Recombinant Photobacterium profundum Elongation factor P (efp)

What is Elongation Factor P (EFP) and what is its functional significance in bacteria?

Elongation Factor P (EFP) is a specialized translation factor that stimulates the peptidyltransferase activity of fully assembled 70S prokaryotic ribosomes. EFP specifically enhances the synthesis of certain dipeptides initiated by N-formylmethionine and prevents ribosomal stalling during the synthesis of proteins containing consecutive proline residues (such as PPG, PPP, or longer proline clusters) . This function is evolutionarily conserved, with homologous proteins found in eukaryotes (eIF5A) and archaea (aIF5A) .

Research has demonstrated that EFP is essential for bacterial viability and protein synthesis, making it a critical component of the bacterial translation machinery . The efficient and coordinated synthesis of proteins containing polyproline stretches is required for various bacterial functions including growth, motility, virulence, and stress response mechanisms .

How can Photobacterium profundum EFP be characterized compared to other bacterial EFPs?

Photobacterium profundum is a deep-sea bacterium that grows optimally at 28 MPa (approximately 280 atmospheres) and 15°C . This piezophilic (pressure-loving) nature makes its cellular components, including EFP, potentially adapted to high-pressure environments. P. profundum SS9 serves as a model organism for studying piezophily due to its ability to grow under both atmospheric and high-pressure conditions, allowing for easier genetic manipulation and culture compared to obligate piezophiles .

While the general structure and function of EFP are conserved across bacterial species, potential adaptations in P. profundum EFP might contribute to protein synthesis efficiency under high hydrostatic pressure conditions. Comparative studies examining the structural and functional properties of P. profundum EFP versus EFPs from non-piezophilic bacteria would provide insights into pressure-adaptive mechanisms in translation factors.

What expression systems are typically used for recombinant EFP production?

Recombinant Elongation Factor P can be expressed and purified from various host systems, each offering distinct advantages:

What are the optimal conditions for expressing recombinant P. profundum EFP in E. coli systems?

When expressing recombinant P. profundum EFP in E. coli, several parameters should be optimized:

Temperature: Since P. profundum is a psychrophilic organism, expressing its proteins at lower temperatures (15-18°C) rather than standard E. coli growth temperatures (37°C) may improve proper folding and solubility. Evidence from similar cold-adapted bacteria suggests that cultivation at 18°C can yield higher amounts of recombinant protein than at 4°C, while still ensuring proper folding .

Induction conditions: For T7-based systems, IPTG concentration should be optimized (typically 0.1-1.0 mM), with induction at lower cell densities (OD600 of 0.2-0.8) followed by extended expression periods (12h at 18°C) .

Codon optimization: Considering the GC content differences between P. profundum and E. coli, codon optimization of the efp gene sequence may improve expression levels.

What purification strategies yield the highest purity and activity of recombinant P. profundum EFP?

A multi-step purification approach is recommended:

• Affinity chromatography: A His-tag fusion construct allows for initial purification using Ni-NTA or IMAC columns. Imidazole gradient elution (20-250 mM) should be optimized to prevent co-purification of contaminants.

• Ion exchange chromatography: As a second step, anion or cation exchange (depending on the calculated pI of P. profundum EFP) can remove remaining impurities.

• Size exclusion chromatography: A final polishing step to ensure homogeneity and remove any aggregates.

Throughout purification, maintaining conditions that preserve the native state is crucial. For P. profundum proteins, consider:

• Including stabilizing agents (glycerol 5-10%)

• Maintaining lower temperatures during purification (4°C)

• Using buffers that mimic physiological conditions

• Testing the effects of moderate pressure during storage

Activity assays should be performed after each purification step to ensure functionality is maintained. For EFP, this typically involves in vitro translation assays measuring the synthesis of polyproline-containing peptides.

How can post-translational modifications of P. profundum EFP be identified and characterized?

Post-translational modifications (PTMs) of bacterial EFPs include β-lysinylation, rhamnosylation, or 5-aminopentanolyation, which contribute to their catalytic proficiency . To identify PTMs in P. profundum EFP:

Mass spectrometry-based approaches:

• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) after tryptic digestion

• Top-down proteomics using intact protein mass spectrometry

• Electron transfer dissociation (ETD) for PTM site localization

Biochemical assays:

• Specific antibodies against known EFP modifications

• Chemical labeling strategies for specific PTM types

• Comparative analysis with known modified and unmodified EFP variants

It's worth noting that some bacterial EFPs belonging to the PGKGP-subfamily remain unmodified yet functional . Determining whether P. profundum EFP requires modification or functions in an unmodified state would provide valuable insights into its mechanism of action under high-pressure conditions.

How does hydrostatic pressure affect the structure and function of P. profundum EFP?

As a piezophilic organism, P. profundum has evolved molecular adaptations to function optimally under high hydrostatic pressure. To investigate pressure effects on EFP:

Structural analysis under pressure:

• High-pressure NMR spectroscopy to monitor conformational changes

• High-pressure X-ray crystallography

• Molecular dynamics simulations incorporating pressure effects

Functional assays under pressure:

• In vitro translation systems equipped with high-pressure cells

• Comparative activity measurements at atmospheric versus high pressure (28 MPa)

• Assessment of EFP-ribosome interactions under varying pressure conditions

Previous proteomic analyses of P. profundum grown at different pressures have shown differential expression of proteins involved in various metabolic pathways . Similar approaches could reveal whether EFP itself is differentially expressed or modified in response to pressure changes, providing insights into its role in pressure adaptation.

