Recombinant Oligotropha carboxidovorans Elongation factor Tu (tuf)

What is Oligotropha carboxidovorans and why is it scientifically significant?

Oligotropha carboxidovorans is an aerobic gram-negative bacterium characterized by its chemolithoautotrophic metabolism, most notably its ability to utilize carbon monoxide (CO) as a sole energy and carbon source. This unique metabolic capability makes O. carboxidovorans a significant model organism for studying carbon fixation pathways and bacterial adaptation to nutrient-limited environments. The organism oxidizes CO through a specialized CO dehydrogenase (CODH) enzyme containing a unique bimetallic [CuSMoO₂] cluster in its active site that matures posttranslationally after integration into the completely folded apoenzyme . This distinctive enzymatic mechanism has drawn considerable research attention for potential biotechnological applications in carbon capture and bioremediation processes. The complete genome of O. carboxidovorans OM5 has been sequenced, revealing 3,582 genes, including 3,482 protein-coding genes distributed across its 3,745,629 base pair genome .

What is Elongation factor Tu (tuf) and what is its function in bacterial systems?

Elongation factor Tu (EF-Tu), encoded by the tuf gene, is a highly conserved GTP-binding protein essential for protein synthesis in all bacteria. In bacterial translation, EF-Tu forms a ternary complex with GTP and aminoacyl-tRNA, delivering the aminoacyl-tRNA to the ribosomal A-site during the elongation phase of protein synthesis. Upon correct codon-anticodon pairing, EF-Tu hydrolyzes GTP and dissociates from the ribosome, allowing for the incorporation of the amino acid into the growing peptide chain. Given its central role in protein synthesis, EF-Tu typically constitutes 5-10% of total cellular protein and is often expressed at high levels during active growth phases. While specific information about O. carboxidovorans EF-Tu is limited in the provided research materials, comparative analysis with other bacterial systems suggests it likely plays similar fundamental roles in translation while potentially harboring species-specific structural adaptations that could reflect the organism's unique ecological niche and metabolic adaptations.

Why would researchers be interested in recombinant expression of O. carboxidovorans Elongation factor Tu?

Researchers pursue recombinant expression of O. carboxidovorans Elongation factor Tu for multiple scientific objectives. First, as a model chemolithoautotroph with unique metabolic capabilities, O. carboxidovorans potentially harbors molecular adaptations in its translation machinery that could provide insights into protein synthesis under specialized growth conditions. Second, recombinant expression enables structure-function studies through site-directed mutagenesis, allowing researchers to investigate specific amino acid residues important for EF-Tu activity in this organism. Third, heterologous expression systems facilitate large-scale protein production for crystallographic and biophysical characterization, which would be challenging to achieve with native purification from O. carboxidovorans cultures. The establishment of successful heterologous expression systems for other O. carboxidovorans proteins, such as CO dehydrogenase in E. coli, demonstrates the feasibility and scientific value of this approach . These recombinant systems have proven instrumental in enabling detailed kinetic and spectroscopic studies of O. carboxidovorans proteins and facilitate active-site variants generation for mechanistic investigations.

What expression systems are most suitable for recombinant production of O. carboxidovorans Elongation factor Tu?

The selection of an optimal expression system for recombinant O. carboxidovorans Elongation factor Tu requires careful consideration of multiple factors including codon optimization, post-translational requirements, and protein solubility. Based on successful heterologous expression of other O. carboxidovorans proteins, several systems merit consideration:

• E. coli expression systems: The successful expression of O. carboxidovorans CO dehydrogenase (CODH) in E. coli demonstrates that this organism can serve as an effective host for O. carboxidovorans proteins . For EF-Tu expression, E. coli BL21(DE3) or its derivatives would be appropriate due to their reduced protease activity and tight regulation of expression via the T7 promoter system. Codon optimization may be necessary, as O. carboxidovorans (GC content ~64.5%) codon usage might differ from E. coli.

