Recombinant Pseudomonas putida UPF0271 protein PP_2920 (PP_2920)

What is the UPF0271 protein PP_2920 in Pseudomonas putida?

The UPF0271 protein PP_2920 is an uncharacterized protein family member encoded in the Pseudomonas putida genome. It belongs to the UPF0271 protein family, which is typically characterized by proteins with unknown functions. In P. putida, this protein is encoded by the PP_2920 gene locus. Based on bioinformatic analysis, it shares structural features with other UPF0271 family members across bacterial species, though its precise biological function remains to be fully elucidated through experimental approaches .

What expression systems are recommended for recombinant production of PP_2920?

For recombinant expression of PP_2920, several expression systems have been successfully employed with P. putida proteins. Both constitutive and inducible expression systems can be used, with inducible systems generally yielding higher protein production. Based on research with P. putida, the XylS/Pm expression system inducible by 3-methylbenzoate (3-mBz) and the RhaRS/PrhaBAD module inducible by rhamnose have shown tight inducer-dependent regulation and high levels of gene expression . When comparing constitutive versus inducible expression, studies with other P. putida proteins have demonstrated that inducible systems typically provide better control and often result in higher protein yields .

What are the common challenges in expressing PP_2920 in heterologous hosts?

Expression of P. putida proteins in heterologous hosts often faces several challenges. These include:

• Codon bias differences between P. putida and common expression hosts like E. coli

• Potential toxicity of the expressed protein to the host cell

• Protein misfolding or aggregation in the cytoplasm

• Formation of inclusion bodies requiring refolding protocols

• Low expression levels or unstable protein products

To address these challenges, researchers typically optimize codon usage for the expression host, test different induction conditions (temperature, inducer concentration, and induction time), and explore various solubility-enhancing fusion tags such as MBP, SUMO, or GST. Additionally, co-expression with molecular chaperones may improve proper folding and solubility of the recombinant PP_2920 protein .

How can I detect recombinant PP_2920 expression in P. putida?

Detection of recombinant PP_2920 can be achieved through several complementary methods:

• Western blotting using antibodies against the protein or any fusion tags

• Mass spectrometry-based protein identification

• Fluorescence in situ hybridization (FISH) targeting both 23S rRNA of P. putida and the mRNA of the recombinant gene

• Confocal microscopy visualization if the protein is tagged with a fluorescent marker

For FISH-based detection, probes can be designed specifically targeting P. putida 23S rRNA sequences (labeled with one fluorophore) and the recombinant gene sequence (labeled with a different fluorophore). This dual-labeling approach enables specific detection of the recombinant strain expressing the target protein. The hybridization temperature should be optimized to ensure probe specificity with minimal non-specific binding .

What strategies can be employed to characterize the function of the uncharacterized PP_2920 protein?

Characterizing the function of uncharacterized proteins like PP_2920 requires a multifaceted approach:

• Structural analysis: Determination of protein structure through X-ray crystallography, NMR, or cryo-EM to identify structural motifs that may suggest function

• Comparative genomics: Analysis of genomic context and conservation patterns across different bacterial species

• Protein-protein interaction studies: Yeast two-hybrid, pull-down assays, or co-immunoprecipitation to identify interaction partners

• Gene knockout/knockdown studies: CRISPR-Cas9 or homologous recombination to create PP_2920 deletion mutants and observe phenotypic changes

• Transcriptomics and proteomics: RNA-seq and quantitative proteomics to identify genes/proteins with correlated expression patterns

• Biochemical activity screening: Testing the protein against libraries of potential substrates to identify enzymatic activities

• Localization studies: Fluorescent protein tagging to determine subcellular localization

A combination of these approaches typically yields the most comprehensive functional characterization .

How does the solvent tolerance of P. putida affect the expression and stability of recombinant PP_2920?

P. putida is known for its exceptional solvent tolerance, making it an excellent host for expressing proteins that may be involved in pathways generating potentially toxic compounds. Research has shown that P. putida can grow in the presence of high concentrations of solvents like 2-pentanone (up to 25-50 g/L), significantly outperforming other bacterial hosts like E. coli .

