Recombinant Pseudomonas putida Maf-like protein PP_0936 (maf-1)

What expression systems are most effective for recombinant production of PP_0936 (maf-1)?

For recombinant production of P. putida proteins like PP_0936 (maf-1), several expression systems have demonstrated effectiveness:

• E. coli-based expression systems: Similar to the approach used for MAF-1 from Musca domestica, prokaryotic expression vectors like pET-28a(+) with BL21(DE3) competent cells can be utilized . This system allows for His-tag fusion protein production, facilitating subsequent purification.

• P. putida KT2440 as a homologous expression host: Recent genetic tools developed specifically for P. putida enable reliable gene expression within its native context . This approach may preserve natural protein folding and post-translational modifications.

• Inducible promoter systems: When using P. putida as an expression host, modified IPTG-inducible promoters with optimized LacI repressor expression can achieve up to 80-fold induction, significantly improving controlled expression of target proteins .

How can the solubility and stability of recombinant PP_0936 (maf-1) be optimized during expression?

To optimize solubility and stability of recombinant PP_0936 (maf-1):

• Temperature modulation: Lowering induction temperature (16-25°C) often increases the proportion of soluble protein by slowing expression rate and allowing proper folding.

• Co-expression with chaperones: Introduction of molecular chaperones can assist in proper protein folding.

• Fusion partners selection: Beyond simple His-tags, larger solubility-enhancing fusion partners (MBP, SUMO, or TrxA) can significantly improve soluble protein yields.

• Media optimization: Complex media supplements (like tryptone or yeast extract) at 1-5% concentrations can increase protein stability.

• Buffer optimization during purification: Including stabilizing agents (glycerol 5-10%, reducing agents) in purification buffers helps maintain protein integrity.

Experimental determination of optimal conditions through small-scale expression trials is essential before scaling up production.

What are the most effective purification strategies for recombinant PP_0936 (maf-1)?

For efficient purification of recombinant PP_0936 (maf-1), a multi-step approach is recommended:

• Affinity chromatography: If expressed with a His-tag, purification using Ni-NTA affinity chromatography represents an effective initial capture step, similar to methods used for other recombinant proteins . Typical binding conditions include 20-50 mM imidazole in the binding buffer, with elution achieved using 250-500 mM imidazole.

• Secondary purification: Following affinity purification, ion exchange chromatography (selecting the appropriate resin based on the protein's theoretical pI) and/or size exclusion chromatography can remove remaining contaminants and aggregates.

• Tag removal considerations: If protein function might be affected by the presence of purification tags, inclusion of a specific protease cleavage site (TEV or PreScission) allows tag removal under native conditions.

The purification protocol should be validated using SDS-PAGE and Western blotting to confirm identity and purity at each stage.

What analytical methods are most informative for characterizing the structural properties of purified PP_0936 (maf-1)?

For comprehensive structural characterization of purified PP_0936 (maf-1):

• Circular Dichroism (CD) Spectroscopy: Provides information on secondary structure composition (α-helices, β-sheets) and can assess thermal stability through temperature-dependent measurements.

• Dynamic Light Scattering (DLS): Determines the hydrodynamic radius and oligomeric state in solution, while also identifying potential aggregation.

• Limited Proteolysis coupled with Mass Spectrometry: Identifies stable domains and flexible regions within the protein structure.

• NMR Spectroscopy: For detailed atomic-level structural characterization if isotopically labeled protein can be produced.

• X-ray Crystallography: Provides high-resolution structural data if diffraction-quality crystals can be obtained.

These complementary techniques collectively provide a comprehensive structural profile that can inform subsequent functional studies.

How can researchers verify the identity and integrity of purified recombinant PP_0936 (maf-1)?

To verify identity and integrity of purified recombinant PP_0936 (maf-1):

• Western Blotting: Using antibodies specific to either the protein itself or the fusion tag (e.g., anti-His antibodies) . A polyclonal antibody approach similar to that used for MAF-1 fusion protein can be employed.

• Mass Spectrometry Analysis:

  • Intact protein MS to confirm molecular weight

  • Peptide mass fingerprinting following tryptic digestion to confirm sequence identity

  • Top-down proteomics to identify post-translational modifications

• N-terminal Sequencing: Edman degradation to confirm the N-terminal sequence matches the expected sequence.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): Verifies protein homogeneity and molecular weight in solution.

A combination of these techniques provides robust validation of protein identity and integrity before proceeding to functional studies.

