Recombinant Pseudomonas putida UPF0345 protein PP_4248 (PP_4248)

What expression systems are most effective for recombinant production of PP_4248?

Recombinant PP_4248 can be expressed in various host systems, each offering distinct advantages depending on research objectives. E. coli and yeast systems generally provide higher yields and faster turnaround times, making them suitable for initial characterization studies . For applications requiring post-translational modifications, insect cells with baculovirus or mammalian expression systems may be preferable to ensure proper protein folding and biological activity retention .

When selecting an expression system, consider the following comparative factors:

For optimal heterologous expression in P. putida itself, strong native promoters should be considered. The rRNA gene promoters have demonstrated effective expression of heterologous genes in P. putida . The TREX expression system has also proven useful for establishing biosynthesis of secondary metabolites in heterologous hosts based on T7 RNA polymerase-dependent gene expression .

What purification strategies are most suitable for recombinant PP_4248?

Based on available protocols for similar recombinant proteins from P. putida, a general purification workflow would include:

• Initial cell lysis through sonication or mechanical disruption in an appropriate buffer system

• Clarification of lysate by centrifugation (typically 10,000-15,000 × g for 30 minutes)

• Affinity chromatography if the recombinant protein contains an affinity tag

• Size exclusion chromatography for further purification

• Concentration determination using established spectrophotometric methods

When working with recombinant PP_4248, it's recommended to briefly centrifuge the vial prior to opening to bring contents to the bottom . For reconstitution, using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL is advised, potentially with 5-50% glycerol addition for long-term storage .

How should recombinant PP_4248 be stored to maintain stability and activity?

The shelf life of recombinant proteins like PP_4248 is influenced by multiple factors including storage state, buffer composition, temperature, and the intrinsic stability of the protein itself . Standard storage recommendations include:

• Liquid formulations typically maintain stability for approximately 6 months at -20°C/-80°C

• Lyophilized forms generally remain stable for 12 months at -20°C/-80°C

• Working aliquots can be stored at 4°C for up to one week

• Repeated freezing and thawing should be avoided to prevent protein degradation

For optimal stability, consider aliquoting the protein with 50% glycerol as a cryoprotectant for long-term storage at -80°C .

What approaches can be used to determine the function of PP_4248 in P. putida's cellular processes?

While the specific function of PP_4248 remains to be fully characterized, several methodological approaches can elucidate its role:

• Comparative genomic analysis: Examining similar UPF0345 family proteins across Pseudomonas species to identify conserved domains and potential functions. This approach has successfully identified novel small proteins in P. putida KT2440 through bioinformatic and proteomic approaches .

• Gene knockout and phenotypic analysis: Systematically removing the gene encoding PP_4248 from P. putida and examining resulting phenotypic changes. This can reveal whether the protein is essential and what cellular processes it might influence.

• Protein-protein interaction studies: Using pull-down assays, yeast two-hybrid systems, or co-immunoprecipitation to identify binding partners of PP_4248. Validated small proteins in P. putida have been found to be located adjacent to annotated genes on the same strand and in close proximity to genes with known functions, including ABC transporter operons and transcriptional regulators involved in biofilm formation and biosynthesis pathways .

• Expression pattern analysis: Examining when and under what conditions PP_4248 is expressed can provide functional clues. Transcriptomic analysis has revealed that genome reduction in P. putida has global effects on transcript levels and gene expression modulation in response to environmental changes .

• Structural analysis: Determining the three-dimensional structure of PP_4248 through X-ray crystallography or NMR spectroscopy to gain insights into potential binding partners or enzymatic functions.

How can genetic engineering tools be optimized for manipulating PP_4248 expression in P. putida?

Several genetic tools have been developed specifically for heterologous protein expression in P. putida that can be applied to PP_4248 studies:

• Plasmid-based expression systems: Multiple plasmid systems have been developed specifically for P. putida, including pRGPDuo series vectors that offer different antibiotic resistance markers (gentamicin, kanamycin) and promoter options .

• Chromosomal integration: Random chromosomal integration using Tn5-based transposons can identify optimal genomic locations for gene expression. Notably, integration near ribosomal RNA genes has proven particularly effective for expressing heterologous genes in P. putida .

• Promoter selection: For constitutive expression, rRNA promoters have demonstrated high efficiency . For inducible expression, several options are available:

  • T7 RNA polymerase-dependent expression systems

  • Tac promoter (IPTG-inducible)

  • Tet promoter (tetracycline-inducible)

  • Bad promoter (arabinose-inducible)

• RBS optimization: Different ribosome binding site (RBS) variants have been tested in P. putida with significant impacts on protein expression levels. Several optimized RBS sequences are available for maximizing expression in P. putida KT2440 and related strains .

What strain engineering considerations are important when working with PP_4248 in P. putida?

The choice of P. putida strain can significantly impact recombinant protein expression and experimental outcomes:

The following table compares key characteristics of wild-type KT2440 and the genome-reduced EM42 strain:

What cultivation parameters should be optimized for maximum recombinant PP_4248 production?

Based on optimized protocols developed for other recombinant proteins in P. putida, the following cultivation parameters should be considered:

• Temperature: While P. putida typically grows optimally at 30°C, lower temperatures (20-25°C) often improve recombinant protein production by reducing aggregation and proteolytic degradation .