• Baculovirus expression systems: For cases requiring eukaryotic post-translational modifications or improved protein folding, baculovirus expression in insect cells has proven effective for other O. carboxidovorans proteins as demonstrated in the production of the UPF0317 protein OCAR_7359/OCA5_c07590 . This system might be particularly valuable if E. coli expression yields insoluble protein.

• Pichia pastoris (Komagataella phafii): For large-scale production, the methylotrophic yeast P. pastoris offers advantages including growth to high cell densities, strong promoters, and effective secretion capabilities. While not directly mentioned in the search results for O. carboxidovorans proteins, P. pastoris has been genetically modified for improved recombinant protein expression through approaches like OCH1 gene knockout and introduction of various glycosylation enzymes .

The final selection should balance expression level, protein solubility, post-translational requirements, and downstream purification considerations. Initial pilot experiments in E. coli with different fusion tags (His6, GST, MBP) would provide valuable data to guide system optimization.

What are the critical factors for optimizing expression of recombinant O. carboxidovorans EF-Tu in E. coli?

Optimizing recombinant expression of O. carboxidovorans EF-Tu in E. coli requires systematic attention to multiple parameters:

Expression vector selection and genetic elements:

• Promoter strength and inducibility (T7, tac, or araBAD promoters)

• Codon optimization to align with E. coli codon usage preferences

• Inclusion of appropriate fusion tags (N- or C-terminal) to enhance solubility and facilitate purification

• Incorporation of protease cleavage sites for tag removal with minimal residual amino acids

Expression conditions:

• Induction timing (typically at mid-logarithmic phase, OD₆₀₀ = 0.6-0.8)

• Inducer concentration (IPTG at 0.1-1.0 mM for T7-based systems)

• Post-induction temperature (lower temperatures of 16-25°C often enhance solubility)

• Media composition (rich media like TB or minimal media with specific supplements)

Host strain considerations:

• BL21(DE3) derivatives optimized for different purposes:

  • Rosetta for rare codon supplementation

  • C41/C43 for potentially toxic proteins

  • SHuffle for disulfide bond formation

Post-harvest processing:

• Cell lysis methods (sonication, high-pressure homogenization)

• Buffer composition, including:

  • pH optimization (typically 7.0-8.0 for EF-Tu)

  • Salt concentration (100-500 mM NaCl)

  • Stabilizing additives (glycerol at 5-10%)

  • Protease inhibitors

Drawing from the successful heterologous expression of O. carboxidovorans CO dehydrogenase in E. coli , a promising initial approach would employ a pET-based expression vector with an N-terminal His6-tag in BL21(DE3) cells, followed by induction with 0.2-0.5 mM IPTG at OD₆₀₀ = 0.8 and expression at 20°C for 16-18 hours to maximize soluble protein yield.

How can researchers assess and optimize the folding and activity of recombinant O. carboxidovorans EF-Tu?

Assessing proper folding and functional activity of recombinant O. carboxidovorans EF-Tu requires a multi-faceted approach:

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy to analyze secondary structure composition and thermal stability

• Intrinsic tryptophan fluorescence to evaluate tertiary structure

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to confirm appropriate oligomeric state

• Limited proteolysis to detect properly folded domains resistant to proteolytic digestion

Functional activity assays:

• GTP binding capacity using fluorescent GTP analogs (mant-GTP) or filter-binding assays with radiolabeled [γ-³²P]GTP

• GTPase activity measurement via:

  • Colorimetric phosphate release assays (malachite green)

  • HPLC-based methods for guanine nucleotide quantification

• Aminoacyl-tRNA binding assays using filter binding or fluorescence anisotropy methods

• In vitro translation assays using purified ribosomes and translation components

Comparative analysis approach:
Researchers should benchmark the recombinant O. carboxidovorans EF-Tu against well-characterized EF-Tu proteins from model organisms like E. coli. This comparative approach allows for verification of expected properties while potentially identifying unique characteristics of the O. carboxidovorans protein. For instance, the CO dehydrogenase from O. carboxidovorans expressed in E. coli was validated by comparing its kinetic and spectroscopic properties with those of the native enzyme purified from O. carboxidovorans . A similar strategy would be appropriate for EF-Tu characterization.