What roles does EFP play in the stress response and virulence of P. profundum?

Elongation Factor P facilitates the translation of proteins containing polyproline stretches, many of which are involved in bacterial stress response and virulence mechanisms . To investigate these roles in P. profundum:

Genetic approaches:

• Construction of efp gene disruption mutants using techniques similar to those employed for recD mutants in P. profundum

• Complementation studies with wild-type and mutant efp genes

• Conditional expression systems to control EFP levels

Phenotypic analyses:

• Growth curves under various stressors (pressure, temperature, pH, oxidative stress)

• Biofilm formation and motility assays

• Comparative proteomics between wild-type and efp mutants under stress conditions

Understanding the specific proteins whose translation depends on EFP in P. profundum would provide a mechanistic link between this translation factor and the organism's ability to adapt to extreme deep-sea environments.

How does recombinant P. profundum EFP compare functionally to EFP from non-piezophilic bacteria in heterologous systems?

Comparative functional analysis:

Cross-complementation experiments would be particularly informative, testing whether P. profundum EFP can functionally replace EFP in mesophilic bacteria (like E. coli) and vice versa, especially under different pressure and temperature conditions.

What are common pitfalls in expressing recombinant P. profundum proteins and how can they be addressed?

Expression challenges for psychrophilic and piezophilic proteins include:

Protein misfolding: The cold-adapted nature of P. profundum proteins may lead to misfolding at standard E. coli growth temperatures.

• Solution: Lower expression temperatures (15-18°C), co-expression with cold-adapted chaperones, or using psychrophilic expression hosts like Shewanella livingstonensis Ac10 .

Low solubility: Hydrostatic pressure adaptations may affect protein solubility under atmospheric conditions.

• Solution: Addition of stabilizing agents (glycerol, specific ions), fusion tags that enhance solubility (MBP, SUMO), or expression under mild pressure conditions.

Codon usage bias: Differences in codon preference between P. profundum and expression hosts.

• Solution: Codon optimization or use of strains with supplementary tRNAs for rare codons.

Plasmid stability issues: Some recombinant constructs may show reduced stability in standard hosts.

• Solution: Consider using hosts with recD mutations that improve plasmid stability, similar to those described for P. profundum itself .

How can functional assays for EFP activity be optimized for P. profundum EFP?

Standard EFP functional assays measure the ability to enhance translation of polyproline-containing peptides. For P. profundum EFP, these assays should be adapted:

Temperature considerations:

• Conduct assays at lower temperatures (4-15°C) in addition to standard temperatures

• Compare activity profiles across temperature ranges

Pressure adaptations:

• Develop high-pressure in vitro translation systems

• Compare activity at atmospheric pressure versus 28 MPa (optimal growth pressure)

Substrate selection:

• Identify P. profundum proteins with polyproline stretches as natural substrates

• Design reporter constructs containing these specific sequences

Control selections:

• Include EFPs from mesophilic and other psychrophilic bacteria as comparative controls

• Test chimeric EFPs to identify pressure/cold-adaptive domains

What strategies can overcome heterogeneity in post-translational modifications of recombinant P. profundum EFP?

Heterogeneity in post-translational modifications can compromise experimental reproducibility:

Co-expression with modification enzymes:

• Identify and co-express the genes responsible for EFP modification in P. profundum

• Optimize stoichiometry between EFP and modifying enzymes

Homogeneous preparation strategies:

• In vitro enzymatic modification of purified unmodified EFP

• Separation of modification variants using chromatographic techniques

• Site-directed mutagenesis to create modification-mimicking variants

If P. profundum EFP belongs to the unmodified PGKGP-subfamily , focus should shift to ensuring proper folding rather than modification state. In this case, structural characterization (circular dichroism, limited proteolysis) would be more relevant than PTM analysis.

How might comparative genomics and proteomics inform our understanding of P. profundum EFP evolution?

Evolutionary analyses can provide insights into pressure adaptation mechanisms:

Sequence analysis approaches:

• Phylogenetic comparison of EFP sequences across marine bacteria from different depths

• Identification of positively selected residues correlated with pressure adaptation

• Domain architecture analysis comparing EFP across the pressure gradient

Proteomics strategies:

• Quantitative proteomics of P. profundum grown at different pressures to assess EFP expression levels and modification states

• Interactome studies to identify pressure-specific EFP binding partners

• Analysis of polyproline-containing proteins differentially expressed under pressure

These approaches could reveal whether P. profundum EFP has undergone specific adaptations for high-pressure environments and identify which features are conserved versus divergent compared to EFPs from surface-dwelling bacteria.

What potential biotechnological applications might emerge from studies of P. profundum EFP?

Understanding the pressure-adaptive features of P. profundum EFP could lead to several applications:

Enhanced protein expression systems:

• Development of pressure-tolerant cell-free translation systems incorporating P. profundum EFP

• Improved expression of difficult-to-produce proteins containing polyproline stretches

• Creation of expression hosts with optimized translation machinery for psychrophilic proteins

Biocatalyst development:

• Design of pressure-stable enzymes using principles derived from P. profundum proteins

• Creation of chimeric translation factors with enhanced stability under non-standard conditions

Antimicrobial strategies:

• Since EFP modification enzymes are absent in higher eukaryotes, they represent potential targets for antimicrobial development

• Comparative studies between P. profundum and pathogenic bacteria could identify specific inhibitors of EFP function or modification