What approaches can be used to determine the function of PP_0936 (maf-1) in P. putida?

To elucidate the function of PP_0936 (maf-1) in P. putida:

• Comparative genomics and bioinformatics analysis: Identifying conserved domains, structural motifs, and phylogenetic relationships with functionally characterized Maf proteins.

• Gene knockout/knockdown studies: Creating deletion mutants using λRed/Cas9 recombineering methods developed for P. putida KT2440 . This scarless mutation approach allows assessment of phenotypic changes without marker interference.

• Protein-protein interaction studies:

  • Pull-down assays using tagged PP_0936

  • Bacterial two-hybrid systems

  • Co-immunoprecipitation followed by mass spectrometry

• Transcriptomics analysis: RNA-seq comparing wild-type and ΔPP_0936 mutant strains under various conditions to identify affected pathways.

• Metabolomics profiling: Identifying metabolic changes in knockout mutants that might indicate biochemical pathways involving PP_0936.

The integration of multiple approaches provides the most comprehensive functional characterization.

How does the oxygen sensitivity of PP_0936 (maf-1) compare to other regulatory proteins in P. putida?

While specific oxygen sensitivity data for PP_0936 (maf-1) is limited, insights can be drawn from studies of other P. putida regulatory proteins:

• Comparison with FNR proteins: P. putida contains multiple oxygen-sensing transcription factors with varying oxygen sensitivities. For example, the ANR protein contains [4Fe-4S] clusters that react rapidly with O₂, while PP_3233 and PP_3287 show slower reactivity . These differential oxygen sensitivities allow for graduated responses to changing oxygen levels.

• Experimental approaches to determine oxygen sensitivity:

  • Spectroscopic analysis to detect potential iron-sulfur clusters or other oxygen-sensitive cofactors

  • Activity assays under varying oxygen concentrations

  • Structural stability assessments in aerobic versus anaerobic conditions

• Functional implications: Differential oxygen sensitivity among regulatory proteins enables P. putida to adapt to changing environmental conditions, potentially affecting gene expression patterns relevant to metabolism and stress responses.

Experimental determination of PP_0936 oxygen sensitivity would add valuable insights to understanding its regulatory role in P. putida.

What role might PP_0936 (maf-1) play in P. putida's stress response or metabolic regulation?

Based on analysis of other bacterial Maf proteins and context from P. putida studies:

• Potential roles in stress response:

  • Maf proteins in other bacteria have been implicated in responses to various stressors including oxidative stress and nutrient limitation

  • PP_0936 expression patterns may correlate with specific stress conditions, as suggested by transcriptomic data

• Metabolic regulation connections:

  • P. putida's metabolic versatility requires sophisticated regulatory networks

  • PP_0936 may participate in regulatory cascades affecting carbon metabolism or aromatic compound degradation pathways, which are hallmarks of P. putida metabolism

• Biofilm formation and cell morphology:

  • Some Maf proteins affect cell division and morphology

  • PP_0936 might influence biofilm development, an important stress adaptation mechanism

• Experimental approaches to test these hypotheses:

  • Phenotypic characterization of ΔPP_0936 mutants under various stress conditions

  • Transcriptomic profiling to identify genes co-regulated with PP_0936

  • Metabolic flux analysis comparing wild-type and mutant strains

The exact role requires experimental validation using these complementary approaches.

How can researchers optimize codon usage for heterologous expression of PP_0936 (maf-1)?

For optimal heterologous expression of PP_0936 (maf-1):

• Codon optimization strategy:

  • Analyze the codon adaptation index (CAI) of the native PP_0936 sequence

  • Optimize codons based on the preferred codon usage of the expression host

  • Avoid rare codons particularly at the N-terminus of the protein

  • Maintain any potential regulatory secondary structures in the mRNA

• GC content considerations:

  • P. putida has a high GC content (~61.5%)

  • When expressing in E. coli, moderate the GC content to avoid expression issues

  • Balance GC content particularly in the 5' region to prevent strong secondary structures

• Experimental validation:

  • Compare expression levels of native versus optimized sequences

  • Analyze mRNA levels to determine if limitations occur at transcriptional or translational levels

• Tools and resources:

  • JCat, OPTIMIZER, or commercial services for codon optimization

  • mRNA structure prediction tools to identify potential expression barriers

These optimization strategies can significantly improve heterologous expression yields.

