Recombinant Pyrococcus furiosus UPF0252 protein PF1496 (PF1496)

What expression systems are most effective for producing recombinant PF1496 protein?

Multiple expression systems can be used for PF1496 protein production, each with distinct advantages:

• E. coli expression system: Most commonly used due to:

  • Higher protein yields

  • Shorter production timeframes

  • Well-established protocols

  • Cost-effectiveness

• Yeast expression system:

  • Offers good yields

  • Provides some post-translational modifications

  • Maintains protein solubility

• Insect/Baculovirus expression system:

  • Provides more complex post-translational modifications

  • May improve protein folding

  • Better for functional studies requiring specific modifications

• Mammalian cell expression:

  • Most sophisticated post-translational modifications

  • Closest to native protein structure

  • May preserve activity requiring complex modifications

For standard structural studies and initial characterization, the E. coli system using strains like Rosetta 2(DE3)pLysS has shown successful expression of P. furiosus proteins with approximately 75% of recombinant proteins efficiently expressed .

What are the optimal storage conditions for recombinant PF1496 protein to maintain stability?

Based on published protocols, the following storage conditions are recommended for maintaining PF1496 protein stability:

Important recommendations:

• Repeated freezing and thawing is not recommended as it can lead to protein degradation and activity loss

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

• For reconstitution of lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

How can researchers optimize the purification protocol for His-tagged PF1496 protein?

A methodological approach to purifying His-tagged PF1496 includes these key optimizations:

• Lysis optimization:

  • Use anaerobic conditions when possible to prevent oxidation

  • Include protease inhibitors appropriate for thermophilic proteins

  • For thermostable proteins like PF1496, consider heat treatment (70-80°C) of lysate to denature E. coli proteins while preserving target protein

• Affinity chromatography parameters:

  • Use Ni-NTA resin with 20-30 mM imidazole in binding buffer to reduce non-specific binding

  • Perform gradient elution with 50-300 mM imidazole to obtain purer fractions

  • Consider using TALON resin (Co2+-based) for higher specificity if Ni-NTA yields contaminants

• Buffer composition for optimal stability:

  • Maintain pH 8.0 throughout purification

  • Include 6% Trehalose to improve protein stability

  • Consider adding reducing agents if the protein contains cysteine residues

• Additional purification steps:

  • Size exclusion chromatography using Superdex or Sephacryl resins can further improve purity

  • For highest purity requirements, consider ion exchange chromatography as a polishing step

The purification should aim for greater than 90% purity as determined by SDS-PAGE .

What are the challenges in studying the function of an uncharacterized protein like PF1496?

Studying uncharacterized proteins like PF1496 presents several methodological challenges:

• Lack of homology-based function prediction:

  • UPF (Uncharacterized Protein Family) designation indicates limited sequence similarity to characterized proteins

  • Traditional BLAST searches may provide minimal functional insights

  • Advanced approaches like profile-based searches (HHpred) and structural prediction (AlphaFold) may offer better clues

• Expression challenges:

  • Potential toxicity to host cells when overexpressed

  • Improper folding in mesophilic expression systems

  • Formation of inclusion bodies requiring refolding protocols

• Functional assay development:

  • Without predicted function, researchers must design broad screening approaches:

    • Metabolite binding assays using differential scanning fluorimetry

    • Activity screens against diverse substrate libraries

    • Pull-down experiments to identify interaction partners

• Biological context considerations:

  • The hyperthermophilic nature of P. furiosus requires special consideration for assay conditions

  • Standard enzymatic assays may need to be performed at elevated temperatures (80-100°C)

  • Interacting partners from the original organism may be required for function

Researchers addressing these challenges typically employ iterative approaches combining computational predictions, high-throughput screening, and targeted biochemical characterization .

How does the thermostability of PF1496 compare to other proteins from P. furiosus?

While specific thermostability data for PF1496 is not directly reported in the provided sources, we can draw comparisons based on what is known about P. furiosus proteins generally:

The following methodological approaches can be used to determine the thermostability of PF1496:

• Differential Scanning Calorimetry (DSC) to determine the melting temperature (Tm)

• Activity assays at increasing temperatures (once function is determined)

• Circular Dichroism (CD) spectroscopy to monitor structural changes with increasing temperature

• Limited proteolysis at different temperatures to assess structural integrity

P. furiosus proteins generally display exceptional thermostability due to several adaptations:

• Increased number of ion pairs

• More hydrophobic core interactions

• Decreased loop regions

• Higher proportion of charged amino acids on the surface

What methodologies are most effective for determining the three-dimensional structure of PF1496 protein?

