Recombinant Pseudomonas putida Chaperone protein htpG (htpG), partial

What is the function of chaperone protein htpG in Pseudomonas putida?

Chaperone protein htpG in Pseudomonas putida functions as a molecular chaperone that assists in protein folding and maintains protein homeostasis, particularly under stress conditions. As a member of the heat shock protein 90 (Hsp90) family, htpG possesses ATPase activity that drives its chaperoning functions . The protein helps maintain cellular integrity by preventing protein misfolding and aggregation, especially during environmental stresses like elevated temperatures. In P. putida, which is known for its metabolic versatility and stress resistance, htpG likely plays a crucial role in adapting to changing environmental conditions by ensuring proper protein folding and function throughout various stress responses.

What expression systems are recommended for recombinant production of P. putida htpG protein?

• Chromosomal integration using Tn5-based transposons for stable expression

• Selection of strong native promoters (such as ribosomal RNA gene promoters) for constitutive expression

• Unidirectional gene expression with proper consideration of transcriptional terminators

This approach allows for stable, high-level expression without requiring external inducers like T7 RNA polymerase, which may be particularly advantageous for larger proteins like htpG .

How can I confirm the purity and identity of recombinant htpG protein?

To confirm the purity and identity of recombinant htpG protein, employ a systematic multi-method approach:

• SDS-PAGE analysis: Run the purified protein sample on a discontinuous SDS-PAGE gel (typically using a 5% enrichment gel and 15% separation gel) to verify the expected molecular weight and assess purity. A purity level of >85% is generally considered acceptable for most research applications .

• Western blotting: Use antibodies specific to htpG or to any tags incorporated into the recombinant protein construct.

• Mass spectrometry: For definitive identification, perform peptide mass fingerprinting or tandem mass spectrometry (MS/MS) analysis.

• ATPase activity assay: Since htpG possesses intrinsic ATPase activity, a functional assay measuring ATP hydrolysis can confirm not only identity but also proper folding and activity .

• N-terminal sequencing: Confirm the correct sequence of the first 5-10 amino acids to verify proper translation initiation and processing.

For recombinant htpG protein with high purity, typically the major band on SDS-PAGE should appear at the expected molecular mass, with minimal contaminating bands. Records should document the exact range of amino acids expressed (e.g., for partial constructs) and the expression host used.

What strategies can optimize chromosomal integration of the htpG gene in P. putida?

Optimizing chromosomal integration of the htpG gene in P. putida requires strategic approaches that balance expression levels with genomic stability. Based on successful strategies with other recombinant proteins, the following methodology is recommended:

• Transposon-based integration: Utilize Tn5-based transposons for random chromosomal integration, following the workflow established for other heterologous genes in P. putida . This method enables creation of a library of integration variants with potentially different expression levels.

• Integration site selection: While random integration via transposons creates variability, targeted approaches can also be employed. The genomic locations adjacent to ribosomal RNA genes have proven particularly effective for high-level constitutive expression in P. putida .

• Screening methodology: Develop an efficient screening method to identify optimal integration variants. For htpG, this could involve:

  • Monitoring protein production levels via fluorescently tagged htpG

  • Assessing ATPase activity as a functional readout

  • Measuring growth characteristics under stress conditions where htpG function is critical

• Verification of integration sites: For promising clones, employ a plasmid rescue strategy to identify the precise genomic location of integration. This involves:

  • Isolating genomic DNA

  • Restriction digestion with appropriate enzymes (e.g., MluI or NcoI)

  • Ligation and transformation into E. coli

  • Sequencing using outward-facing primers from the integrated construct

• Post-integration analysis: Assess transcriptional efficiency from different integration sites using RT-qPCR to determine which genomic contexts provide optimal expression levels.

This comprehensive approach enables identification of integration variants with stable, high-level expression without requiring continuous antibiotic selection, making it ideal for long-term research applications.

How does the structure-function relationship of P. putida htpG compare to other bacterial heat shock proteins?

The structure-function relationship of P. putida htpG aligns with other bacterial heat shock proteins of the Hsp90 family but may exhibit unique adaptations related to P. putida's environmental versatility. While specific structural data for P. putida htpG is limited, comparative analysis with homologous proteins suggests:

• Domain organization: P. putida htpG likely contains three functional domains:

  • N-terminal domain (NTD) with ATPase activity

  • Middle domain (MD) involved in client protein binding

  • C-terminal domain (CTD) responsible for dimerization

• Sequence conservation: Key functional regions show high conservation across bacterial species. The N-terminal ATPase domain typically contains the ATP-binding pocket with conserved residues essential for nucleotide binding and hydrolysis .

