Recombinant Persephonella marina Elongation factor Ts (tsf)
Product Specs
Target Background
KEGG: pmx:PERMA_0232
STRING: 123214.PERMA_0232
Q&A
What are the optimal storage and handling conditions for recombinant P. marina tsf?
Based on manufacturer specifications and standard protocols for thermostable proteins:
For experiments requiring extended stability at higher temperatures, the inherent thermostability of P. marina tsf provides an advantage compared to mesophilic homologs.
What expression systems are most effective for producing recombinant P. marina tsf?
While specific optimization studies for P. marina tsf expression are not reported in the literature, research on other thermophilic proteins from P. marina provides valuable methodological guidance:
Research on P. marina carbonic anhydrase demonstrated that cytoplasmic expression in E. coli BL21(DE3) using pET vectors yields soluble protein, though expression levels may be 20% lower than for some other thermophilic proteins .
What purification strategy yields the highest purity and activity of recombinant P. marina tsf?
A multi-step purification approach is recommended:
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Heat treatment: Exploiting the thermostability of P. marina tsf to denature E. coli host proteins (60-70°C for 15-20 minutes)
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Immobilized Metal Affinity Chromatography (IMAC): Using Ni-NTA resin to capture His-tagged tsf
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Size exclusion chromatography: For further purification and buffer exchange
Comparative analysis with purification of other P. marina proteins suggests this approach routinely yields >85% purity . For highest activity retention, purification buffers should contain:
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50 mM Tris-HCl (pH 7.5-8.0)
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150-300 mM NaCl
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5-10 mM MgCl₂ (important for nucleotide exchange activity)
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1-5 mM β-mercaptoethanol or DTT (if cysteine residues are present)
How can researchers assess the purity and functionality of purified P. marina tsf?
| Assessment Method | Parameter Measured | Technical Approach |
|---|---|---|
| SDS-PAGE | Purity and molecular weight (expected ~27 kDa) | Coomassie or silver staining |
| Western blot | Identity confirmation | Anti-His antibody or tsf-specific antibody |
| Circular Dichroism | Secondary structure integrity | Far-UV spectrum (190-260 nm) |
| Nucleotide Exchange Assay | Functional activity | Monitoring EF-Tu·mantGDP to EF-Tu·mantGTP conversion by fluorescence |
| Thermal Shift Assay | Thermostability | Differential scanning fluorimetry |
For nucleotide exchange activity measurement, stopped-flow kinetic analysis monitoring the fluorescence of either tryptophan residues in EF-Tu or mant-GDP provides quantitative assessment of function similar to methods used for E. coli tsf .
How does the mechanism of P. marina tsf-catalyzed nucleotide exchange compare with other bacterial species?
Studies of E. coli EF-Ts provide a framework for understanding the likely mechanism of P. marina tsf:
| Phase | E. coli Mechanism | Potential P. marina Adaptations |
|---|---|---|
| Initial Contact | "Base-side-first" mechanism - contacts between helix D of EF-Tu and N-terminal domain of EF-Ts weaken binding around guanine base | May include thermostable modifications to maintain contact interface integrity at high temperatures |
| Phosphate Release | Contacts with phosphate binding side promote release of phosphate moiety | Potentially strengthened interactions to compensate for increased molecular motion at high temperatures |
| GTP Binding | Facilitated by conformational changes in EF-Tu | Likely preserved mechanism with thermostable structural elements |
The "base-side-first" mechanism identified in E. coli differs from mechanisms described for other GTPase·GEF complexes where interactions at the phosphate side of the nucleotide are released first . Confirmation of this mechanism in P. marina tsf would require specific kinetic studies at elevated temperatures.
What protein-protein interactions define the functional network of P. marina tsf?
STRING database analysis reveals a conserved interaction network for P. marina tsf :
| Interaction Partner | Interaction Score | Functional Relationship |
|---|---|---|
| rpsB (Ribosomal protein S2) | 0.999 | Part of translation machinery |
| pyrH (UMP kinase) | 0.995 | Nucleotide metabolism |
| tuf_1/tuf_2 (EF-Tu) | 0.992 | Primary functional partner in translation |
| frr (Ribosome recycling factor) | 0.990 | Translation termination |
| ACO04597.1 (Ribosomal protein S01) | 0.971 | Translation machinery component |
These interaction scores suggest a highly conserved role of tsf in the translation apparatus, even in thermophilic organisms. The extremely high confidence scores (>0.97) indicate that these interactions are essential for cellular function and have been maintained despite adaptation to extreme environments.