For determining the structure of PF1496, researchers should consider these methodological approaches, with specific considerations for this archaeal protein:

• X-ray Crystallography:

  • Crystallization optimization: Screen thermophilic crystallization conditions (higher temperatures, higher salt concentrations)

  • Data collection: Consider room temperature data collection to capture native conformation

  • Phase determination: Use selenomethionine labeling for MAD/SAD phasing

  • Advantages: Highest resolution potential (potentially sub-2.0 Å)

  • Challenges: Obtaining diffraction-quality crystals

• Cryo-Electron Microscopy (Cryo-EM):

  • Sample preparation: Test detergent screening if PF1496 has predicted transmembrane regions

  • Data processing: Implement 3D classification to identify conformational states

  • Advantages: No crystallization required, visualization of multiple conformations

  • Challenges: May require larger protein complexes for reliable reconstruction

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Sample requirements: 15N/13C isotopic labeling in minimal media

  • Experimental approach: Start with 2D HSQC to assess feasibility

  • Advantages: Solution structure, dynamics information

  • Challenges: Limited by protein size (39.9 kDa may be challenging)

• Integrative structural biology approaches:

  • Combine AlphaFold2 predictions with experimental validation

  • Use small-angle X-ray scattering (SAXS) for envelope determination

  • Apply cross-linking mass spectrometry to identify distance constraints

For membrane-associated proteins like PF1496 (which has predicted transmembrane regions), structural determination presents additional challenges that might require specialized approaches similar to those used for the membrane-bound hydrogenase (MBH) from P. furiosus .

How can researchers investigate potential protein-protein interactions involving PF1496 in the context of P. furiosus biology?

Investigating PF1496's protein-protein interactions requires specialized approaches considering its archaeal origin and thermophilic nature:

• Co-immunoprecipitation under thermophilic conditions:

  • Use His-tag pull-down with thermostable buffers

  • Perform experiments at elevated temperatures (60-80°C)

  • Cross-link interacting proteins before cell lysis

  • Identify partners by mass spectrometry

• Yeast two-hybrid adaptations:

  • Use thermotolerant yeast strains

  • Consider splitting the 338 aa protein into domains to avoid folding issues

  • Create a P. furiosus-specific cDNA library for comprehensive screening

• In vitro reconstitution approaches:

  • Express potential interacting partners identified through genomic context

  • Perform binding assays at elevated temperatures

  • Use techniques like isothermal titration calorimetry (ITC) or microscale thermophoresis

• Proximity labeling in heterologous systems:

  • Express PF1496 fused to BioID or APEX2 in thermophilic hosts

  • Perform labeling at highest temperatures compatible with the system

  • Identify biotinylated proteins by streptavidin pull-down and MS

• Genomic context analysis:

  • Analyze the genomic neighborhood of PF1496 for potential functional partners

  • Examine gene co-occurrence patterns across archaeal genomes

  • Look for conserved operonic structures

Similar approaches have been successfully used to characterize other P. furiosus proteins, such as the RNase P complex and the membrane-bound hydrogenase complex , where recombinant expression and reconstitution enabled functional characterization of multi-protein assemblies.

What computational approaches can predict functional domains and potential enzymatic activities of PF1496?

Advanced computational approaches can help predict the function of uncharacterized proteins like PF1496:

• Structure-based function prediction:

  • Generate AlphaFold2 or RoseTTAFold structural models

  • Use structure comparison tools (DALI, VAST) to identify similar folds

  • Apply ProFunc or COFACTOR for binding site and function prediction

  • Examine surface electrostatics for potential nucleic acid binding regions

• Sequence-based deep learning approaches:

  • Apply ESM (Evolutionary Scale Modeling) to detect distant homologies

  • Use protein language models to identify functional motifs

  • Implement DeepFRI for function recognition from sequence information

• Integrated genomic context analysis:

  • Examine gene neighborhood conservation across Thermococcales

  • Apply STRING database for functional association networks

  • Use phylogenetic profiling to identify co-evolving proteins

• Substrate docking and molecular dynamics:

  • Perform virtual screening of potential metabolites

  • Conduct molecular dynamics simulations at elevated temperatures (80-100°C)

  • Identify stable binding pockets and potential catalytic residues

• Transmembrane topology analysis:

  • PF1496 contains predicted transmembrane regions

  • Use TMHMM, TOPCONS, and DeepTMHMM for topology prediction

  • Analyze amphipathic helices that might interact with membranes

A careful examination of PF1496's sequence reveals several interesting features that might hint at function:

• Multiple transmembrane domains suggesting membrane association

• Conserved cysteine residues that might be involved in metal coordination

• Motifs consistent with transporter or channel functionality

What are the key considerations when designing experiments to determine the biochemical function of PF1496?

