Recombinant Enterobacteria phage I2-2 Attachment protein G3P (III)

What is the biological function of Enterobacteria phage Attachment protein G3P (III)?

Attachment protein G3P (III) plays essential roles in both the infection and extrusion processes of bacteriophages. During infection, G3P mediates adsorption of the phage to its primary receptor, typically the tip of the host pilus. Subsequent interaction with the host entry receptor (such as TolA) induces penetration of the viral DNA into the host cytoplasm. In the extrusion process, G3P mediates the release of the membrane-anchored virion from the cell via its C-terminal domain .

The protein is a minor coat protein found at one end of filamentous phage particles, with each phage particle containing approximately 3-5 copies of g3p in close proximity . This arrangement may contribute to cooperative binding effects during host cell interaction.

What is the molecular structure of Recombinant Enterobacteria phage G3P protein?

The G3P protein consists of multiple functional domains, with the N-terminal domains (g3p-N) being responsible for host recognition and binding. These domains interact with the host receptor (F pilus in many cases) and co-receptor (TolA), while the C-terminal domain serves as a structural element of the phage coat . The N-terminal domains have been crystallized and found to be structurally similar to homologous proteins from other filamentous phage .

For reference, the full-length mature Enterobacteria phage M13 G3P protein spans amino acids 19-424 and has a molecular weight of approximately 44.6 kDa . While specific structural details of I2-2 G3P may vary, it likely shares similar domain organization given the functional conservation among phage attachment proteins.

How does Recombinant Enterobacteria phage G3P differ from other phage attachment proteins?

Each bacteriophage has evolved attachment proteins specific to its host range. For example, filamentous phages like M13 and If1 have g3p proteins with different N-terminal domains that determine binding specificity. M13 phage g3p binds to F pili, while If1 phage g3p binds to I pili .

The specificity of these interactions is demonstrated by experiments where replacing the N-terminal domains of M13 g3p with those from If1 phage creates a chimeric phage that loses the ability to infect F+ cells but gains the ability to interact with I plasmid-containing cells . This specificity is primarily determined by the amino acid sequence and structural features of the N-terminal domains.

What are the optimal expression systems for producing Recombinant Enterobacteria phage I2-2 Attachment protein G3P?

Based on established protocols for similar phage proteins, recombinant G3P proteins can be successfully expressed in bacterial systems, particularly E. coli . For Enterobacteria phage I2-2 Gene 1 protein, expression in E. coli with an N-terminal His tag has proven effective . Yeast expression systems have also been successfully employed for producing recombinant phage proteins, as seen with Enterobacteria phage M13 G3P .

When expressing G3P proteins, consider the following recommendations:

• Use expression vectors with strong, inducible promoters (T7, tac)

• Include affinity tags (His, GST) for purification, preferably at the N-terminus

• Optimize codon usage for the expression host

• Control expression temperature (typically 16-30°C) to enhance solubility and proper folding

What purification strategies yield the highest purity and activity of Recombinant G3P proteins?

For His-tagged G3P proteins, immobilized metal affinity chromatography (IMAC) is the primary purification method. Based on protocols for similar proteins, a multi-step purification strategy is recommended:

• Initial capture using IMAC with Ni²⁺ or Co²⁺ resins

• Further purification by ion exchange chromatography

• Final polishing step using size exclusion chromatography

This approach typically yields protein with >90% purity as determined by SDS-PAGE . For optimal results, all purification steps should be performed at 4°C with protease inhibitors to prevent degradation. The purified protein should be stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 to maintain stability.

How should researchers store and reconstitute lyophilized Recombinant G3P to maintain activity?

Lyophilized G3P protein should be stored at -20°C to -80°C upon receipt. For reconstitution:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot for long-term storage at -20°C to -80°C

It's important to note that repeated freeze-thaw cycles should be avoided to maintain protein activity. Working aliquots can be stored at 4°C for up to one week . When using the protein for binding studies, reconstitution in a buffer system that maintains physiological pH and ionic strength is critical for preserving native conformation and activity.

How can Recombinant G3P be used to study bacterial conjugation inhibition mechanisms?

Recombinant G3P, particularly the soluble N-terminal domains (g3p-N), has been shown to effectively inhibit bacterial conjugation at low nanomolar concentrations. This inhibition occurs through binding to the F pilus, which prevents the transmission of plasmids encoding antibiotic resistance genes .