This solvent tolerance may affect recombinant protein expression and stability in several ways:

• Enhanced protein stability: Proteins expressed in P. putida may possess inherent stability characteristics adapted to function in the presence of solvents

• Modified membrane composition: P. putida's adaptive membrane modifications in response to solvents may create a more favorable environment for membrane-associated proteins

• Stress response mechanisms: The activation of stress response pathways in P. putida may lead to increased expression of chaperones that aid in proper protein folding

• Post-translational modifications: Solvent exposure may influence post-translational modifications of the recombinant protein

The table below summarizes the growth of P. putida compared to E. coli in the presence of different concentrations of 2-pentanone:

This exceptional solvent tolerance makes P. putida especially valuable for expressing proteins that may be involved in pathways producing potentially toxic compounds .

What are the optimal conditions for long-term stability and activity of purified recombinant PP_2920?

While specific data for PP_2920 is limited, research on other P. putida proteins suggests several strategies for maintaining long-term stability and activity:

• Buffer optimization: Testing various buffer compositions (pH, ionic strength, additives) to identify conditions that maximize protein stability

• Storage conditions: Evaluating different temperatures (-80°C, -20°C, 4°C) and the impact of freeze-thaw cycles

• Stabilizing additives: Testing the effects of glycerol, reducing agents (DTT, β-mercaptoethanol), and specific ligands or co-factors

• Lyophilization: Assessing whether lyophilization with appropriate excipients preserves activity

• Engineering approaches: Introduction of disulfide bonds or surface mutations to enhance thermostability

For many recombinant proteins from P. putida, storage at -80°C in buffer containing 10-25% glycerol and appropriate reducing agents typically provides good stability. Activity assays should be performed periodically to monitor potential loss of function during storage .

How can I design a minicell-based system for studying PP_2920 function?

P. putida minicells represent an innovative platform for studying protein function, particularly for proteins that may be involved in toxic metabolic pathways. Based on recent advances with P. putida minicells, the following approach could be implemented for PP_2920:

• Generation of minicell-producing P. putida: Modify the strain to produce chromosome-free bacterial vesicles through genetic engineering approaches targeting cell division genes

• PP_2920 expression optimization: Test both constitutive and inducible expression systems (such as XylS/Pm or RhaRS/PrhaBAD) to identify the optimal expression conditions in minicells

• Minicell purification: Implement a 3-step purification protocol to isolate minicells containing the recombinant PP_2920 protein

• Functional assays: Design assays to test potential functions in the isolated minicells

• Long-term stability assessment: Evaluate the functional stability of PP_2920 in minicells over extended timeframes

P. putida minicells have been shown to remain catalytically functional for over 4 months in some studies, making them excellent vehicles for long-term studies of protein function. This approach is particularly valuable for proteins that may be involved in pathways generating toxic compounds, as the minicells provide a contained environment without the constraints of maintaining cell viability .

What approaches can be used to resolve contradictory data in PP_2920 functional studies?

When faced with contradictory results in PP_2920 functional studies, researchers should implement a systematic approach:

• Replication with standardized protocols: Ensure methods are standardized across laboratories with detailed protocol sharing

• Independent validation: Have multiple laboratories test the same hypothesis using different techniques

• Strain and construct verification: Confirm the genetic background of all strains and the sequence of all constructs used

• Controlled environmental conditions: Test whether different growth conditions influence results

• Hypothesis refinement: Consider whether contradictory results might actually reflect different aspects of a more complex function

• Statistical analysis: Apply rigorous statistical methods to determine significance of observed differences

• Meta-analysis: Compile all available data to identify patterns and potential sources of variation

The table below outlines a step-by-step approach for resolving contradictory data:

This systematic approach ensures that contradictions are addressed methodically rather than being dismissed or ignored .

What purification protocol is recommended for recombinant PP_2920?

A comprehensive purification protocol for recombinant PP_2920 typically involves the following steps:

• Expression optimization: Test different expression systems with variable induction parameters (temperature, inducer concentration, duration)

• Cell lysis: Compare mechanical methods (sonication, French press) with chemical lysis methods to identify the approach that maintains protein integrity

• Initial clarification: Centrifugation at 10,000-15,000 × g for 30-45 minutes to remove cell debris

• Affinity chromatography: If the protein contains an affinity tag (His, GST, MBP), use the appropriate affinity resin

• Tag removal: If applicable, cleave the affinity tag using a specific protease (TEV, PreScission, etc.)