• Functional differences: Compared to other bacterial Hsp90 homologs, P. putida htpG may exhibit:

  • Modified temperature responsiveness aligned with P. putida's environmental adaptability

  • Potentially broader client specificity to accommodate P. putida's diverse metabolic functions

  • Altered ATP hydrolysis rates optimized for its ecological niche

The partial htpG construct (amino acids 1-326) would likely encompass the NTD and portions of the MD, capturing the ATPase activity but potentially lacking complete client binding and dimerization capabilities .

Understanding these structure-function relationships is crucial for designing partial constructs with specific activities and for interpreting functional assays on recombinant htpG proteins.

What experimental approaches can resolve contradictory data on htpG function in stress response pathways?

Resolving contradictory data on htpG function in stress response pathways requires systematic experimental approaches that integrate multiple levels of analysis:

• Genetic approaches:

  • Generate clean deletion mutants of htpG in P. putida using allelic exchange or CRISPR-Cas9

  • Create conditional expression strains with titratable promoters

  • Perform complementation studies with wild-type and mutant htpG variants

  • Conduct genetic interaction studies with other stress response genes

• Biochemical characterization:

  • Compare the ATPase activity of htpG under various stress conditions (temperature, pH, oxidative stress)

  • Identify client proteins through co-immunoprecipitation followed by mass spectrometry

  • Analyze post-translational modifications of htpG during different stress responses

  • Perform in vitro chaperone activity assays with model substrate proteins

• Structural biology:

  • Obtain crystal structures or use cryo-EM to determine conformational changes under different conditions

  • Employ hydrogen-deuterium exchange mass spectrometry to map dynamic regions during client binding

• Systems biology approaches:

  • Perform transcriptomics to compare global expression changes in wild-type vs. htpG mutant strains

  • Use proteomics to identify changes in protein abundance and stability

  • Employ metabolomics to detect metabolic shifts resulting from htpG dysfunction

• In vivo validation:

  • Develop fluorescent reporters to monitor htpG activity in real-time

  • Use microfluidics combined with time-lapse microscopy to track single-cell responses

  • Perform competition assays under various stress conditions

By integrating these approaches, researchers can develop a comprehensive model of htpG function that reconciles seemingly contradictory observations and places htpG within the broader context of cellular stress response networks in P. putida.

What is the optimal protocol for purifying recombinant P. putida htpG protein?

The optimal protocol for purifying recombinant P. putida htpG protein involves a multi-step chromatographic approach designed to maximize yield while ensuring high purity and preserved activity:

• Expression system selection:

  • Express in either P. putida KT2440 (for native-like folding) or E. coli (for higher yields)

  • Incorporate an affinity tag (6xHis or Strep-tag) at either the N- or C-terminus

  • Include a TEV protease cleavage site if tag removal is desired

• Cell lysis:

  • Harvest cells during late logarithmic phase

  • Resuspend in buffer containing:

    • 50 mM Tris-HCl, pH 7.5

    • 150 mM NaCl

    • 10% glycerol

    • 5 mM MgCl₂ (to stabilize nucleotide binding)

    • 1 mM DTT or 2 mM β-mercaptoethanol

    • Protease inhibitor cocktail

  • Lyse cells using sonication or high-pressure homogenization

• Initial affinity purification:

  • Apply clarified lysate to appropriate affinity resin

  • For His-tagged constructs, use IMAC (Ni-NTA or Co-based resins)

  • Wash extensively with buffer containing 20-30 mM imidazole

  • Elute with buffer containing 250-300 mM imidazole

• Secondary purification:

  • Perform ion-exchange chromatography (typically anion exchange using Q-Sepharose)

  • Apply sample diluted to reduce salt concentration

  • Elute using a gradient of 0-500 mM NaCl

• Final polishing:

  • Size-exclusion chromatography using Superdex 200

  • Buffer composition: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Quality control:

  • Assess purity by SDS-PAGE (target >85% purity)

  • Verify identity via mass spectrometry

  • Confirm activity through ATPase assays

  • Evaluate oligomeric state using native PAGE or analytical SEC

This protocol typically yields 5-10 mg of purified protein per liter of bacterial culture with >85% purity, suitable for most biochemical and structural studies .