If initial expression produces inactive protein, optimization strategies include co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE), adjustment of expression conditions to slower growth rates, or exploration of alternative expression systems as outlined in FAQ 2.1.

What is the optimal purification strategy for recombinant O. carboxidovorans EF-Tu?

A comprehensive purification strategy for recombinant O. carboxidovorans EF-Tu should balance high recovery, purity, and preservation of biological activity. Based on established protocols for similar proteins, the following multi-step approach is recommended:

Step 1: Initial capture

• Immobilized Metal Affinity Chromatography (IMAC) for His-tagged EF-Tu using Ni-NTA or Co-TALON resins

• Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol, with imidazole gradient elution (10-250 mM)

• Alternative: GTP-agarose affinity chromatography exploiting EF-Tu's natural nucleotide-binding capability

Step 2: Intermediate purification

• Ion Exchange Chromatography (IEX) to separate based on surface charge distribution

  • Typical conditions: Q-Sepharose at pH 7.5-8.0 with 50-500 mM NaCl gradient

• Tag removal (if desired) using specific proteases (TEV, PreScission, or thrombin) followed by reverse IMAC

Step 3: Polishing

• Size Exclusion Chromatography (SEC) to achieve final purity and separate oligomeric states

  • Recommended column: Superdex 75 or 200 in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, 5% glycerol

Critical considerations:

• Include GDP or GTP (typically 50-100 μM) in purification buffers to stabilize the protein

• Maintain reducing conditions with 1-5 mM DTT or 0.5-2 mM TCEP to prevent oxidation of cysteine residues

• Monitor protein stability and quality throughout purification via activity assays and biophysical methods

The optimal storage conditions typically involve flash-freezing aliquots in liquid nitrogen and storing at -80°C in a buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, 50 μM GDP, 5% glycerol, and 1 mM DTT. The purification approach should be validated by SDS-PAGE, western blotting, mass spectrometry, and activity assays to ensure consistent protein quality.

What analytical methods are most informative for structural characterization of O. carboxidovorans EF-Tu?

Comprehensive structural characterization of O. carboxidovorans EF-Tu requires a multi-technique approach spanning various resolution levels:

Primary structure confirmation:

• Mass spectrometry (MS) analyses:

  • Intact mass determination using ESI-MS or MALDI-TOF

  • Peptide mapping with LC-MS/MS after tryptic digestion to verify sequence coverage

  • Post-translational modification identification

• N-terminal sequencing by Edman degradation to confirm processing

Secondary and tertiary structure analysis:

• Circular dichroism (CD) spectroscopy to assess secondary structure composition and thermal stability

• Fourier-transform infrared spectroscopy (FTIR) for complementary secondary structure information

• Nuclear magnetic resonance (NMR) spectroscopy for solution structure determination:

  • ¹H-¹⁵N HSQC experiments to evaluate folding quality

  • Complete structure determination for smaller domains or the entire protein if feasible

• X-ray crystallography for high-resolution structure determination:

  • Crystallization screening with various nucleotides (GDP, GTP, GTP analogs)

  • Co-crystallization with aminoacyl-tRNA and/or ribosomal components

Conformational dynamics:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to monitor protein dynamics

• Small-angle X-ray scattering (SAXS) for solution-state conformational analysis

• Cryo-electron microscopy (cryo-EM) for structural analysis of EF-Tu in complex with larger components

Comparative structure analysis:

• Homology modeling against known bacterial EF-Tu structures

• Molecular dynamics simulations to predict conformational changes during the GTPase cycle

The near-atomic resolution structure (1.1Å) achieved for O. carboxidovorans CO dehydrogenase demonstrates that high-resolution structural determination is feasible for proteins from this organism. Similar techniques could be applied to EF-Tu, with particular attention to capturing different conformational states associated with its GTPase cycle (GTP-bound, GDP-bound, and nucleotide-free states).

How can researchers effectively characterize the functional properties of recombinant O. carboxidovorans EF-Tu?

