PBPRA1738)

What is Photobacterium profundum PBPRA1738 and how is it classified?

PBPRA1738 is classified as a UPF0178 family protein from the deep-sea bacterium Photobacterium profundum strain SS9. This bacterium belongs to the family Vibrionaceae and is a model organism for studying piezophily (pressure adaptation) . The UPF0178 designation indicates it belongs to a protein family with unknown function, though its conservation suggests biological significance. Sequence analyses and comparative genomics approaches with other characterized proteins from P. profundum, such as PBPRA1750 (phosphotransferase), can provide initial insights into potential functions .

What are the optimal storage conditions for recombinant PBPRA1738?

Based on storage protocols for similar recombinant proteins from P. profundum, PBPRA1738 should be stored at -20°C/-80°C, with expected shelf life of approximately 6 months for liquid formulations and 12 months for lyophilized preparations . Critical storage factors include:

• Buffer composition: Buffer ingredients significantly impact protein stability

• Aliquoting: Working aliquots should be stored at 4°C for up to one week

• Freeze-thaw cycles: Repeated freezing and thawing should be avoided

• Glycerol concentration: Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

For optimal results, centrifuge vials briefly before opening to bring contents to the bottom, and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

What expression systems are most suitable for PBPRA1738 production?

The following expression systems have been utilized for recombinant P. profundum proteins:

For PBPRA1738 specifically, baculovirus expression systems have been successfully employed . When designing expression constructs, consider that tag type will be determined during the manufacturing process, and optimization may be required for specific applications .

What purification strategies yield the highest purity for recombinant PBPRA1738?

Based on purification protocols for similar P. profundum proteins:

• Aim for purity >85% as verified by SDS-PAGE

• Consider affinity chromatography based on the tag incorporated during expression

• Implement additional purification steps (ion exchange, size exclusion) as needed

• Verify final purity using analytical techniques such as SDS-PAGE

When designing purification protocols, consider that PBPRA1738 may have specific biochemical properties related to its adaptation to high-pressure environments that might affect binding and elution conditions .

How does PBPRA1738 expression change under different pressure conditions?

While specific data for PBPRA1738 is limited, proteomic studies of P. profundum have shown significant pressure-dependent regulation of protein expression. Using label-free quantitative proteomic analysis methodology:

• Culture P. profundum SS9 at different pressures (typically 0.1 MPa vs. 28 MPa)

• Extract proteins and perform shotgun proteomic analysis

• Use label-free quantitation and mass spectrometry analysis

• Identify differentially expressed proteins with statistical significance (p<0.05)

Previous studies have identified numerous proteins that are differentially expressed at high pressure (28 MPa) compared to atmospheric pressure (0.1 MPa) . To study PBPRA1738 specifically:

• Compare its expression ratio between high and low pressure conditions

• Determine p-values associated with quantitation (aim for p<0.05)

• Analyze its regulation pattern in context with other known pressure-responsive proteins

Recent proteomics studies have shown that proteins involved in specific metabolic pathways (like glycolysis/gluconeogenesis) are up-regulated at high pressure, while others (like oxidative phosphorylation) are up-regulated at atmospheric pressure .

What methodologies can assess PBPRA1738's role in pressure adaptation?

To investigate potential roles in pressure adaptation:

• Gene Disruption Studies:

  • Construct gene disruption mutants using internal gene fragments amplified by PCR

  • Clone fragments into vectors like pMUT100

  • Introduce into P. profundum via conjugation

  • Test growth at different pressures to identify pressure-sensitive phenotypes

• Complementation Analysis:

  • Isolate genomic DNA from complemented strains

  • Digest with restriction enzymes (BglII, HpaI, KpnI, SacI, or XbaI)

  • Circularize digested DNA with T4 ligase

  • Transform into E. coli and select for resistance markers

  • Test complemented strains for pressure growth phenotypes

• Proteomic Analysis Under Pressure:

  • Perform comparative proteomic analysis at different pressures

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

  • Correlate with transcriptomic data when available

How might PBPRA1738 interact with the ToxR regulatory system in P. profundum?

The ToxR regulatory system plays a crucial role in pressure-responsive gene expression in P. profundum. To investigate potential interactions between PBPRA1738 and the ToxR pathway:

• RNA Arbitrarily Primed PCR (RAP-PCR):

  • Compare expression in wild-type and toxR mutant strains

  • Verify ToxR regulation of candidate genes

  • Previous studies identified seven ToxR-activated and one ToxR-repressed transcripts

• Comparative Expression Analysis:

  • Culture wild-type and toxR mutant strains under identical conditions

  • Extract RNA and perform gene expression analysis

  • Look for differential expression patterns suggesting ToxR regulation

• Pressure Regulation Characterization:

  • Characterize transcript levels at various hydrostatic pressures

  • Note that ToxR activation/repression cannot predict pressure response patterns

Previous studies have shown that ToxR-regulated genes often fall into categories related to membrane structure modification or starvation response .

How can computational approaches predict functional domains in PBPRA1738?