• Secondary purification: Ion exchange chromatography based on the predicted isoelectric point of PP_2920

• Polishing step: Size exclusion chromatography to achieve high purity and remove aggregates

• Quality control: SDS-PAGE, Western blot, and mass spectrometry to confirm identity and purity

• Activity verification: Functional assays based on predicted or experimentally determined activities

For difficult-to-express proteins, co-expression with molecular chaperones may improve solubility and yield. Additionally, optimizing buffer conditions (pH, salt concentration, additives) at each step can significantly improve purification outcomes .

How can I develop a specific antibody against PP_2920 for immunodetection?

Developing specific antibodies against PP_2920 involves the following methodological approach:

• Antigen preparation: Options include:

  • Full-length recombinant PP_2920 protein (if soluble)

  • Specific peptide fragments (15-20 amino acids) from hydrophilic, surface-exposed regions

  • Fusion proteins containing PP_2920 epitopes

• Immunization strategy:

  • Select appropriate animal model (rabbit, mouse, guinea pig) based on quantity of antibody needed

  • Design immunization schedule with primary and multiple boost injections

  • Use appropriate adjuvants to enhance immune response

• Antibody purification:

  • For polyclonal antibodies: Affinity purification using immobilized PP_2920

  • For monoclonal antibodies: Hybridoma screening and clonal selection

• Validation:

  • Western blot against recombinant PP_2920 and P. putida lysates

  • Immunoprecipitation to confirm specificity

  • Immunofluorescence to verify recognition of native protein

  • Testing against knockout/knockdown strains as negative controls

• Cross-reactivity assessment:

  • Test against closely related proteins or P. putida strains with different genetic backgrounds

  • Evaluate specificity across different bacterial species

Optimizing antibody conditions (dilution, incubation time, blocking agents) for each application is crucial for successful immunodetection of PP_2920 .

What methods are most effective for studying protein-protein interactions involving PP_2920?

Multiple complementary approaches should be employed to robustly identify and characterize protein-protein interactions involving PP_2920:

• In vivo approaches:

  • Bacterial two-hybrid systems adapted for P. putida

  • Split-protein complementation assays (e.g., split-GFP)

  • Fluorescence resonance energy transfer (FRET)

  • In vivo crosslinking followed by mass spectrometry

• In vitro approaches:

  • Pull-down assays using tagged recombinant PP_2920

  • Co-immunoprecipitation with PP_2920-specific antibodies

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Computational predictions:

  • Protein-protein interaction predictions based on structural modeling

  • Genomic context and gene neighborhood analyses

  • Co-evolution analysis to identify potential interacting partners

• Validation strategies:

  • Reciprocal pull-downs or co-immunoprecipitations

  • Mutational analysis of interaction interfaces

  • Functional assays to test the biological relevance of identified interactions

Each method has specific strengths and limitations, so combining multiple approaches provides more reliable identification of true interaction partners and reduces false positives .

How can I design knockout experiments to investigate PP_2920 function in P. putida?

Designing effective knockout experiments for PP_2920 requires careful planning and multiple controls:

• Knockout strategy selection:

  • Homologous recombination with selection markers

  • CRISPR-Cas9-based genome editing

  • Transposon mutagenesis followed by screening

• Design considerations:

  • Complete gene deletion vs. truncation or point mutations

  • Analysis of potential polar effects on adjacent genes

  • Consideration of genomic context and operon structures

• Validation of knockout strains:

  • PCR verification of deletion/modification

  • RT-PCR to confirm absence of transcript

  • Western blot to confirm absence of protein

  • Whole-genome sequencing to identify potential off-target effects

• Phenotypic characterization:

  • Growth curves under different conditions (carbon sources, stressors)

  • Metabolic profiling to identify altered metabolic pathways

  • Transcriptomic analysis to identify compensatory responses

  • Specific assays based on predicted functions

• Complementation studies:

  • Reintroduction of wild-type PP_2920

  • Expression of PP_2920 homologs from other species

  • Structure-function analysis using mutant variants

This systematic approach helps establish causality between PP_2920 deletion and observed phenotypes, while controlling for potential secondary effects of genetic manipulation .

How can high-throughput approaches be leveraged to characterize PP_2920 function?