Comprehensive functional characterization of recombinant O. carboxidovorans EF-Tu requires multiple assays addressing its three primary activities: GTP binding, GTP hydrolysis, and aminoacyl-tRNA interaction. The following methodological framework provides a systematic approach:

GTP binding characterization:

• Equilibrium binding assays using:

  • Fluorescent nucleotide analogs (mant-GTP, BODIPY-GTP)

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Surface plasmon resonance (SPR) for kinetic binding constants

• Competition assays with unlabeled nucleotides to determine relative affinities for GTP, GDP, and GTP analogs

GTPase activity analysis:

• Steady-state kinetic parameters (k_cat, K_M) determination using:

  • Colorimetric phosphate detection (malachite green assay)

  • HPLC-based nucleotide quantification

  • Enzyme-coupled assays linking phosphate release to NADH oxidation

• Pre-steady-state kinetics using stopped-flow spectroscopy to resolve individual steps

• Influence of factors on GTPase activity:

  • Temperature and pH dependence profiles

  • Effect of monovalent and divalent cations (K⁺, NH₄⁺, Mg²⁺)

  • Activation by ribosomes and aminoacyl-tRNA

Aminoacyl-tRNA interactions:

• Ternary complex formation (EF-Tu·GTP·aa-tRNA) assessed by:

  • Native gel electrophoresis

  • Size exclusion chromatography

  • Fluorescence anisotropy with labeled tRNAs

• Specificity for different aminoacyl-tRNAs using competition assays

Ribosomal interactions:

• Ribosome-stimulated GTPase activity measurements

• Binding studies with isolated ribosomal components

• In vitro translation assays to assess functional incorporation into the translation machinery

The table below presents typical parameters for EF-Tu from various bacterial sources that can serve as benchmarks for O. carboxidovorans EF-Tu characterization:

As demonstrated with the CO dehydrogenase studies , comparing the recombinant enzyme characteristics with those of the native enzyme (if available) provides valuable validation of the recombinant protein's functional integrity.

How can site-directed mutagenesis of O. carboxidovorans EF-Tu inform structure-function relationships?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of O. carboxidovorans EF-Tu. The successful establishment of a functional heterologous expression system for O. carboxidovorans CO dehydrogenase in E. coli has already demonstrated the feasibility of generating active-site variants for mechanistic studies . A similar approach for EF-Tu would provide insights into this protein's unique properties.

Key residues for targeted mutagenesis:

• GTP-binding pocket residues:

  • P-loop motif (GXXXXGK[S/T]) involved in phosphate binding

  • Switch I and Switch II regions that undergo conformational changes upon GTP hydrolysis

  • Magnesium coordination residues

• GTPase catalytic residues:

  • His84 (E. coli numbering) implicated in the GTPase mechanism

  • Asp21 and other residues that position the catalytic water molecule

• Aminoacyl-tRNA interaction interface:

  • Residues in domains 2 and 3 that contact the tRNA acceptor stem

  • Residues interacting with the aminoacyl moiety that contribute to specificity

• Ribosome interaction regions:

  • Residues contacting 23S rRNA

  • Regions interacting with ribosomal proteins

Methodological approach:

• Design phase:

  • Perform multiple sequence alignment of EF-Tu sequences to identify conserved vs. variable residues

  • Analyze available EF-Tu crystal structures to predict critical residues

  • Design mutations: conservative (e.g., Asp→Glu) and non-conservative (e.g., Asp→Ala) substitutions

• Implementation:

  • Use PCR-based site-directed mutagenesis or Gibson Assembly methods

  • Verify mutations by DNA sequencing

  • Express and purify mutant proteins following optimized protocols

• Functional characterization:

  • Assess structural integrity through thermal stability assays (DSF, CD)

  • Determine GTP binding affinities for each mutant

  • Measure intrinsic and ribosome-stimulated GTPase activities

  • Evaluate aminoacyl-tRNA binding capabilities

• Data analysis and interpretation:

  • Compare kinetic parameters (k_cat, K_M, k_cat/K_M) between wild-type and mutant proteins

  • Investigate structure-activity relationships through correlation analysis

  • Develop mechanistic models explaining the roles of specific residues

This approach would be particularly valuable for identifying adaptations in O. carboxidovorans EF-Tu that might relate to the organism's unique chemolithoautotrophic lifestyle. Additionally, comparing the effects of equivalent mutations across EF-Tu proteins from diverse bacteria could reveal species-specific functional adaptations.