How can I design experiments to characterize the client specificity of P. putida htpG?

Designing experiments to characterize the client specificity of P. putida htpG requires a comprehensive approach combining in vitro and in vivo methods:

• Global client identification:

  • Perform co-immunoprecipitation of epitope-tagged htpG followed by mass spectrometry

  • Compare client profiles under normal conditions versus various stresses (heat, oxidative, pH)

  • Validate interactions using reciprocal pull-downs with identified clients

  • Create a network map of htpG interactions using bioinformatics tools

• In vitro binding assays:

  • Develop fluorescence-based assays to quantify htpG-client interactions:

    • Fluorescence anisotropy with labeled model substrates

    • FRET-based assays between tagged htpG and clients

  • Measure binding kinetics using surface plasmon resonance (SPR) or bio-layer interferometry (BLI)

  • Determine the thermodynamics of binding using isothermal titration calorimetry (ITC)

• Functional chaperone assays:

  • Thermal aggregation prevention assays with model substrates (e.g., citrate synthase)

  • Refolding assays to monitor htpG-assisted recovery of denatured proteins

  • ATPase activity modulation in response to client binding

  • Competition assays between different clients to assess preferential binding

• Structural characterization of complexes:

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Cryo-EM analysis of htpG-client complexes

  • Crosslinking mass spectrometry to identify proximal residues

• In vivo validation:

  • Monitor stability of identified clients in wild-type versus htpG deletion strains

  • Use fluorescence microscopy with split-GFP systems to visualize interactions in live cells

  • Perform genetic epistasis analyses between htpG and client-encoding genes

This systematic approach will provide a comprehensive picture of htpG client specificity and the molecular basis for client recognition in P. putida.

What are the key considerations for designing truncated variants of htpG for domain-specific studies?

Designing truncated variants of htpG for domain-specific studies requires careful consideration of structural boundaries, functional elements, and expression characteristics:

• Domain boundary identification:

  • Perform bioinformatic analysis using multiple sequence alignments of htpG homologs

  • Identify conserved domain boundaries based on crystal structures of homologous Hsp90 proteins

  • Use secondary structure prediction to avoid disrupting structural elements

  • Consider intrinsically disordered regions as potential flexible linkers

• Functional elements to preserve:

  • N-terminal domain (NTD): Include the complete ATP-binding pocket (approximately aa 1-220)

  • Middle domain (MD): Maintain intact client-binding regions (approximately aa 221-500)

  • C-terminal domain (CTD): Preserve the dimerization interface (approximately aa 501-670)

  • For partial constructs, ensure the protein segment includes complete functional domains

• Design strategies for optimal expression:

  • Add short linkers (3-5 glycine/serine residues) at truncation points

  • Consider adding stabilizing elements for isolated domains (e.g., dimerization tags for normally dimeric regions)

  • Optimize codon usage for the expression host

  • Include solubility-enhancing tags (MBP, SUMO) for challenging constructs

• Validation of truncated constructs:

  • Assess proper folding using circular dichroism spectroscopy

  • Verify domain-specific activities:

    • NTD constructs: ATP binding and hydrolysis

    • MD constructs: Client protein interaction

    • CTD constructs: Dimerization capacity

  • Compare thermal stability of truncated variants to full-length protein using differential scanning fluorimetry

• Typical truncation points for P. putida htpG:

When designing a partial htpG construct (as described in query), ensure it contains complete functional domains rather than arbitrary fragments. For example, the amino acid range 1-326 would encompass the complete NTD plus a portion of the MD, potentially preserving some but not all client-binding functionality .

How does P. putida htpG differ from other bacterial chaperone systems in terms of stress response coordination?

