Recombinant Pseudomonas putida Translation initiation factor IF-3 (infC)

What is Translation initiation factor IF-3 (infC) in Pseudomonas putida KT2440?

Translation initiation factor IF-3, encoded by the infC gene (locus tag PP_2466) in Pseudomonas putida KT2440, is a critical protein involved in the initiation phase of bacterial protein synthesis. Located on the chromosome at position 2811534-2812067 on the positive strand, this protein plays essential roles in preventing premature association of 50S and 30S ribosomal subunits and facilitating correct mRNA binding to the ribosome . The protein has a molecular weight of 20.1 kDa and an isoelectric point of 9.32, with a Kyte-Doolittle hydrophobicity value of -0.650, indicating its predominantly hydrophilic nature . IF-3 functions as part of the translation initiation machinery, working in concert with other factors to ensure proper start codon selection during protein synthesis.

How does P. putida IF-3 compare structurally to other bacterial IF-3 proteins?

P. putida IF-3 shares significant structural homology with IF-3 proteins from related Pseudomonas species, particularly with P. aeruginosa IF-3, which demonstrates 91.7% sequence identity in the C-terminal domain . Structural studies have been conducted on the P. aeruginosa IF-3 C-terminal domain (PDB: 6VRJ), providing valuable insights into the likely structure of P. putida IF-3 . Like other bacterial IF-3 proteins, P. putida IF-3 likely consists of two globular domains connected by a flexible linker, with the C-terminal domain primarily responsible for ribosome binding activities. AlphaFold 2 protein structure predictions are available for P. putida IF-3, offering computational models of its three-dimensional structure . These structural characteristics inform experimental design for functional studies and recombinant expression strategies.

What expression systems are most effective for recombinant production of P. putida IF-3?

For the recombinant production of P. putida IF-3, several expression systems can be employed with varying advantages depending on research objectives:

• Heterologous expression in E. coli: The most commonly used approach due to its high yield and simplicity. E. coli strains like BL21(DE3) with T7 promoter-based vectors (e.g., pET series) are particularly suitable. These systems can yield 10-20 mg/L of purified protein.

• Homologous expression in Pseudomonas species: P. putida KT2440 itself can be used as an expression host , which may provide advantages for proper folding and native post-translational modifications. Genetic engineering approaches similar to those used for other recombinant proteins in P. putida can be adapted for IF-3 expression .

• Cell-free protein synthesis: For rapid production at smaller scales, particularly useful for preliminary functional studies.

When using heterologous systems, codon optimization may be necessary to account for the different codon usage between E. coli and P. putida, particularly given that the P. putida KT2440 genome has distinct codon preferences within its 6.0 Mb genome .

What purification strategies yield highest purity for recombinant P. putida IF-3?

Purification of recombinant P. putida IF-3 typically follows a multi-step chromatographic approach:

• Initial capture: Affinity chromatography using His-tag (leveraging the N or C-terminus of the protein) provides good initial purification. A typical binding buffer would contain 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10 mM imidazole, with elution using an imidazole gradient (50-250 mM).

• Intermediate purification: Ion exchange chromatography exploiting the basic nature of IF-3 (pI = 9.32) . At physiological pH, IF-3 will carry a net positive charge, making cation exchange chromatography (e.g., SP Sepharose) effective.

• Polishing: Size exclusion chromatography for final purification and buffer exchange, typically using columns like Superdex 75 or Superdex 200.

This stepwise approach typically yields >95% pure protein suitable for structural and functional studies. Throughout purification, protein stability can be maintained by including reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol) and glycerol (5-10%) in all buffers.

How can researchers verify the functional activity of purified recombinant P. putida IF-3?

Functional verification of purified P. putida IF-3 can be accomplished through several complementary approaches:

• 30S ribosomal subunit binding assay: Using fluorescence anisotropy with fluorescently labeled IF-3 to measure binding to isolated 30S subunits from P. putida.

• Anti-association activity: Monitoring the ability of IF-3 to prevent association of 30S and 50S ribosomal subunits using light scattering techniques.

• In vitro translation assay: Testing the ability of purified IF-3 to complement IF-3-depleted cell extracts in a coupled transcription-translation system.

• Structural integrity verification: Using circular dichroism spectroscopy to confirm proper secondary structure content compared to theoretical predictions based on homology models.

Each of these methods provides different and complementary information about the functional state of the purified protein, with the combination providing robust validation of protein activity.

How can three-level factorial design optimize expression conditions for P. putida IF-3?

A three-level factorial design (3ᵏ design) provides a powerful approach for systematically optimizing recombinant P. putida IF-3 expression by investigating potential quadratic effects of key variables . This experimental design allows researchers to examine multiple factors at three levels each (low, intermediate, high) to identify optimal conditions and potential interactions between variables.

For P. putida IF-3 expression optimization, a 3³ design would be appropriate, examining three critical factors:

• Induction temperature (e.g., 18°C, 25°C, 30°C)

• Inducer concentration (e.g., 0.1 mM, 0.5 mM, 1.0 mM IPTG)

• Post-induction time (e.g., 4h, 8h, 16h)

This would result in 27 experimental conditions (3³ = 27) that could be represented pictorially as a three-dimensional cube . The yield and solubility of recombinant IF-3 would serve as response variables.

The mathematical model for such an experiment would include main effects with 2 degrees of freedom each and two-factor interactions . Analysis of the results would reveal not only optimal conditions but also potential curvature in the response surface that simpler two-level designs might miss, ultimately leading to improved production of functional recombinant protein.

What site-directed mutagenesis approaches are most effective for studying P. putida IF-3 functional domains?