Experimental approach for conjugation inhibition studies:

• Prepare donor cells containing a conjugative plasmid (e.g., F plasmid with tetracycline resistance)

• Prepare recipient cells with a different selective marker

• Mix donor and recipient cells at a specific ratio

• Add purified g3p-N at various concentrations (0-100 nM)

• Incubate to allow conjugation to occur

• Plate on selective media to identify transconjugants

• Calculate conjugation efficiency as the ratio of transconjugants to donors

Results can be analyzed using a simple binding equilibrium model between g3p-N and F+ cells. The dissociation constant (Kd) for soluble g3p-N has been determined to be approximately 3 nM , which provides a reference point for interpreting experimental results.

What are the key considerations for designing binding assays with Recombinant G3P and bacterial pili?

When designing binding assays to study interactions between G3P and bacterial pili:

• Protein Presentation Format: Consider that soluble g3p-N and phage-bound g3p may have different binding properties. The Kd of whole phage particles (approximately 2 pM) differs from that of soluble g3p-N (approximately 3 nM) by a factor of ~1000, likely due to differences in binding reversibility and potential avidity effects .

• Buffer Conditions: Ionic strength, pH, and divalent cation concentrations can significantly affect protein-protein interactions. Standard TBS buffer (Tris-buffered saline) has been successfully used for g3p binding assays .

• Controls: Include both positive controls (known interacting partners) and negative controls (non-specific proteins like bovine serum albumin) .

• Detection Methods: Options include:

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • ELISA-based assays for endpoint measurements

  • Fluorescence-based assays using labeled proteins

  • Functional assays measuring inhibition of conjugation or phage infection

• Data Analysis: Apply appropriate binding models that account for potential cooperativity, especially when working with multivalent systems like phage particles with multiple g3p copies.

How can researchers distinguish between specific and non-specific binding in G3P interaction studies?

Distinguishing specific from non-specific binding is critical for accurate characterization of G3P interactions. Recommended approaches include:

• Competitive Binding Assays: Use unlabeled g3p to compete with labeled g3p for binding sites. Specific binding will show concentration-dependent displacement.

• Chimeric Protein Controls: Create chimeric proteins where binding domains are replaced with homologous regions from related phages with different specificities (e.g., replacing M13 g3p N-terminal domains with If1 domains) . This approach can confirm binding specificity as demonstrated in studies where chimeric phage lost ability to bind F+ cells but gained ability to interact with I plasmid-containing cells.

• Mutational Analysis: Introduce point mutations in key residues of g3p based on structural information. Specific binding will be disrupted by mutations in the binding interface.

• Cross-Linking Studies: Use chemical cross-linking followed by mass spectrometry to identify specific interaction sites between g3p and its receptor.

• Control Proteins: Include structurally similar but functionally unrelated proteins as negative controls. For example, bovine serum albumin has been used as a control in g3p binding studies and showed no appreciable inhibition of conjugation even at high concentrations .

What analytical techniques are most effective for confirming the identity and integrity of Recombinant G3P?

A comprehensive characterization of recombinant G3P should employ multiple analytical techniques:

• SDS-PAGE and Western Blotting: For assessing purity, molecular weight, and immunoreactivity. G3P proteins should show >90% purity by SDS-PAGE .

• Mass Spectrometry:

  • Intact mass analysis to confirm full-length protein

  • Peptide mapping for sequence coverage

  • Post-translational modification analysis

• Circular Dichroism (CD) Spectroscopy: To evaluate secondary structure and confirm proper folding.

• Size Exclusion Chromatography (SEC): To assess oligomeric state and detect aggregation.

• Functional Assays: Activity-based assays to confirm biological function, such as:

  • Pilus binding assays

  • Phage infection inhibition assays

  • Bacterial conjugation inhibition assays

How can researchers quantitatively measure the binding affinity of G3P to bacterial pili?