High-throughput approaches offer powerful methods to accelerate the functional characterization of PP_2920:

• Metabolic profiling:

  • Untargeted metabolomics comparing wild-type and PP_2920 knockout strains

  • Stable isotope labeling to track metabolic flux changes

  • Testing growth on Biolog phenotype microarrays covering hundreds of carbon sources

• Transcriptomic analysis:

  • RNA-seq to identify genes differentially expressed in PP_2920 mutants

  • ChIP-seq if PP_2920 is suspected to have DNA-binding properties

  • Time-course experiments to capture dynamic responses

• Proteomic approaches:

  • Quantitative proteomics to identify changes in protein abundance

  • Phosphoproteomics to detect alterations in signaling pathways

  • Thermal proteome profiling to identify potential binding partners

• High-content screening:

  • Fluorescent reporter assays in multi-well format

  • Automated microscopy to detect phenotypic changes

  • CRISPR interference screens to identify genetic interactions

• Computational integration:

  • Network analysis of multi-omics data

  • Machine learning to identify patterns across datasets

  • Structural modeling integrated with experimental data

The table below summarizes key high-throughput approaches and their applications:

Integration of multiple high-throughput datasets typically provides the most comprehensive insights into protein function .

What strategies can be employed to engineer PP_2920 for enhanced function or novel applications?

Engineering PP_2920 for enhanced function or novel applications involves several strategic approaches:

• Rational design based on structural information:

  • Site-directed mutagenesis of key residues identified through structural analysis

  • Domain swapping with homologous proteins

  • Introduction of disulfide bonds for enhanced stability

• Directed evolution approaches:

  • Error-prone PCR to generate libraries of PP_2920 variants

  • DNA shuffling with homologous genes

  • PACE (Phage-Assisted Continuous Evolution) if a selectable phenotype can be established

• Computational design methods:

  • In silico prediction of stabilizing mutations

  • Computational design of new catalytic activities

  • Protein-protein interaction interface design

• Fusion protein strategies:

  • Creation of chimeric proteins with complementary functions

  • Addition of targeting sequences for specific subcellular localization

  • Incorporation of reporter domains for activity monitoring

• Testing and validation:

  • High-throughput screening of variant libraries

  • Detailed biochemical characterization of promising variants

  • In vivo testing in different P. putida strains and conditions

These engineering approaches can be applied iteratively, with each round of modifications informed by the results of previous experiments. The most successful engineering strategies typically combine rational design with directed evolution approaches .

How can systems biology approaches integrate PP_2920 function into broader metabolic networks?

Systems biology offers powerful frameworks for understanding how PP_2920 functions within broader cellular networks:

• Genome-scale metabolic modeling:

  • Integration of PP_2920 into existing P. putida metabolic models

  • Flux balance analysis to predict metabolic impacts of PP_2920 modulation

  • Identification of synthetic lethal interactions through in silico gene knockouts

• Multi-omics data integration:

  • Correlation analysis across transcriptomic, proteomic, and metabolomic datasets

  • Bayesian network analysis to infer causal relationships

  • Identification of co-regulated gene clusters suggesting functional relationships

• Regulatory network reconstruction:

  • ChIP-seq or DNA affinity purification sequencing if PP_2920 has DNA-binding properties

  • Analysis of transcription factor binding sites in PP_2920 promoter region

  • Inference of regulatory relationships from time-course expression data

• Protein interaction networks:

  • Placement of PP_2920 within the broader protein-protein interaction network

  • Identification of functional modules containing PP_2920

  • Analysis of network perturbations upon PP_2920 deletion or overexpression

• Experimental validation of predictions:

  • Testing of computationally predicted interactions or functions

  • Targeted metabolic engineering based on model predictions

  • Iterative refinement of models based on experimental results

Systems biology approaches are particularly valuable for understanding proteins with complex or multifunctional roles that cannot be fully characterized through reductionist approaches alone .

What considerations are important when scaling up recombinant PP_2920 production for structural studies?

Scaling up PP_2920 production for structural studies requires careful optimization at each stage:

• Expression system optimization:

  • Comparison of different P. putida strains as expression hosts

  • Evaluation of alternative hosts (E. coli, yeast) if P. putida presents challenges

  • Testing different promoters, including inducible systems like XylS/Pm and RhaRS/PrhaBAD that have shown high expression levels in P. putida

• Culture conditions:

  • Optimization of media composition (carbon sources, nitrogen sources, trace elements)

  • Testing different induction parameters (timing, concentration, temperature)

  • Scale-up considerations from shake flasks to bioreactors

• Protein solubility enhancement:

  • Fusion tags specifically selected for structural biology applications

  • Co-expression with molecular chaperones

  • Addition of specific solubilizing agents during expression

• Purification process development:

  • Multi-step purification strategy optimization

  • Scale-appropriate chromatography approaches

  • Quality control metrics specific to structural biology requirements

• Sample preparation for structural studies:

  • Buffer screening for optimal protein stability

  • Concentration methods that avoid aggregation

  • Assessment of sample homogeneity by dynamic light scattering

• Crystallization or NMR sample optimization:

  • For X-ray crystallography: Crystallization condition screening

  • For NMR: Isotope labeling strategies (15N, 13C, 2H)

  • For cryo-EM: Grid preparation and vitrification conditions

Careful attention to each of these aspects is essential for producing the high-quality, homogeneous protein samples required for successful structural studies .