How can molecular dynamics simulations enhance our understanding of P. marina tsf thermostability?
Molecular dynamics (MD) simulations offer valuable insights into the thermal adaptation mechanisms of P. marina tsf:
| Simulation Type | Research Question | Methodological Approach |
|---|---|---|
| Temperature ramping | Unfolding pathway identification | Gradually increasing temperature from 300K to 500K |
| Salt bridge analysis | Electrostatic network mapping | Tracking distance between charged residues over simulation time |
| Water coordination | Hydration shell dynamics | Radial distribution function analysis of water molecules |
| Principal component analysis | Essential dynamics at high temperatures | Identification of major conformational motions |
Similar approaches with other thermophilic proteins have revealed that increased salt bridge networks, reduced surface loop flexibility, and optimized hydrophobic cores contribute to thermostability . For P. marina tsf, these simulations would be particularly valuable in identifying structural features that allow nucleotide exchange activity to be maintained at high temperatures.
What structural adaptations contribute to the thermostability of proteins from deep-sea thermophiles like P. marina?
Research on P. marina carbonic anhydrase and other thermophilic proteins reveals several key adaptations that likely apply to tsf as well:
Analysis of other deep-sea proteins shows that adaptations to high pressure often overlap with thermal adaptations, with both focusing on strengthening the protein's structural integrity .
How can researchers experimentally measure and compare the thermostability of P. marina tsf?
Multiple complementary methods can assess thermostability:
| Method | Parameter Measured | Experimental Design |
|---|---|---|
| Differential Scanning Calorimetry (DSC) | Melting temperature (Tm) | Heating rate of 1°C/min, 25-100°C range |
| Circular Dichroism (CD) | Secondary structure retention | Monitoring at 222 nm during temperature ramping |
| Activity Assays at Various Temperatures | Functional thermostability | Nucleotide exchange rate measurement at 30-80°C |
| Thermal Shift Assays | Unfolding transition | SYPRO Orange fluorescence monitoring during heating |
| Long-term Stability Tests | Practical stability | Incubation at elevated temperatures (40-70°C) with periodic activity testing |
For comparative stability assessment, researchers can use temperature cycling conditions (e.g., 4-minute cycles between 40°C and 77°C) to mimic conditions used in industrial applications, similar to approaches used with thermostable carbonic anhydrases .
What structural information can be inferred about P. marina tsf from homologous proteins?
While no crystal structure of P. marina tsf is reported in the literature, structural insights can be inferred from homologous proteins:
Homology modeling using E. coli EF-Ts as a template, followed by molecular dynamics equilibration at elevated temperatures, could provide a reasonable structural model for P. marina tsf. Key differences would likely include increased surface charge, reduced loop lengths, and additional stabilizing interactions.
How can P. marina tsf be used to enhance in vitro translation systems for high-temperature applications?
P. marina tsf offers several advantages for high-temperature translation systems:
| Application | Methodological Approach | Expected Benefit |
|---|---|---|
| Thermostable Cell-Free Protein Synthesis | Incorporation of P. marina tsf, EF-Tu and other translation factors | Extended operational temperature range (50-70°C) |
| PCR-Coupled In Vitro Translation | Direct protein production from PCR products at elevated temperatures | Reduced RNA secondary structure interference |
| Continuous Translation Systems | Long-duration protein synthesis at moderate-high temperatures | Improved stability of translation machinery |
| Translation of Thermophilic Proteins | Matched translation conditions to native protein folding environment | Enhanced folding and solubility of target proteins |
For implementation, researchers should reconstitute translation systems using a combination of P. marina translation factors (tsf, EF-Tu, EF-G) while optimizing ribosome composition based on the operational temperature range.
What comparative kinetic studies would reveal adaptation mechanisms in P. marina tsf?