What approaches can be used to study the interaction of O. carboxidovorans EF-Tu with the translational machinery?

Investigating the interactions between O. carboxidovorans EF-Tu and components of the translational machinery requires a multi-faceted approach combining biochemical, biophysical, and structural methods:

Reconstituted in vitro translation systems:

• Develop a hybrid translation system using:

  • Purified O. carboxidovorans EF-Tu

  • Ribosomes and translation factors from model organisms (E. coli)

  • Various aminoacyl-tRNAs to assess specificity

• Measure translation efficiency using reporter systems:

  • Firefly luciferase for quantitative output

  • Fluorescent proteins for real-time monitoring

Direct binding and kinetic studies:

• Surface Plasmon Resonance (SPR) to determine:

  • Association and dissociation rates with ribosomes

  • Binding constants with isolated ribosomal components

• Microscale Thermophoresis (MST) for interaction studies in solution

• Fluorescence-based approaches:

  • FRET pairs between labeled EF-Tu and ribosomal components

  • Single-molecule FRET to capture dynamic interactions

Crosslinking and mass spectrometry:

• Chemical crosslinking followed by mass spectrometry (XL-MS) to identify:

  • Residues at interaction interfaces

  • Conformational changes upon complex formation

• Site-specific incorporation of photoreactive amino acids for precise contact mapping

Structural analysis of complexes:

• Cryo-electron microscopy of:

  • EF-Tu bound to ribosomes at different functional states

  • Ternary complex interactions with the ribosomal A-site

• X-ray crystallography of sub-complexes where feasible

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

Comparative systems approach:
Compare the interaction properties of O. carboxidovorans EF-Tu with those of EF-Tu from:

• Model organisms (E. coli, B. subtilis)

• Other chemolithoautotrophs

• Phylogenetically related bacteria

This comparative approach could reveal adaptations specific to O. carboxidovorans that might relate to protein synthesis under its unique metabolic conditions, such as during carbon monoxide utilization. The successful heterologous expression of O. carboxidovorans proteins in E. coli suggests that development of these interaction assays should be feasible with recombinant components.

How might O. carboxidovorans EF-Tu differ from EF-Tu proteins in other bacterial species?

O. carboxidovorans EF-Tu likely exhibits both conserved features essential for translational function and unique adaptations reflecting the organism's specialized metabolism and ecological niche. While specific information about O. carboxidovorans EF-Tu is limited in the search results, a predictive analysis can be constructed based on knowledge of bacterial EF-Tu proteins and O. carboxidovorans biology:

Potential adaptations in primary structure:

• Codon usage and GC content: O. carboxidovorans has a high GC content genome (approximately 64.5%) , which would influence codon usage in the tuf gene. This might result in amino acid composition biases compared to EF-Tu from organisms with different GC content.

• Thermal stability adaptations: As a soil bacterium capable of chemolithoautotrophic growth, O. carboxidovorans may encounter variable temperature conditions. Its EF-Tu might contain adaptations affecting thermal stability, potentially including:

  • Altered surface charge distribution

  • Modified hydrophobic core packing

  • Differential salt bridge and hydrogen bond networks

• Post-translational modifications: The pattern of post-translational modifications on EF-Tu could differ, potentially including unique phosphorylation, methylation, or acetylation sites that regulate activity in response to metabolic states specific to carbon monoxide utilization.