P. putida htpG exhibits distinctive features in stress response coordination compared to other bacterial chaperone systems, reflecting this organism's remarkable environmental adaptability:

• Integration with stress-specific responses:

  • Unlike E. coli where the heat shock response dominates htpG regulation, P. putida htpG likely responds to a broader range of stressors (solvent exposure, oxidative stress, temperature fluctuations)

  • P. putida's htpG may have evolved specialized coordination with detoxification and biodegradative pathways that are central to this organism's ecological niche

• Regulatory network differences:

  • While most bacterial htpG proteins are regulated by the σ32 heat shock sigma factor, P. putida likely incorporates additional regulatory inputs from stress-responsive pathways

  • The promoter architecture of P. putida htpG may contain binding sites for additional transcription factors related to xenobiotic stress responses

• Functional cooperation with other chaperones:

  • P. putida contains an expanded repertoire of stress-response proteins compared to many bacteria

  • The htpG system likely shows specialized cooperation with DnaK/DnaJ/GrpE and GroEL/GroES systems tailored to P. putida's environmental challenges

  • Unique co-chaperones may exist that direct htpG activity toward specific client subsets

• Metabolic integration:

  • P. putida htpG may play a more significant role in maintaining the integrity of metabolic enzymes involved in aromatic compound degradation and other specialized pathways

  • The chaperone likely contributes to the rapid metabolic adaptability that P. putida exhibits when shifting between carbon sources

This specialized adaptation makes P. putida htpG an interesting model for studying how molecular chaperones evolve to support specific ecological strategies, particularly in bacteria that occupy challenging and variable environmental niches.

What are the implications of using P. putida as a host for heterologous htpG expression compared to E. coli?

Using P. putida as a host for heterologous htpG expression offers several distinct advantages and considerations compared to the more conventional E. coli expression system:

• Expression efficiency and protein folding:

  • P. putida may provide a more native-like folding environment for other Pseudomonad htpG proteins

  • The GRAS (Generally Recognized As Safe) status of P. putida KT2440 makes it an attractive host for producing proteins for downstream applications

  • P. putida's robust stress tolerance mechanisms may support higher quality protein production, especially for stress-responsive proteins like htpG

• Genetic stability and expression consistency:

  • Chromosomal integration methods in P. putida provide exceptional stability without antibiotic selection

  • The random integration approach using transposons allows screening for optimal expression loci

  • Strong native promoters (like those from ribosomal RNA genes) enable consistent, constitutive expression without requiring induction systems

• Post-translational modifications and interactions:

  • P. putida may provide more appropriate post-translational modifications for htpG proteins from related species

  • The presence of native P. putida co-chaperones might assist in proper folding of heterologous htpG

  • Potential interference from endogenous P. putida htpG must be considered, possibly requiring background deletion

• Technical considerations and yields:

  • While E. coli systems are more established with higher initial yields, optimized P. putida systems can achieve comparable productivity

  • P. putida grows more slowly than E. coli, potentially extending production timelines

  • Extraction and purification protocols may require optimization specific to P. putida

For researchers prioritizing authentic folding and long-term stability for Pseudomonad htpG proteins, P. putida represents an excellent expression platform, particularly when employing chromosomal integration strategies .

How can advanced structural biology techniques be applied to study htpG conformational dynamics during the chaperone cycle?

Advanced structural biology techniques provide powerful approaches to decipher the complex conformational dynamics of htpG during its chaperone cycle:

• Time-resolved cryo-electron microscopy (cryo-EM):

  • Capture transient conformational states by rapid freezing at defined time points after ATP addition

  • Employ image classification algorithms to sort heterogeneous particle populations

  • Reconstruct the complete conformational landscape throughout the ATP hydrolysis cycle

  • Visualize client protein interactions and their impact on htpG conformation

• Single-molecule Förster resonance energy transfer (smFRET):

  • Introduce fluorescent probe pairs at strategic locations across htpG domains

  • Monitor real-time distance changes between domains during ATP binding, hydrolysis, and client interaction

  • Quantify the kinetics of conformational transitions and identify rate-limiting steps

  • Detect rare or transient conformational states missed by ensemble methods

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map regions of conformational flexibility and solvent accessibility

  • Compare exchange patterns in different functional states:

    • Apo state

    • ATP-bound

    • ADP-bound

    • Client-bound

  • Identify allosteric communication networks between domains

• Nuclear magnetic resonance (NMR) spectroscopy:

  • For domain-specific studies, use truncated htpG constructs amenable to NMR analysis

  • Employ methyl-TROSY techniques for studying larger assemblies

  • Characterize μs-ms timescale dynamics using relaxation dispersion experiments

  • Map client binding interfaces through chemical shift perturbation

• Molecular dynamics (MD) simulations:

  • Develop atomistic models of P. putida htpG based on homologous structures

  • Simulate conformational transitions during the ATPase cycle

  • Investigate water networks and ion coordination in catalytic sites

  • Model client protein interactions and induced conformational changes

• Integrative structural biology approach:

By integrating these complementary approaches, researchers can construct a comprehensive model of htpG's conformational cycle, mapping the structural basis for ATP-driven chaperone activity and client protein interaction specificity.

