Recombinant Photobacterium profundum Elongation factor 4 (lepA), partial

What is Elongation factor 4 (lepA) in Photobacterium profundum and what are its primary functions?

Elongation factor 4 (EF-4), also known as Ribosomal back-translocase LepA, is a GTPase (EC 3.6.5.n1) that plays critical roles in protein synthesis fidelity. This protein facilitates the back-translocation of ribosomes along mRNA, essentially allowing for a "proofreading" mechanism during translation. In P. profundum, a deep-sea bacterium adapted to high-pressure environments, LepA likely contributes to maintaining translational accuracy under extreme conditions. The protein has been associated with stress response mechanisms in various bacteria, suggesting it may play a role in P. profundum's adaptation to high-pressure environments, although specific pressure-related functions have not been fully characterized in the available literature .

How should recombinant P. profundum LepA be stored for optimal stability and activity?

Storage conditions significantly impact protein stability and activity. For recombinant P. profundum LepA:

The shelf life depends on multiple factors including buffer ingredients and the intrinsic stability of the protein itself. Centrifuge vials briefly before opening to ensure contents are at the bottom .

What is the recommended reconstitution protocol for lyophilized P. profundum LepA?

For optimal reconstitution of lyophilized recombinant P. profundum LepA:

• Centrifuge the vial briefly to collect all material at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is commonly used)

• Prepare small aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted protein at -20°C to -80°C for long-term storage

This protocol helps maintain protein stability and activity by preventing degradation from repeated temperature changes and providing cryoprotection through glycerol addition .

What expression systems are most suitable for producing recombinant P. profundum LepA?

While P. profundum is a deep-sea bacterium adapted to high-pressure environments, the recombinant LepA protein is typically produced using mammalian cell expression systems . This approach may offer advantages for proper folding and post-translational modifications.

When designing an expression system, consider:

• Codon optimization for the host system

• Addition of appropriate tags for purification (tag type may vary based on manufacturing process)

• Signal peptides for secretion or cellular localization

• Temperature adjustments to account for the psychrophilic nature of the source organism

For researchers exploring alternative expression systems, the adaptation mechanisms of P. profundum to high pressure may provide insights. The bacterium's ability to modify membrane composition under pressure stress involves specific genetic regulation systems that might influence protein expression strategies .

How can purity and activity of recombinant P. profundum LepA be assessed?

Standard purity assessment for recombinant P. profundum LepA employs SDS-PAGE analysis, with commercial preparations typically achieving >85% purity . For more comprehensive quality assessment:

For activity assessment, measuring the GTPase activity using colorimetric phosphate release assays or the ability to catalyze ribosomal back-translocation in a reconstituted in vitro translation system would provide functional verification.

What strategies can mitigate the effects of pressure sensitivity when working with P. profundum proteins?

Drawing from P. profundum's natural adaptations to high pressure, consider these approaches when working with its proteins:

• Buffer optimization: Include osmolytes or pressure-protective compounds

• Temperature adjustments: Work at lower temperatures (4-15°C) to mimic deep-sea conditions

• Membrane mimetics: If studying membrane interactions, incorporate pressure-adapted lipid compositions (higher unsaturated fatty acid content)

• Pressure-controlled experimental setup: For truly accurate studies, conduct experiments under native pressure conditions (approximately 30 MPa)

P. profundum adapts to high pressure through various mechanisms, including modifications to membrane composition (increased unsaturated fatty acids like cis-vaccenic acid) and expression of pressure-specific outer membrane proteins like OmpH . These adaptations suggest that pressure sensitivity is a critical consideration when working with proteins from this organism.

How does P. profundum LepA compare structurally and functionally to LepA homologs from non-piezophilic bacteria?

While the search results don't provide specific structural comparisons for P. profundum LepA, comparative analysis with homologs from non-piezophilic bacteria would examine:

• Amino acid composition: Potential enrichment in flexible residues (glycine) or pressure-resistant amino acids

• Domain organization: Conservation of GTPase domains and potential pressure-adaptive regions

• Surface characteristics: Changes in hydrophobicity or charge distribution that might confer pressure resistance

• Functional differences: Potential altered GTPase activity rates or ribosome interactions

P. profundum demonstrates various adaptations to high pressure, including modifications to membrane-associated proteins and fatty acid composition . These adaptations suggest that cytoplasmic proteins like LepA may also contain pressure-specific modifications. Studies on ribosomes from piezophilic bacteria have shown structural adaptations to pressure, and as LepA interacts directly with ribosomes, it likely possesses complementary adaptations.

How might P. profundum LepA function in the context of high-pressure stress response pathways?