Potential functional adaptations:

• Nucleotide binding and hydrolysis: The GTP binding pocket might show adaptations affecting:

  • Binding affinities for GTP/GDP

  • Intrinsic GTPase activity rates

  • Response to cellular energy status

• Aminoacyl-tRNA interactions: Modifications in domains 2 and 3 could alter:

  • Selectivity for different aminoacyl-tRNAs

  • Binding kinetics and stability of ternary complexes

  • Sensitivity to amino acid starvation conditions

• Ribosome interactions: Adaptations at the ribosome-binding interface might affect:

  • Affinity for ribosomes

  • Rates of accommodation and GTP hydrolysis

  • Sensitivity to translation-targeting antibiotics

Experimental approaches to identify differences:

• Comparative sequence and structure analysis:

  • Multiple sequence alignment with diverse bacterial EF-Tu sequences

  • Homology modeling based on known EF-Tu structures

  • Phylogenetic analysis to identify lineage-specific adaptations

• Differential functional assays:

  • Comparing temperature and pH activity profiles

  • Assessing stability under various stress conditions

  • Measuring aminoacyl-tRNA selectivity patterns

• Cross-complementation studies:

  • Testing whether O. carboxidovorans EF-Tu can functionally replace EF-Tu in model organisms

  • Evaluating growth phenotypes under various stress conditions

Understanding these differences would provide insights into how translation machinery adapts to specialized metabolic lifestyles and could potentially reveal novel regulatory mechanisms specific to chemolithoautotrophic bacteria.

What are common challenges in recombinant expression of O. carboxidovorans proteins and how can they be addressed?

Recombinant expression of O. carboxidovorans proteins presents several challenges, as evidenced by the need for specialized approaches in previous studies . Below are common issues and strategic solutions:

Challenge 1: Codon usage bias and rare codons

• Problem: The high GC content of O. carboxidovorans genome (~64.5%) creates codon usage patterns that differ significantly from typical expression hosts like E. coli.

• Solutions:

  • Use codon-optimized synthetic genes adapted to the expression host

  • Select E. coli strains supplemented with rare tRNAs (e.g., Rosetta, CodonPlus)

  • Reduce expression temperature to allow slower, more accurate translation

Challenge 2: Protein solubility and folding

• Problem: Heterologous expression often leads to inclusion body formation due to rapid expression and different folding environments.

• Solutions:

  • Employ solubility-enhancing fusion partners (MBP, SUMO, or TrxA)

  • Optimize induction conditions (lower IPTG concentration, reduced temperature)

  • Co-express molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Screen multiple expression constructs with varied N- and C-termini

Challenge 3: Metal coordination and cofactor incorporation

• Problem: O. carboxidovorans proteins often require specific metal cofactors, as seen with the [CuSMoO₂] cluster in CO dehydrogenase .

• Solutions:

  • Supplement expression media with required metals

  • Consider post-expression metal reconstitution protocols

  • Explore periplasmic expression for proteins requiring disulfide bonds

Challenge 4: Post-translational modifications

• Problem: Bacterial expression systems may lack machinery for specific post-translational modifications.

• Solutions:

  • Identify essential modifications through mass spectrometry of native protein

  • Select expression systems capable of performing required modifications

  • Consider chemical or enzymatic modification of purified protein

Challenge 5: Protein stability during purification

• Problem: Recombinant proteins may exhibit reduced stability outside their native environment.

• Solutions:

  • Optimize buffer conditions (pH, salt concentration, additives)

  • Include stabilizing ligands (e.g., nucleotides for EF-Tu)

  • Determine aggregation propensity using dynamic light scattering

  • Add protease inhibitors and reducing agents during purification

Practical approach based on previous successes:

The successful heterologous expression of O. carboxidovorans CO dehydrogenase in E. coli provides a valuable template. This study demonstrated that functional expression of complex O. carboxidovorans proteins is achievable with appropriate optimization. For EF-Tu expression, a similar systematic approach would involve:

• Initial screening of multiple expression constructs with various tags

• Testing expression in different E. coli strains at varied temperatures

• Optimization of lysis and purification conditions to maintain nucleotide binding

• Functional validation through comparison with EF-Tu from model organisms

This methodical approach addresses the specific challenges of O. carboxidovorans protein expression while leveraging established protocols from previous successful studies.

How can researchers validate that recombinant O. carboxidovorans EF-Tu maintains native-like properties?