How can cutting-edge imaging techniques be applied to study PP_2920 localization and dynamics in P. putida?

Advanced imaging techniques offer powerful tools for studying PP_2920 localization and dynamics:

• Fluorescent protein tagging strategies:

  • C-terminal vs. N-terminal fusions to minimize functional disruption

  • Selection of appropriate fluorescent proteins (mScarlet, mNeonGreen, mTurquoise2)

  • Sandwich fusions if terminal tagging affects function

• Super-resolution microscopy approaches:

  • Stimulated emission depletion (STED) microscopy

  • Single-molecule localization microscopy (PALM/STORM)

  • Structured illumination microscopy (SIM)

• Live-cell imaging techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure mobility

  • Single-particle tracking to follow individual protein molecules

  • Fluorescence correlation spectroscopy (FCS) for concentration and diffusion measurements

• Multi-color imaging for co-localization:

  • Simultaneous labeling of PP_2920 and potential interaction partners

  • Organelle markers to determine subcellular localization

  • Quantitative co-localization analysis

• Advanced fluorescence techniques:

  • Fluorescence resonance energy transfer (FRET) to detect protein-protein interactions

  • Fluorescence lifetime imaging microscopy (FLIM) for quantitative FRET measurements

  • Split fluorescent protein complementation to visualize protein interactions

• Integration with other techniques:

  • Correlative light and electron microscopy (CLEM)

  • Fluorescence in situ hybridization (FISH) to correlate protein localization with mRNA

  • Optogenetic approaches to manipulate protein function with light

These imaging approaches can reveal dynamic aspects of PP_2920 function that are inaccessible through biochemical or genetic approaches alone, providing crucial insights into its cellular role .

What are the most promising future research directions for PP_2920?

Based on current understanding of P. putida proteins and uncharacterized protein families, several promising research directions for PP_2920 emerge:

• Structural genomics approaches: Determining the three-dimensional structure of PP_2920 would provide crucial insights into its potential function and evolutionary relationships.

• Systems-level characterization: Integration of PP_2920 into genome-scale metabolic models and protein interaction networks of P. putida to understand its role in cellular physiology.

• Comparative genomics: Comprehensive analysis of PP_2920 homologs across different bacterial species to identify conserved features and potential functional clues.

• Application in biotechnology: Exploration of PP_2920's potential roles in P. putida's exceptional environmental stress tolerance and metabolic versatility.

• Genetic interaction mapping: Systematic creation of double mutants combining PP_2920 deletion with other genes to identify functional relationships.

These research directions complement each other and would collectively advance our understanding of this uncharacterized protein and potentially reveal new aspects of P. putida biology that could be leveraged for biotechnological applications .

How might PP_2920 contribute to the unique properties of P. putida as a biotechnological chassis?

P. putida has emerged as a valuable biotechnological chassis due to its metabolic versatility, stress tolerance, and ability to grow on diverse carbon sources. PP_2920 may contribute to these properties in several ways:

• Solvent tolerance mechanisms: If PP_2920 is involved in P. putida's exceptional tolerance to organic solvents (where it can grow in concentrations of chemicals that are lethal to other bacteria) , it could be a valuable target for enhancing bioproduction of toxic compounds.

• Metabolic robustness: PP_2920 might play a role in the metabolic flexibility that allows P. putida to efficiently utilize diverse carbon sources, including organic acids like butyrate .

• Stress response pathways: If PP_2920 participates in stress response mechanisms, understanding its function could lead to engineering more robust strains for industrial applications.

• Novel enzymatic activities: As an uncharacterized protein, PP_2920 may possess novel enzymatic activities that could be harnessed for biocatalysis applications.

• Minicell functionality: If PP_2920 influences minicell formation or function, it might be relevant to the development of P. putida minicell technology, which has shown promise for long-term (>4 months) biocatalytic applications .