Quantitative measurement of G3P-pili interactions can be achieved through several methods:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified pili or pili-expressing bacteria on a sensor chip

  • Flow different concentrations of G3P over the surface

  • Determine association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD = kd/ka)

• Microscale Thermophoresis (MST):

  • Label G3P or pili with a fluorescent tag

  • Measure changes in thermophoretic mobility upon binding

  • Determine KD from binding curves

• Isothermal Titration Calorimetry (ITC):

  • Directly measure heat changes associated with binding

  • Determine KD, stoichiometry, and thermodynamic parameters (ΔH, ΔS)

• Functional Inhibition Assays:

  • Measure inhibition of conjugation at different G3P concentrations

  • Fit data to binding models to determine apparent KD

  • This approach has yielded a KD of approximately 3 nM for soluble g3p-N binding to F+ cells

What are the critical quality attributes that should be monitored for Recombinant G3P in research applications?

For research applications, monitor these critical quality attributes:

What are common pitfalls in G3P functional studies and how can they be avoided?

Common challenges in G3P functional studies include:

• Protein Inactivation During Storage/Handling:

  • Store at -20°C to -80°C and avoid repeated freeze-thaw cycles

  • Add 6% trehalose as a stabilizer in storage buffer

  • Maintain working aliquots at 4°C for no more than one week

• Non-specific Binding Effects:

  • Include appropriate negative controls (e.g., BSA)

  • Use blocking agents to minimize non-specific interactions

  • Validate specificity through competition assays

• Variability in Host Cell Receptor Expression:

  • Standardize culture conditions for bacterial cells

  • Confirm pilus expression through microscopy or functional assays

  • Use well-characterized bacterial strains with stable F plasmid expression

• Aggregation of Recombinant Protein:

  • Optimize buffer conditions (pH, ionic strength, additives)

  • Filter solutions before use

  • Monitor aggregation by dynamic light scattering or SEC

• Discrepancies Between Different Binding Assays:

  • Consider format-dependent effects (e.g., soluble vs. phage-bound g3p)

  • Account for potential avidity effects with multivalent interactions

  • Validate results using multiple, complementary assay formats

How should researchers design experiments to investigate G3P-mediated inhibition of bacterial conjugation?

For robust experimental design to study G3P-mediated conjugation inhibition:

• Experimental Setup:

  • Use a mating pair system with well-characterized F+ donor and F- recipient strains

  • Include appropriate antibiotic resistance markers for selection

  • Maintain consistent cell densities and ratios between experiments

  • Test a range of g3p concentrations (0.1-100 nM) based on the reported KD of 3 nM

• Controls:

  • Positive control: mating mixture without g3p

  • Negative control: non-conjugating donor or recipient strain

  • Specificity control: unrelated protein (e.g., BSA) at equivalent concentrations

  • System validation control: known conjugation inhibitor (e.g., sodium azide)

• Analysis Framework:

  • Calculate conjugation efficiency as the ratio of transconjugants to donors

  • Plot inhibition curves as a function of g3p concentration

  • Fit data to appropriate binding models (e.g., single-site binding equilibrium)

  • Consider time-dependence by sampling at multiple time points

• Additional Considerations:

  • Test both soluble g3p-N and intact phage particles for comparison

  • Investigate potential strain-dependent effects with different F+ bacteria

  • Examine the impact of growth conditions on susceptibility to inhibition

What strategies can overcome expression and solubility challenges with Recombinant G3P?

For researchers facing expression and solubility challenges:

• Optimization of Expression Conditions:

  • Test different E. coli strains (BL21(DE3), Rosetta, Origami)

  • Evaluate various induction temperatures (16°C, 25°C, 30°C)

  • Optimize inducer concentration and induction duration

  • Consider auto-induction media for gentle protein expression

• Construct Design Strategies:

  • Express individual domains separately if full-length protein is problematic

  • Test different affinity tags (His, GST, MBP) for improved solubility

  • Optimize codon usage for expression host

  • Remove potential problematic regions (predicted disorder, hydrophobic patches)

• Solubilization Approaches:

  • Screen different buffer conditions using a factorial design approach

  • Test additives such as trehalose (6%) , glycerol (5-10%), and low concentrations of non-ionic detergents

  • Evaluate the effect of salt concentration (100-500 mM NaCl)

  • Consider stabilizing agents like arginine or proline

• Refolding Strategies (if inclusion bodies are formed):

  • Solubilize inclusion bodies in 6-8 M urea or guanidine HCl

  • Remove denaturant by dialysis or dilution

  • Use a redox system (GSH/GSSG) to facilitate disulfide bond formation

  • Consider step-wise dialysis with decreasing denaturant concentration

How does the binding mechanism of G3P differ between various bacteriophages and how can this inform protein engineering approaches?