Validating the native-like properties of recombinantly expressed O. carboxidovorans EF-Tu requires a comprehensive comparison with the native protein or established benchmarks. The following multi-faceted approach ensures that the recombinant protein accurately represents the natural counterpart:

Structural validation:

• Mass spectrometry characterization:

  • Intact mass analysis to confirm correct primary structure

  • Peptide mapping to verify sequence coverage and identify any unexpected modifications

  • Comparison with theoretical mass based on the genomic sequence

• Secondary and tertiary structure analysis:

  • Circular dichroism (CD) spectroscopy to confirm expected secondary structure content

  • Intrinsic fluorescence spectroscopy to assess tertiary folding

  • Thermal denaturation profiles to determine stability parameters

• Oligomeric state verification:

  • Size exclusion chromatography to confirm monomeric state

  • Dynamic light scattering to assess homogeneity

  • Analytical ultracentrifugation for definitive molecular weight determination

Functional validation:

• Core biochemical activities:

  • Nucleotide binding affinities (GTP and GDP)

  • Intrinsic and ribosome-stimulated GTPase activities

  • Aminoacyl-tRNA binding capabilities

• Comparative benchmarking:

  • Side-by-side comparison with EF-Tu from well-characterized organisms

  • Assessment of activity under varied conditions (temperature, pH, ionic strength)

  • Evaluation of stability and activity retention during storage

• Integration into translation systems:

  • Functional complementation in translation assays

  • Ability to support poly(Phe) synthesis in minimal translation systems

  • Interaction with known EF-Tu binding partners

Validation strategy from previous O. carboxidovorans protein studies:

The successful heterologous expression of O. carboxidovorans CO dehydrogenase provides an informative precedent. In that study, researchers validated the recombinant enzyme by showing that it was "purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans" . A similar approach for EF-Tu would involve:

• Direct comparison with native EF-Tu isolated from O. carboxidovorans (if feasible)

• Kinetic parameter comparison with established values for bacterial EF-Tu proteins

• Functional complementation tests in systems where EF-Tu activity can be directly measured

The table below outlines typical validation parameters that should be assessed and their expected values based on bacterial EF-Tu literature:

This systematic validation approach ensures that the recombinant protein accurately represents the native O. carboxidovorans EF-Tu and provides a reliable foundation for subsequent research applications.

What are the most promising future research directions involving recombinant O. carboxidovorans EF-Tu?

The development of recombinant expression systems for O. carboxidovorans proteins has opened significant avenues for research, as demonstrated by the successful heterologous expression of CO dehydrogenase in E. coli . Several promising research directions for recombinant O. carboxidovorans EF-Tu include:

• Comparative translation systems biology: Investigating how EF-Tu from a chemolithoautotroph like O. carboxidovorans differs from heterotrophic counterparts could reveal adaptations in translation machinery that support specialized metabolic lifestyles. This comparative approach could identify novel regulatory mechanisms that coordinate protein synthesis with carbon monoxide utilization pathways.

• Structural biology of specialized adaptations: High-resolution structural studies of O. carboxidovorans EF-Tu, similar to the near-atomic resolution achieved for its CO dehydrogenase (1.1Å) , could reveal unique structural features. Of particular interest would be structures capturing different nucleotide-bound states and interactions with aminoacyl-tRNAs.

• Translation regulation under extreme conditions: O. carboxidovorans thrives in environments where it utilizes toxic carbon monoxide as a carbon and energy source. Studies examining how EF-Tu function is maintained under these challenging conditions could provide insights into translation resilience mechanisms.

• Evolution of translation machinery in specialized metabolic niches: Phylogenetic and functional analyses comparing O. carboxidovorans EF-Tu with homologs from related organisms could illuminate the evolutionary trajectory of translation components during adaptation to chemolithoautotrophic metabolism.

• Synthetic biology applications: The unique properties of translation components from specialized organisms like O. carboxidovorans might be harnessed for synthetic biology applications, such as developing translation systems optimized for extreme conditions or specialized metabolic engineering contexts.