What emerging technologies could enhance the study of P. putida htpG function in stress adaptation?

Emerging technologies offer exciting new avenues to investigate P. putida htpG function in stress adaptation with unprecedented precision and scope:

• CRISPR interference (CRISPRi) and activation (CRISPRa) systems:

  • Develop P. putida-optimized CRISPRi systems for tunable repression of htpG

  • Apply CRISPRa for controlled overexpression under specific conditions

  • Create libraries targeting htpG regulators to map the complete regulatory network

  • Implement multiplexed CRISPR systems to simultaneously modulate htpG and client proteins

• Single-cell technologies:

  • Employ microfluidic devices combined with time-lapse microscopy to track htpG activity in real-time

  • Apply single-cell RNA-seq to capture cell-to-cell variability in htpG expression during stress responses

  • Develop fluorescent sensors to monitor htpG conformational states in living cells

  • Utilize nano-sampling techniques to extract proteins from individual cells for proteomic analysis

• Proximity labeling proteomics:

  • Employ engineered ascorbate peroxidase (APEX) or TurboID fused to htpG to identify transient interactions

  • Map the dynamic changes in htpG interactome across different stress conditions

  • Combine with pulse-chase approaches to determine client residence times

  • Develop split proximity labeling systems to identify complexes containing specific combinations of chaperones

• Synthetic biology approaches:

  • Engineer orthogonal htpG variants with altered client specificity

  • Develop synthetic genetic circuits to probe htpG function in defined stress response networks

  • Create chimeric htpG proteins incorporating domains from different species to investigate domain-specific functions

  • Design minimal synthetic clients with defined characteristics to dissect recognition principles

• Advanced imaging techniques:

  • Apply super-resolution microscopy (PALM/STORM) to track htpG localization with nanometer precision

  • Employ lattice light-sheet microscopy for rapid 3D imaging with reduced phototoxicity

  • Develop correlative light and electron microscopy workflows to connect htpG dynamics to cellular ultrastructure

  • Utilize expansion microscopy to visualize chaperone-client complexes at enhanced resolution

These emerging technologies promise to transform our understanding of htpG function by enabling analyses with improved spatiotemporal resolution, sensitivity, and throughput, ultimately revealing how this molecular chaperone contributes to P. putida's remarkable environmental adaptability.

How might findings from P. putida htpG research contribute to biotechnological applications?

Findings from P. putida htpG research have significant potential to advance various biotechnological applications, leveraging the unique properties of this chaperone system:

• Enhanced recombinant protein production:

  • Co-expression of optimized htpG variants could improve folding and yield of difficult-to-express proteins

  • Development of specialized expression systems where htpG expression is dynamically coordinated with target protein production

  • Engineering of htpG variants with broadened client specificity to assist folding of non-native proteins

  • Creation of synthetic chaperone networks incorporating htpG for complex protein production challenges

• Bioremediation applications:

  • Enhanced stability of biodegradative enzymes through targeted htpG chaperoning

  • Development of P. putida strains with modified htpG systems optimized for function in contaminated environments

  • Engineering stress-responsive htpG variants to protect cellular machinery during exposure to toxic compounds

  • Creation of biosensors utilizing htpG-client interactions to detect environmental pollutants

• Synthetic biology tools:

  • Utilization of htpG-based protein quality control modules in synthetic circuits

  • Development of conditional protein degradation systems based on htpG recognition

  • Creation of protein solubility switches regulated by engineered htpG variants

  • Design of post-translational regulatory systems utilizing modified htpG-client interactions

• Biomaterial production:

  • Enhanced production of biodegradable polymers through stabilization of biosynthetic enzymes

  • Development of protein-based materials with htpG-assisted folding

  • Improvement of enzyme cascade stability for continuous bioproduction processes

  • Creation of self-healing biomaterials incorporating engineered htpG systems

• Potential applications in heterologous protein production:

The strategic integration of htpG chaperone systems into biotechnological processes represents a promising approach to enhance the robustness and efficiency of various bioprocesses, particularly those involving challenging environmental conditions or complex protein production demands .