Different bacteriophages have evolved various G3P binding mechanisms specific to their host range. Comparative analysis reveals:

Bacteriophage M13 G3P binds specifically to F pili and subsequently interacts with TolA, while If1 phage G3P binds to I pili . These specificity differences are primarily determined by the N-terminal domains.

This natural diversity provides a foundation for protein engineering approaches:

• Domain Swapping: Creating chimeric proteins by exchanging N-terminal domains between phages with different specificities has successfully altered receptor specificity . This approach can be used to redirect phage tropism for specific bacterial targets.

• Rational Design: Based on structural knowledge of G3P-receptor interfaces, targeted mutations can be introduced to enhance binding affinity or modify specificity. Phage display technologies can accelerate this process through directed evolution.

• Novel Applications: Engineered G3P variants could be developed as:

  • Targeted antimicrobials that inhibit conjugation in specific bacterial species

  • Diagnostic tools for detecting specific bacterial strains

  • Delivery vehicles for introducing genetic material into specific bacteria

• Structure-Function Relationships: The differential binding properties of soluble G3P (KD ~3 nM) versus phage-bound G3P (KD ~2 pM) suggest that presentation context dramatically affects function, providing insights for optimal protein engineering strategies.

What role might G3P play in the development of novel anti-conjugation therapeutics to combat antimicrobial resistance?

G3P proteins show significant potential as anti-conjugation therapeutics based on their ability to inhibit bacterial conjugation at nanomolar concentrations . This mechanism addresses a fundamental process in horizontal gene transfer that spreads antibiotic resistance.

Key considerations for developing G3P-based therapeutics include:

• Mechanism of Action: G3P inhibits conjugation primarily through occlusion of the conjugative pilus , preventing the transmission of antibiotic resistance genes without directly killing bacteria. This non-bactericidal approach may reduce selective pressure for resistance development.

• Therapeutic Potential:

  • Anti-resistance adjuvant: Co-administration with antibiotics to prevent resistance spread

  • Prophylactic use in high-risk settings (hospitals, animal husbandry)

  • Targeted therapy for patients colonized with multi-drug resistant organisms

• Delivery Challenges:

  • Protein stability in physiological conditions

  • Targeted delivery to sites of bacterial colonization

  • Potential immunogenicity of phage-derived proteins

• Development Considerations:

  • Engineering enhanced variants with improved stability and potency

  • Formulation strategies to protect protein activity in vivo

  • Combination approaches with other anti-virulence or antibiotic therapies

• Experimental Support: Research has shown that exogenous addition of soluble g3p-N results in nearly complete inhibition of conjugation at low nanomolar concentrations , providing proof-of-concept for this therapeutic approach.

How can structural biology approaches enhance our understanding of G3P interaction with bacterial receptors?

Advanced structural biology techniques can provide critical insights into G3P-receptor interactions:

• Cryo-Electron Microscopy (Cryo-EM):

  • Visualize the interaction between G3P and pili at near-atomic resolution

  • Capture different states of the binding process

  • Examine the structural arrangement of multiple g3p molecules on phage particles

• X-ray Crystallography:

  • Determine high-resolution structures of G3P in complex with receptor fragments

  • Identify specific residues at the binding interface

  • Guide structure-based drug design for anti-conjugation therapeutics

• NMR Spectroscopy:

  • Characterize dynamics of G3P-receptor interactions in solution

  • Map binding interfaces through chemical shift perturbation analysis

  • Study conformational changes upon binding

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Probe conformational dynamics and solvent accessibility changes upon binding

  • Identify regions that undergo structural rearrangements during receptor engagement

  • Complement higher-resolution techniques with solution-phase dynamics information

• Molecular Dynamics Simulations:

  • Model the energetics and dynamics of G3P-receptor interactions

  • Predict effects of mutations on binding affinity and specificity

  • Simulate the complete infection process including pilus retraction

• Integrative Structural Biology:

  • Combine multiple techniques for a comprehensive understanding

  • Build models that capture the entire phage-host interaction process

  • Inform rational design of improved inhibitors or engineered phages