• Media composition: Rich media generally supports higher protein expression, but defined media may be necessary for specific experimental requirements. Nitrogen limitation can trigger certain metabolic pathways in P. putida .

• Aeration: High aeration conditions typically enhance recombinant protein production in P. putida. Standard shake flask cultures should maintain a filling volume/flask capacity ratio of approximately 1/10 .

• Induction parameters: For inducible promoter systems, the concentration of inducer and timing of induction significantly impact protein yields. For instance, with IPTG-inducible systems, concentrations between 0.1-1.0 mM are typically used, with induction occurring at mid-log phase.

• Batch vs. fed-batch cultivation: Fed-batch cultivation often yields higher recombinant protein concentrations. For example, in studies with recombinant P. putida, fed-batch cultures maintained substrate concentrations at ~20 g/L, resulting in significantly higher protein production compared to batch cultures .

How can structural and functional characterization of PP_4248 be approached?

A comprehensive characterization workflow for PP_4248 would include:

• Sequence analysis and bioinformatics:

  • Analysis of primary sequence for conserved domains

  • Homology modeling based on related structures if available

  • Prediction of potential binding partners or enzymatic functions

• Biochemical characterization:

  • Determination of oligomeric state (size exclusion chromatography, analytical ultracentrifugation)

  • Assessment of secondary structure (circular dichroism spectroscopy)

  • Thermal stability analysis (differential scanning fluorimetry)

  • Binding studies with potential cofactors or substrates

• Structural determination:

  • X-ray crystallography for high-resolution structure

  • NMR spectroscopy for solution structure and dynamics

  • Cryo-electron microscopy for large assemblies or membrane associations

• Functional assays:

  • In vitro enzymatic activity assays (if enzymatic function is predicted)

  • Protein-protein interaction studies (pull-downs, surface plasmon resonance)

  • In vivo complementation studies in knockout strains

What advanced genetic engineering approaches can be used to study PP_4248 function in vivo?

Beyond basic gene knockout approaches, several sophisticated genetic tools can provide insights into PP_4248 function:

• Conditional expression systems: Using tightly controlled inducible promoters to modulate PP_4248 expression levels and observe resulting phenotypic changes.

• Reporter fusions: Creating translational fusions with fluorescent proteins to monitor PP_4248 localization, expression patterns, and potential interactions with cellular structures.

• CRISPR-Cas9 genome editing: Precise modification of PP_4248 sequence to alter specific domains or residues, allowing structure-function relationship studies without complete protein removal.

• Transposon mutagenesis: Systematic disruption of genes to identify genetic interactions with PP_4248. This approach has been successfully used to identify novel small proteins in P. putida KT2440 .

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data to build comprehensive models of how PP_4248 fits into cellular networks. This approach has revealed that genome reduction in P. putida has global effects on transcript levels relative to wild-type strains .

How might PP_4248 be relevant to biotechnological applications of P. putida?

While specific functions of PP_4248 are still being investigated, small proteins in P. putida often play important roles in cellular processes relevant to biotechnology:

• Metabolic engineering: If PP_4248 is involved in metabolic regulation, understanding its function could enable more efficient production of valuable compounds in P. putida. For example, P. putida has been engineered to produce various substances from renewable resources, including polyhydroxyalkanoates from sucrose and prodigiosin .

• Stress response modulation: Small proteins often function in stress response pathways. If PP_4248 contributes to P. putida's notable stress tolerance, engineered variants might enhance the strain's performance in challenging industrial conditions.

• Biofilm formation: Some small proteins in P. putida are associated with transcriptional regulators involved in biofilm formation . If PP_4248 functions in this area, it could be relevant to applications where biofilm formation is either beneficial (e.g., bioremediation) or detrimental (e.g., biofouling).

• Synthetic biology applications: As a small protein with potentially diverse functions, engineered variants of PP_4248 might serve as modular components in synthetic biology applications, such as biosensors or cellular logic gates.

What methodological approaches can resolve conflicting data in PP_4248 research?

When faced with contradictory results in PP_4248 research, consider the following methodological approaches:

• Strain-specific variations: Confirm that identical P. putida strains were used across studies. Different strains (KT2440, EM42, W619, etc.) may exhibit divergent behaviors . Always verify the exact strain used and consider repeating key experiments in multiple strains.

• Expression system differences: Expression levels, fusion tags, and host backgrounds can dramatically affect recombinant protein properties. Standardize expression constructs when making direct comparisons, and consider testing multiple tag positions or tag-free constructs.

• Cultivation condition discrepancies: Subtle differences in media composition, temperature, or aeration can significantly impact P. putida physiology and protein expression. Develop standardized protocols with precise parameter reporting to ensure reproducibility.

• Technical variation in purification: Differences in purification protocols can yield protein preparations with varying levels of contaminants, denaturation, or aggregation. Employ multiple orthogonal purification methods and confirm protein homogeneity through analytical techniques like SEC-MALS or native PAGE.

• Functional context dependencies: Protein function may differ depending on cellular context or experimental conditions. Use complementary in vitro and in vivo approaches to build a comprehensive understanding of PP_4248 function under diverse conditions.