What are the specific considerations for expressing PP_0936 (maf-1) in P. putida versus E. coli?

When choosing between P. putida and E. coli as expression hosts for PP_0936 (maf-1):

Key considerations:

• Expression level control: When using IPTG-inducible systems in P. putida, the promoter driving lacI expression must be weakened to achieve good dynamic range (up to 80-fold induction compared to poor induction with standard constructs) .

• Plasmid stability: Due to higher copy numbers in P. putida, expression constructs may impose greater metabolic burden, potentially affecting stability .

• Induction conditions: The XylS/Pm system (induced by 3-methylbenzoate) and RhaRS/PrhaBAD (induced by rhamnose) show tight regulation in P. putida and can provide higher expression levels than constitutive systems .

The choice depends on specific experimental goals, with P. putida offering advantages for functional studies in the native context.

How do different fusion tags affect the solubility and activity of recombinant PP_0936 (maf-1)?

Different fusion tags can significantly impact recombinant PP_0936 (maf-1) properties:

Recommendations for PP_0936 (maf-1):

• Initial screening: Test multiple fusion constructs in parallel small-scale expression trials:

  • N-terminal His-tag

  • N-terminal MBP-His fusion

  • N-terminal SUMO-His fusion

• Comparing solubility and yield: SDS-PAGE analysis of soluble versus insoluble fractions for each construct.

• Activity assessment: Develop functional assays to determine if the fusion tag interferes with protein activity.

• Tag removal evaluation: For tags affecting activity, include a protease cleavage site and test protein stability after tag removal.

Similar to MAF-1 fusion protein from Musca domestica, His-tag fusions can provide a good starting point, with subsequent optimization based on initial results .

How can genomic integration techniques be used to study PP_0936 (maf-1) function in P. putida?

For genomic manipulation of PP_0936 (maf-1) in P. putida:

• λRed/Cas9 recombineering method:

  • Recently developed for P. putida KT2440, enabling scarless mutations without traditional two-step integration and marker removal protocols

  • Can generate precise deletions, insertions, or point mutations in the PP_0936 gene

• Implementation approach:

  • Design guide RNA targeting PP_0936

  • Create donor DNA with desired modifications flanked by homology regions

  • Express λRed recombination proteins and Cas9/sgRNA in P. putida

  • Select for recombinants and verify by sequencing

• Genomic integration sites:

  • Utilize identified "landing pads" in P. putida KT2440 genome for stable integration of constructs

  • These sites ensure minimal disruption of native cellular processes

• Applications:

  • Gene knockout for loss-of-function studies

  • Promoter replacements to study expression regulation

  • Point mutations to identify critical residues

  • Reporter fusions to study localization and expression patterns

This approach allows for precise genome modifications without antibiotic marker interference, providing cleaner experimental systems for functional studies.

What crystallization approaches are most promising for structural determination of PP_0936 (maf-1)?

For crystallization of PP_0936 (maf-1):

• Pre-crystallization optimization:

  • Surface entropy reduction (SER): Identify and mutate clusters of high-entropy residues (Lys, Glu) to alanines

  • Construct optimization: Create truncated constructs based on limited proteolysis results

  • Thermal stability screening: Use differential scanning fluorimetry to identify stabilizing buffer conditions

• Initial screening strategy:

  • Implement sparse matrix screens at multiple protein concentrations (5-15 mg/mL)

  • Test at different temperatures (4°C, 18°C)

  • Include commercial screens specifically designed for bacterial proteins

• Optimization approaches:

  • Grid screens around promising conditions

  • Additive screens to improve crystal quality

  • Seeding techniques to promote crystal growth

• Alternative approaches if traditional crystallization fails:

  • Co-crystallization with binding partners identified through interaction studies

  • Crystallization with antibody fragments (Fab, nanobody)

  • LCP (Lipidic Cubic Phase) crystallization if membrane associations are suspected

• Complementary structural methods:

  • Cryo-EM for structural determination if crystallization proves challenging

  • SAXS for low-resolution envelope of the protein in solution

These approaches provide multiple avenues for structural characterization when initial crystallization attempts are unsuccessful.

How can researchers identify and characterize protein-protein interactions involving PP_0936 (maf-1)?