To predict functional domains and potential roles:

• Sequence Analysis:

  • Compare with the complete amino acid sequence of similar proteins (like PBPRA1750)

  • Search for conserved domains and motifs

  • Analyze for potential signal sequences or transmembrane regions

• Structural Prediction:

  • Generate structural models using homology modeling

  • Identify potential binding sites or catalytic residues

  • Compare with known structures of UPF0178 family proteins

• Genomic Context Analysis:

  • Examine neighboring genes for functional clues

  • Look for gene clusters that might suggest functional relationships

  • Compare genomic organization across related species

• Phylogenetic Analysis:

  • Construct phylogenetic trees of UPF0178 family proteins

  • Identify evolutionary relationships that might suggest function

  • Compare between piezophilic and non-piezophilic bacteria

What role might PBPRA1738 play in fatty acid biosynthesis pathways?

P. profundum is known for its unique fatty acid composition, particularly the presence of polyunsaturated fatty acids (PUFAs) that are important for high-pressure adaptation. To investigate potential roles of PBPRA1738 in fatty acid metabolism:

• Comparative Analysis with Known Pathways:

  • P. profundum has two distinct pathways for fatty acid synthesis:

    • Classical type II fatty acid synthase for monounsaturated and saturated fatty acids

    • Hybrid polyketide/fatty acid synthase (encoded by pfa genes) for omega-3 PUFAs

• Suppressor Mutation Analysis:

  • Generate mutants with defects in fatty acid biosynthesis

  • Look for suppressor phenotypes that restore growth

  • Analyze PBPRA1738 expression in these suppressor strains

• Protein-Protein Interaction Studies:

  • Investigate potential interactions with fatty acid biosynthesis enzymes

  • Look for co-regulation with known fatty acid biosynthesis genes

  • Analyze expression in response to fatty acid supplementation

Recent research has demonstrated that mutations in fatty acid biosynthesis genes can lead to compensatory increases in PUFA production, suggesting complex regulatory networks controlling membrane lipid composition .

How can CRISPR-Cas systems be optimized for studying PBPRA1738 function?

P. profundum genomes contain various CRISPR-Cas systems that could be adapted for genetic manipulation. Based on comparative genomic studies:

• CRISPR-Cas System Selection:

  • P. profundum strains have CRISPR-Cas systems similar to those in Yersinia pestis, Escherichia coli, and Desulfovibrio vulgaris

  • Select appropriate system based on efficiency and specificity

• Guide RNA Design:

  • Design specific guide RNAs targeting PBPRA1738

  • Consider genomic context and potential off-target effects

  • Optimize for the specific CRISPR-Cas system being used

• Delivery Methods:

  • Optimize transformation or conjugation protocols for P. profundum

  • Consider pressure conditions that might affect transformation efficiency

  • Use appropriate selection markers for P. profundum genetics

• Phenotypic Analysis:

  • Analyze mutants under various pressure conditions

  • Look for effects on growth, membrane composition, and stress responses

  • Compare with other characterized genes in P. profundum

When implementing CRISPR-Cas systems, note that P. profundum strains may have varying numbers of CRISPR array spacers (from 1 to 64 in the same array), indicating different histories of phage infection or horizontal gene transfer .

How does PBPRA1738 compare to related proteins in other piezophilic bacteria?

To perform comparative analysis:

• Phylogenetic Distribution:

  • Compare across the Photobacterium genus, which is the second largest in the Vibrionaceae family

  • Look at distribution in other piezophilic bacteria

  • Analyze correlation with depth/pressure adaptation

• Evolutionary Analysis:

  • Examine evidence of horizontal gene transfer

  • The Photobacterium genus shows high genomic diversity with evidence of genetic exchange through transposable elements, phage infection, or conjugative plasmids

  • Consider the role of genomic islands in functional acquisition

• Structural Comparison:

  • Compare predicted structures with proteins from related bacteria

  • Identify conserved domains that might be pressure-responsive

  • Look for unique features in piezophilic variants

• Expression Pattern Comparison:

  • Compare expression patterns under pressure across species

  • Look for conserved regulatory elements in promoter regions

  • Analyze correlation with ecological niche and depth distribution

This comparative approach can provide insights into how PBPRA1738 might contribute to the specific adaptations that allow P. profundum to thrive under high-pressure conditions.

What special considerations apply when assessing enzymatic activity of PBPRA1738 under pressure?

Working with proteins from piezophilic organisms requires specialized approaches:

• High-Pressure Equipment:

  • Use pressure vessels capable of maintaining 28 MPa

  • Consider temperature control (typically 15°C for P. profundum)

  • Ensure proper sealing and safety measures

• Activity Assays:

  • Design assays that can be performed under pressure

  • Compare activity at atmospheric vs. high pressure

  • Consider control proteins from non-piezophilic organisms

• Stability Assessment:

  • Evaluate protein stability under various pressure conditions

  • Analyze effect of pressure on protein folding and oligomerization

  • Consider the role of specific amino acids in pressure adaptation

For reference, P. profundum α-carbonic anhydrase (PprCA) exhibits maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic ranges, and shows salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions . Similar biochemical characterization approaches could be applied to PBPRA1738.