Systematic kinetic analysis comparing P. marina tsf with mesophilic homologs would reveal thermal adaptation mechanisms:
| Kinetic Parameter | Experimental Approach | Expected Thermophilic Adaptation |
|---|---|---|
| Association rate (kon) | Stopped-flow with fluorescent nucleotides | Potentially reduced at low temperatures, maintained at high temperatures |
| Dissociation rate (koff) | Chase experiments with excess unlabeled substrate | Temperature dependence differing from mesophilic homologs |
| Temperature dependence | Arrhenius plots of rate constants | Lower activation energy for thermophilic proteins |
| Stability of protein-protein complexes | Isothermal titration calorimetry | Enhanced enthalpy-entropy compensation |
Studying these parameters across a temperature range (20-80°C) would reveal how P. marina tsf maintains functionality under conditions where mesophilic proteins would denature. Based on studies of E. coli EF-Ts, key rate constants to measure include GDP dissociation from EF-Tu and the rate of EF-Ts binding to EF-Tu·GDP .
What mutagenesis strategies could identify key residues responsible for P. marina tsf thermostability?
Targeted mutagenesis approaches can identify critical thermostability determinants:
| Mutagenesis Approach | Target Residues | Analytical Method |
|---|---|---|
| Alanine scanning | Charged residues in predicted salt bridge networks | Thermal denaturation monitoring |
| Hydrophobic core mutations | Core residues differing from mesophilic homologs | Activity retention after thermal challenge |
| Ancestral sequence reconstruction | Introducing ancestral (pre-adaptation) residues | Comparative stability analysis |
| Loop modification | Proline substitutions or loop shortening | Thermostability and flexibility assessment |
| Disulfide engineering | Introduction of non-native disulfide bonds | Stability under reducing/non-reducing conditions |
This approach has been successful with other thermostable proteins from P. marina, where targeted mutations in carbonic anhydrase resulted in variants with up to 260% increased stability compared to wild-type .
What are the major methodological challenges in working with recombinant P. marina tsf?
Researchers face several technical challenges when working with this thermophilic protein:
| Challenge | Technical Impact | Potential Solution |
|---|---|---|
| Codon usage bias | Reduced expression in E. coli | Codon optimization or use of Rosetta strains |
| Protein misfolding at low temperatures | Reduced yield of properly folded protein | Expression at elevated temperatures (30-37°C) |
| Assay compatibility at high temperatures | Standard assay limitations | Development of thermostable reagents and protocols |
| Structural determination challenges | Difficulty obtaining crystal structures | Exploration of cryo-EM as alternative approach |
| Compatibility with mesophilic components | Functional mismatch in reconstituted systems | Systematic replacement with thermophilic counterparts |
Experience with other P. marina proteins suggests that expression can be enhanced through optimization of induction conditions and choice of appropriate host strains .
How might P. marina tsf be engineered for specific research applications?
Strategic protein engineering can adapt P. marina tsf for various applications:
| Engineering Goal | Approach | Potential Application |
|---|---|---|
| Enhanced thermostability | Introduction of additional salt bridges or disulfide bonds | Ultra-high temperature translation systems |
| Fluorescent labeling | Introduction of surface-exposed cysteine residues | Real-time monitoring of translation dynamics |
| Altered substrate specificity | Modification of EF-Tu binding interface | Study of heterologous translation systems |
| Immobilization compatibility | Addition of terminal tags for directed attachment | Reusable translation factors for biotechnology |
| pH tolerance expansion | Surface charge modification | Combined pH and temperature tolerance |
Similar engineering approaches with P. marina carbonic anhydrase have successfully created variants with enhanced stability for carbon capture applications , suggesting the feasibility of engineering tsf for specialized applications.
What emerging technologies could advance research on P. marina tsf and other thermophilic translation factors?
Several cutting-edge approaches show promise for advancing our understanding:
| Technology | Application to P. marina tsf Research | Potential Insight |
|---|---|---|
| Single-molecule FRET | Real-time observation of nucleotide exchange dynamics | Conformational changes during catalysis |
| Cryo-electron microscopy | Structure determination of tsf-EF-Tu complexes | Binding interface adaptations |
| Hydrogen-deuterium exchange mass spectrometry | Mapping flexible regions | Dynamic properties at different temperatures |
| Deep mutational scanning | Comprehensive mutational landscape | Identification of all stability-determining residues |
| AlphaFold2 and other AI structure prediction | Model generation and refinement | Structure prediction without crystallization |
These technologies could overcome current limitations in studying thermophilic proteins and provide unprecedented insights into how P. marina tsf functions under extreme conditions.