To identify and characterize PP_0936 (maf-1) interaction partners:

• In vivo approaches:

  • Bacterial two-hybrid screening using PP_0936 as bait

  • In vivo crosslinking followed by immunoprecipitation and mass spectrometry

  • Fluorescence resonance energy transfer (FRET) with potential interaction partners

• In vitro approaches:

  • Pull-down assays using tagged recombinant PP_0936

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic characterization

  • Bio-layer interferometry for real-time binding analysis

• Structural characterization of complexes:

  • Co-crystallization of PP_0936 with identified partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cryo-EM of larger complexes

• Validation in P. putida:

  • Co-immunoprecipitation from P. putida lysates

  • Bacterial adenylate cyclase two-hybrid system (BACTH) validation

  • Mutational analysis of predicted interaction interfaces

• Interaction network analysis:

  • Integration of identified interactions into known P. putida protein networks

  • Pathway enrichment analysis to identify biological processes involving PP_0936

These complementary approaches provide a comprehensive characterization of the PP_0936 (maf-1) interactome.

How does PP_0936 (maf-1) compare with Maf-like proteins in other bacterial species?

Comparative analysis of PP_0936 (maf-1) with other bacterial Maf proteins:

• Phylogenetic distribution and conservation:

  • Maf proteins are widely distributed across bacterial phyla

  • Conservation patterns may reveal functional specialization in different bacterial lineages

  • P. putida contains multiple Maf-like proteins (PP_0936, PP_1909) indicating potential functional diversification

• Domain architecture comparison:

  • Core Maf domain conservation analysis

  • Identification of additional domains that may confer specific functions

  • Comparison with functionally characterized Maf proteins from model organisms

• Structural homology modeling:

  • Using solved structures of Maf proteins as templates

  • Identification of conserved catalytic or binding residues

  • Prediction of potential functional sites specific to PP_0936

• Functional context comparison:

  • Genomic neighborhood analysis across species

  • Co-expression patterns with conserved gene clusters

  • Association with specific metabolic or regulatory pathways

This comparative approach provides evolutionary context for understanding PP_0936 function in P. putida.

What techniques can be used to investigate the potential involvement of PP_0936 (maf-1) in P. putida stress response?

To investigate PP_0936 (maf-1) involvement in stress response:

• Expression analysis under stress conditions:

  • qRT-PCR analysis of PP_0936 expression under various stressors (oxidative, osmotic, temperature, nutrient limitation)

  • Reporter fusions (GFP/luciferase) to monitor expression dynamics in real-time

  • Western blotting to assess protein levels under stress conditions

• Phenotypic characterization of mutants:

  • Growth curve analysis of ΔPP_0936 mutants under stress conditions

  • Survival assays following acute stress exposure

  • Competitive fitness assays with wild-type under fluctuating conditions

• Omics approaches:

  • Transcriptomics comparing wild-type and ΔPP_0936 responses to stress

  • Proteomics to identify differentially regulated proteins

  • Metabolomics to detect alterations in stress-related metabolites

• Physiological assessments:

  • Measurement of reactive oxygen species levels

  • Membrane integrity assessment

  • Biofilm formation quantification

• Complementation studies:

  • Expression of PP_0936 from controlled promoters to restore wild-type phenotypes

  • Expression of homologs from other species to assess functional conservation

These multi-faceted approaches can establish connections between PP_0936 and specific stress response pathways in P. putida.

How might PP_0936 (maf-1) be involved in Pseudomonas putida's metabolic versatility?

Investigating PP_0936 (maf-1) in P. putida's metabolic network:

• Metabolic phenotyping:

  • Biolog phenotype microarrays comparing wild-type and ΔPP_0936 mutants

  • Growth characterization on diverse carbon and nitrogen sources

  • Oxygen consumption rates under various metabolic conditions

• Enzyme activity assays:

  • In vitro enzymatic analysis of purified PP_0936

  • Metabolite profiling to identify accumulated or depleted compounds in mutants

  • Isotope labeling studies to track carbon flux through central metabolic pathways

• Integration with systems biology data:

  • Correlation analysis with expression profiles of metabolic genes

  • Regulatory network reconstruction incorporating PP_0936

  • Flux balance analysis incorporating PP_0936 constraints

• Specialized metabolism connections:

  • Assessment of secondary metabolite production in ΔPP_0936 mutants

  • Analysis of aromatic compound degradation capabilities

  • Connection to stress-induced metabolic adaptations

P. putida's remarkable metabolic versatility, including its ability to degrade aromatic compounds and tolerate various stressors, requires sophisticated regulatory networks in which PP_0936 may play an important role .