P. profundum has evolved specialized mechanisms to thrive under high-pressure conditions. LepA may play crucial roles in these adaptations:

• Translational fidelity maintenance: Ensuring accurate protein synthesis under pressure-induced conformational stress

• Integration with pressure-sensing regulatory networks: Potential interaction with pressure-responsive transcription factors

• Coordination with membrane adaptations: Possible synchronized regulation with systems that modify membrane fluidity

• Cold adaptation synergy: Coordination with cold-shock response, as deep-sea environments combine both pressure and cold stress

Research on P. profundum has identified several pressure-responsive pathways, including the ToxR regulon that controls outer membrane protein expression . While direct evidence linking LepA to these pathways is not present in the search results, its role in translational processes suggests it could be a downstream effector in pressure adaptation.

What techniques can be employed to study the interaction between P. profundum LepA and ribosomes under varying pressure conditions?

Studying LepA-ribosome interactions under pressure requires specialized approaches:

P. profundum has been studied under high pressure conditions using specialized equipment that maintains pressure during growth and experimentation . Similar approaches could be adapted for biochemical interaction studies, potentially revealing pressure-specific conformational changes or binding dynamics not observable under standard conditions.

What are common issues when working with recombinant P. profundum LepA and how can they be addressed?

For protein sourced from a deep-sea piezophile like P. profundum, consider that standard laboratory conditions (1 atmosphere pressure) represent extreme low-pressure stress from the protein's evolutionary perspective. This might necessitate specialized handling beyond conventional protein work.

How should experimental protocols be modified when comparing LepA activity from piezophilic versus non-piezophilic bacteria?

When conducting comparative studies:

• Establish equivalent baseline conditions: Standardize protein concentration, buffer composition, and assay parameters

• Pressure gradient analysis: Test activity across pressure ranges (1-50 MPa) with appropriate controls

• Temperature compensation: Account for psychrophilic nature of deep-sea bacteria by testing at lower temperatures

• Substrate optimization: Ensure ribosome substrates or other interacting partners are compatible with both proteins

• Data normalization: Develop appropriate reference points for comparison, considering the proteins' evolutionary adaptations to different environments

P. profundum demonstrates different growth capabilities at varying pressures, with strain-specific adaptations . Similarly, protein activities may show pressure optima that differ significantly from mesophilic homologs, requiring careful experimental design to make valid comparisons.

What considerations are important when designing site-directed mutagenesis experiments to investigate pressure-adaptive features of P. profundum LepA?

For mutational analysis targeting pressure adaptation:

• Identify potential pressure-sensing regions by comparative sequence analysis with non-piezophilic homologs

• Target residues involved in:

  • GTP binding and hydrolysis (conserved motifs)

  • Ribosome interaction interfaces

  • Regions with unusual amino acid composition compared to mesophilic homologs

• Design mutations that:

  • Convert to mesophilic-type residues to potentially reduce pressure adaptation

  • Enhance features hypothesized to confer pressure resistance

• Test mutants across pressure ranges to establish pressure-activity profiles

P. profundum has demonstrated genetic adaptations for high-pressure growth, including specific genes like RecD that are required for growth under elevated pressure . Similar functional dependencies may exist within the structure of LepA, which could be identified through targeted mutagenesis approaches.

How might structural studies of P. profundum LepA inform our understanding of protein adaptation to extreme environments?

Detailed structural analysis of P. profundum LepA could reveal:

• Pressure-adaptive structural features: Identification of domains or motifs that maintain function under pressure

• Conformational flexibility: Regions that allow necessary structural changes without loss of function

• Comparative insights: Structural differences from mesophilic homologs that explain pressure tolerance

• Evolutionary patterns: Conservation of pressure-adaptive features across piezophilic species

Such insights could extend beyond LepA to inform broader principles of protein adaptation to extreme environments. P. profundum has been shown to modify various cellular components in response to pressure, including membrane composition and flagellar systems , suggesting comprehensive adaptation strategies that likely include translational machinery components like LepA.

What role might P. profundum LepA play in cold and pressure co-adaptation mechanisms?

Deep-sea environments combine high pressure with low temperature, creating multiple stressors:

• Translational thermal compensation: LepA may help maintain translation rates at low temperatures under pressure

• Synergistic adaptation pathways: Potential integration with cold-shock response systems

• Conformational stability balance: Maintenance of necessary flexibility at low temperature while resisting pressure-induced compaction

• Regulatory crossover: Shared regulatory elements between cold and pressure response systems

Studies on P. profundum have shown adaptations to both cold and pressure, including polyunsaturated fatty acid synthesis systems that respond to both stressors . The combined pressure-temperature adaptation strategies may involve LepA in maintaining translational fidelity under these challenging conditions.

How could insights from P. profundum LepA be applied to enhance protein function in biotechnological applications?

Understanding pressure adaptation in LepA could inform:

• Engineered enzymes with enhanced pressure tolerance for industrial bioprocessing

• Improved protein expression systems for high-pressure bioreactors

• Novel approaches to stabilizing proteins for storage and transport

• Biomimetic designs for pressure-resistant synthetic biology applications

The mechanisms that allow P. profundum to thrive under pressure, including specific genes like RecD and membrane adaptations involving FabF , represent evolutionary solutions to extreme conditions that could inspire biotechnological innovations beyond their natural context.