Designing experiments to elucidate PF1496's function requires addressing several methodological considerations:

• Temperature optimization:

  • Conduct experiments at multiple temperatures (37°C, 60°C, 80°C, 95°C)

  • Use thermostable buffers and reaction components

  • Include controls with thermolabile and thermostable proteins

• Substrate screening approach:

  • Develop a hierarchical screening strategy:

    1. Start with broad substrate classes (nucleotides, sugars, amino acids, lipids)

    2. Narrow down to specific compound families based on initial hits

    3. Perform detailed kinetic analysis on promising candidates

  • Consider substrate stability at high temperatures

• Activity assay development:

  • Use coupling enzymes with proven thermostability

  • Implement direct detection methods when possible (spectrophotometric, fluorescence)

  • Consider membrane reconstitution for transport activity assessment

• Physicochemical condition matrix:

  Parameter	Range to Test	Intervals	Notes
  Temperature	37-100°C	10-20°C	Include controls at each temperature
  pH	5.0-9.0	0.5 pH units	Use thermostable buffers
  Salt concentration	0-500 mM	100 mM	Test different cations (Na+, K+)
  Metal cofactors	Various	-	Prioritize Fe, Ni, Zn based on P. furiosus metalloproteome
  Reducing conditions	0-10 mM DTT	1-2 mM	Monitor cysteine oxidation states

• Protein modification analysis:

  • Test activity with/without His-tag

  • Examine post-translational modifications in various expression systems

  • Consider the impact of artificial tags on membrane association

Similar experimental approaches have been successfully employed to characterize other previously uncharacterized proteins from P. furiosus as described in the recombinant expression library project .

How can researchers troubleshoot insolubility or misfolding issues when expressing PF1496 in E. coli systems?

Troubleshooting expression issues for PF1496 requires systematic investigation of expression parameters:

• Expression strain optimization:

  • Test multiple strains including:

    • Rosetta 2(DE3)pLysS for rare codon usage

    • C41/C43(DE3) for membrane/toxic proteins

    • SHuffle or Origami for disulfide bond formation

    • Arctic Express for low-temperature expression

• Induction conditions matrix:

  Parameter	Options to Test	Expected Outcome
  IPTG concentration	0.1, 0.5, 1.0 mM	Lower concentrations may improve folding
  Induction temperature	16, 25, 30, 37°C	Lower temperatures typically improve solubility
  Induction duration	3h, 6h, overnight	Longer at lower temperatures may improve yield
  Media composition	LB, TB, M9	Richer media may improve yields
  Additives	5-10% glycerol, 1% glucose	May prevent leaky expression

• Fusion tag strategies:

  • Test alternative fusion tags known to enhance solubility:

    • MBP (Maltose Binding Protein)

    • SUMO

    • Thioredoxin

    • GST (Glutathione S-Transferase)

  • Include protease cleavage sites for tag removal

• Extraction and solubilization approaches:

  • For membrane-associated proteins like PF1496:

    • Test different detergents (DDM, LDAO, Triton X-100)

    • Try varying detergent concentrations (0.5-2% for extraction, 0.05-0.5% for purification)

    • Consider extracting at elevated temperatures (50-60°C) to leverage thermostability

• Refolding strategies if inclusion bodies form:

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

  • Test gradual vs. rapid dilution refolding

  • Consider on-column refolding during affinity purification

  • Implement temperature-assisted refolding leveraging thermostability

These approaches are based on successful strategies used for other P. furiosus proteins, where expression in E. coli Rosetta 2(DE3)pLysS strain with 0.5 mM IPTG induction at 37°C for 3 hours yielded successful expression for approximately 75% of tested proteins .

What are the specific considerations for functional assays at extreme temperatures when working with proteins from hyperthermophiles?