Site-directed mutagenesis provides a powerful tool for dissecting the structure-function relationships of P. putida IF-3. Based on the available structural information from P. aeruginosa IF-3 (PDB: 6VRJ) , researchers can implement several mutagenesis strategies:

• Alanine scanning of the C-terminal domain: Systematically replacing conserved residues with alanine to identify amino acids critical for ribosome binding. This approach should focus on positively charged residues (Arg, Lys) likely involved in RNA interactions.

• Domain interface mutations: Creating substitutions at the interface between N- and C-terminal domains to investigate interdomain communication. Based on typical IF-3 architecture, these residues would be located in the linker region and adjacent areas.

• Creation of chimeric constructs: Replacing domains of P. putida IF-3 with corresponding regions from related species (e.g., P. aeruginosa, which shares 91.7% identity) to determine species-specific functional elements.

• Fluorescent protein fusions: Creating N- or C-terminal GFP fusions with strategic linkers to enable FRET studies of conformational changes during ribosome binding.

• Introduction of unique cysteines for site-specific labeling: Enabling advanced biophysical studies including single-molecule FRET.

Each mutant should be characterized for structural integrity (CD spectroscopy), thermal stability (differential scanning fluorimetry), and functional activity (ribosome binding and translation initiation assays) to build a comprehensive structure-function map of P. putida IF-3.

How does recombinant P. putida IF-3 interact with ribosomal components during translation initiation?

Investigating the interactions between P. putida IF-3 and ribosomal components requires a multi-technique approach focusing on the temporal and spatial dynamics of these interactions:

• Cryo-electron microscopy studies can reveal the structural basis of IF-3 binding to the 30S ribosomal subunit, highlighting interaction interfaces at near-atomic resolution. This approach has been successful with IF-3 from other species and could be applied to P. putida IF-3 complexed with homologous 30S subunits.

• Chemical cross-linking coupled with mass spectrometry (XL-MS) can identify specific residues involved in protein-protein and protein-RNA contacts. This method involves:

  • Using bifunctional cross-linkers (e.g., BS3, EDC)

  • Enzymatic digestion of cross-linked complexes

  • LC-MS/MS analysis to identify cross-linked peptides

  • Computational modeling to map interactions

• Time-resolved fluorescence studies with strategically labeled IF-3 can monitor conformational changes during binding events, providing kinetic information about the sequential steps of initiation complex formation.

• Ribosome profiling experiments comparing wild-type P. putida with strains expressing mutant IF-3 variants can reveal the genome-wide impact of IF-3 alterations on translation initiation site selection and efficiency.

These approaches collectively would elucidate how P. putida IF-3 functions within the context of the translation initiation machinery, particularly in comparison to the better-studied E. coli system.

What methodological approaches can resolve contradictory data about P. putida IF-3 function?

When faced with contradictory experimental results regarding P. putida IF-3 function, researchers should implement a systematic approach to identify sources of variability and establish consensus:

• Standardization of experimental materials:

  • Use sequence-verified expression constructs deposited in publicly accessible repositories

  • Implement consistent protein purification protocols with defined quality control metrics

  • Establish reference standards for activity assays with defined acceptance criteria

• Orthogonal technique validation:

  • Employ multiple independent methods to measure the same parameter

  • For binding studies: Compare results from surface plasmon resonance, isothermal titration calorimetry, and microscale thermophoresis

  • For functional studies: Combine in vitro reconstitution with in vivo complementation assays

• Systematic analysis of experimental variables:

  • Use Design of Experiments (DoE) approaches, such as three-level factorial designs , to systematically explore experimental conditions

  • Create a matrix of variables (pH, salt concentration, temperature, buffer components) to identify conditions that may cause inconsistent results

• Statistical rigor in data analysis:

  • Implement appropriate statistical tests with consideration of multiple comparisons

  • Establish minimum sample sizes based on power calculations

  • Use Bayesian statistical approaches to evaluate competing models when appropriate

This methodological framework provides a roadmap for resolving contradictions in the literature and establishing a more robust understanding of P. putida IF-3 function.

How can structural studies of P. putida IF-3 inform biodegradation pathway engineering?

While IF-3 is primarily known for its role in translation, insights from its structural and functional studies can inform broader applications in P. putida, particularly for biodegradation pathway engineering:

• Translation efficiency optimization: Understanding P. putida IF-3 function can help optimize translation of heterologous enzymes involved in biodegradation pathways. This is particularly relevant for engineering P. putida strains capable of degrading compounds like difenoconazole , where expression levels of key enzymes are critical.

• Ribosome engineering approaches: Structural information about IF-3-ribosome interactions can guide targeted modifications to enhance translation of biodegradation enzymes, particularly those with non-optimal codon usage or secondary structure.

• Stress response integration: IF-3 function is often modulated during stress responses. Understanding these mechanisms can help design more robust biodegradation strains that maintain pathway expression under environmental stresses typically encountered during bioremediation.

• Synthetic biology applications: Detailed knowledge of P. putida translation machinery, including IF-3, enables more precise genetic circuit design for controllable expression of biodegradation pathways, potentially creating strains with enhanced capabilities similar to those developed for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) production .

• Metabolic burden assessment: Translation factors like IF-3 are central to cellular resource allocation. Understanding their function can help predict and mitigate metabolic burdens when expressing complex biodegradation pathways.

These connections highlight how fundamental studies of translation factors can inform applied biotechnological applications, creating a bridge between basic and applied research in P. putida.

Key biochemical parameters of P. putida Translation initiation factor IF-3

Comparative analysis of IF-3 homologs across bacterial species

Recommended expression conditions for recombinant P. putida IF-3