Working with hyperthermophilic proteins at their physiological temperatures presents unique experimental challenges:

• Equipment and materials adaptation:

  • Use oil baths or specialized high-temperature incubators for reactions

  • Select thermally stable reaction vessels (glass, certain metals)

  • Implement sealed systems to prevent evaporation

  • Consider pressure-resistant containers for reactions above 90°C

• Buffer and reagent stability considerations:

  Component	Thermal Stability Issues	Recommended Alternatives
  Buffers	Degradation at high temperatures	Phosphate, EPPS, MES buffers maintain pH at high temperatures
  Cofactors	NAD(P)H oxidation accelerates at high temperatures	Prepare fresh, use excess, measure decay rates as control
  Substrates	Hydrolysis, oxidation	Test substrate stability separately, account for degradation
  Reducing agents	DTT oxidizes rapidly at high temperatures	Use excess, consider β-mercaptoethanol or TCEP

• Enzyme kinetics considerations:

  • Reaction rates increase dramatically at higher temperatures

  • Use rapid sampling techniques or continuous assays

  • Incorporate temperature controls when comparing enzymes

  • Calculate Q10 temperature coefficients when extrapolating rates

• Controls and calibration:

  • Include thermochemical controls (substrate degradation at temperature)

  • Use internal standards for quantification

  • Perform calibration curves at the same temperature as experiments

  • Include known thermostable enzymes as positive controls

• Data interpretation complexities:

  • Account for spontaneous reaction rates at high temperatures

  • Consider buffer pH shifts at elevated temperatures

  • Analyze both initial rates and endpoint measurements

  • Evaluate enzyme stability during the course of the reaction

These methodological approaches have been successfully applied in studies of other P. furiosus enzymes, such as the characterization of its glutamate dehydrogenase and hydrogenase activities at temperatures up to 100°C.

How might PF1496 contribute to understanding adaptation mechanisms in extremophiles?

Studying PF1496 could provide significant insights into extremophile adaptation through several research avenues:

• Membrane adaptation mechanisms:

  • As a predicted membrane protein, PF1496 may reveal adaptations in membrane protein-lipid interactions at extreme temperatures

  • Comparative studies between PF1496 and mesophilic homologs could identify specific residue substitutions enabling thermostability

  • Analysis of hydrophobic core packing and interfacial residues may reveal principles of membrane protein thermostabilization

• Evolutionary analysis opportunities:

  • Phylogenetic analysis of UPF0252 family across temperature gradients

  • Identification of conserved vs. variable regions correlated with habitat temperature

  • Detection of positive selection signatures in thermophilic lineages

• Structure-function relationship investigations:

  • Determining how protein dynamics are maintained at high temperatures

  • Understanding how conformational changes occur in thermostable proteins

  • Identifying molecular mechanisms that prevent denaturation and aggregation

• Systems biology context:

  • Integration of PF1496 into the broader metabolic network of P. furiosus

  • Understanding its role in the organism's adaptation to hydrothermal vent environments

  • Potential involvement in stress response pathways specific to extreme conditions

• Biotechnological applications:

  • Identification of thermostabilizing motifs for protein engineering

  • Development of robust membrane proteins for biotechnology applications

  • Potential use in high-temperature bioprocesses

These research directions align with the broader field of extremophile biology, where understanding molecular adaptations provides insights into the fundamental principles of protein stability and function across environmental gradients .

What interdisciplinary approaches could accelerate the functional characterization of PF1496?

Accelerating the functional characterization of PF1496 requires integrating multiple scientific disciplines:

• Structural biology and biophysics integration:

  • Combine cryo-EM, NMR, and computational modeling for structural characterization

  • Apply hydrogen-deuterium exchange mass spectrometry to identify flexible regions

  • Use single-molecule FRET to detect conformational changes under various conditions

• Systems biology approaches:

  • Perform transcriptomic analysis of P. furiosus under various stress conditions

  • Conduct metabolomic profiling to identify potential substrates

  • Apply flux balance analysis to predict metabolic contexts where PF1496 might be essential

• Synthetic biology strategies:

  • Create chimeric proteins between PF1496 and characterized homologs

  • Develop minimal synthetic systems to test transport or enzymatic functions

  • Design reporter systems compatible with high-temperature assays

• Comparative genomics with experimental validation:

  • Identify co-evolved gene clusters across archaea

  • Create knockout/knockdown systems in model archaeal hosts

  • Perform complementation studies with homologs from diverse species

• Chemical biology approaches:

  • Design activity-based probes for potential enzymatic functions

  • Perform chemical cross-linking to trap transient interactions

  • Develop small molecule modulators through fragment-based screening

These interdisciplinary approaches would build on successful strategies used for other archaeal proteins, such as those employed in the recombinant expression library project for P. furiosus and the functional characterization of membrane-bound complexes like the hydrogenase system .

How can researchers apply insights from PF1496 to the engineering of thermostable proteins for biotechnological applications?

Translating findings from PF1496 research to protein engineering applications involves several methodological approaches:

These approaches build on the successful engineering of other P. furiosus proteins, such as the widely used Pfu DNA polymerase, which provides superior fidelity in PCR applications due to its thermostability and proofreading capabilities .