Recombinant Thermus thermophilus UPF0447 protein TT_C1352 (TT_C1352)

What is Thermus thermophilus UPF0447 protein TT_C1352 and how is it classified?

TT_C1352 belongs to the Uncharacterized Protein Family (UPF) 0447, encoded in the genome of the thermophilic bacterium Thermus thermophilus. Similar to other UPF proteins in T. thermophilus, such as UPF0365 (TT_C0686) or UPF0102 (TT_C0005), it represents a protein whose structural features have been identified but whose precise biological function remains to be fully elucidated .

The protein is likely cataloged in public databases with a UniProt ID, similar to other T. thermophilus proteins like TT_C0686 (UniProt ID: Q72JT2) . UPF proteins are categorized based on sequence conservation across species rather than functional annotation, making them important targets for structural genomics initiatives.

What expression systems are recommended for producing recombinant TT_C1352 protein?

The most effective expression system for recombinant T. thermophilus proteins, including TT_C1352, is typically E. coli. Based on successful expression of other T. thermophilus proteins:

Methodology:

• Clone the TT_C1352 gene into an expression vector with an N-terminal His-tag for purification

• Transform into an appropriate E. coli strain (preferably one designed for rare codon usage)

• Induce expression (typically with IPTG at OD600 of 0.6-0.8)

• Harvest cells and proceed to purification

What purification protocol is most effective for TT_C1352 recombinant protein?

Based on protocols for similar T. thermophilus proteins, the following multi-step purification approach is recommended:

• Initial Heat Treatment: Exploit the thermostability of T. thermophilus proteins by heating the cell lysate (70-80°C for 10-20 minutes), which denatures most E. coli proteins while leaving the thermophilic target protein intact

• Affinity Chromatography: For His-tagged TT_C1352:

  • Use Ni²⁺ affinity chromatography

  • Wash with Tris/PBS-based buffer containing low concentrations of imidazole

  • Elute with buffer containing 250-500 mM imidazole

• Secondary Purification:

  • Hydrophobic interaction chromatography

  • Anion-exchange chromatography

  • Gel filtration for final polishing and determining oligomerization state

This protocol typically yields protein with >90% purity as determined by SDS-PAGE .

What are the optimal storage conditions for purified TT_C1352 protein?

For maximum stability and activity retention:

Important: Avoid repeated freeze-thaw cycles as they can significantly reduce protein activity. For reconstitution of lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL, then add glycerol to a final concentration of 50% .

How can researchers determine the oligomerization state of TT_C1352?

The oligomerization state of UPF proteins from T. thermophilus varies considerably and may be crucial for their function. Based on studies of similar proteins:

Methodological Approach:

• Gel Filtration Chromatography: Compare retention time with molecular weight standards to estimate native molecular weight

• Analytical Ultracentrifugation: For precise determination of oligomeric state in solution

  • Sedimentation velocity experiments can distinguish between monomers, dimers, and higher-order structures

  • Sedimentation equilibrium provides molecular weight information

• X-ray Crystallography: Can reveal biological assembly in crystal structure

  • TTHA0281 (a UPF0150 protein) forms a homotetramer with specific subunit interactions

  • Interaction surface areas at interfaces can be calculated with programs like AREAIMOL

Many T. thermophilus UPF proteins form specific oligomeric structures essential for their function:

• UPF0150-family proteins form homotetramers

• Some nucleoside phosphorylases form homohexamers, others are monomeric or homodimeric

What experimental design strategies are effective for investigating the potential function of TT_C1352?

A multi-faceted experimental approach is recommended:

• Structural Analysis:

  • Determine crystal structure at high resolution (1.5-3.0 Å)

  • Compare with structural homologs using tools like Dali or VAST

  • Identify potential active sites or binding pockets

• Binding Partner Identification:

  • Pull-down assays with cell extracts

  • Yeast two-hybrid screening

  • Co-immunoprecipitation followed by mass spectrometry

• Nucleic Acid Interaction Analysis:

  • Many UPF proteins from T. thermophilus interact with RNA/DNA

  • RNA/DNA binding assays (EMSA, filter binding)

  • Determine if TT_C1352 shows preference for specific sequences or structures

• Phenotypic Analysis:

  • Generate knockout strains

  • Compare growth under different conditions (temperature, media, stress)

  • Competition experiments between wild-type and knockout strains

A well-designed experimental approach should include proper controls to distinguish specific from non-specific effects, and validation using multiple methodologies.

How do researchers assess the thermostability of TT_C1352 and its potential applications?

Methodological approach:

• Thermal Denaturation Analysis:

  • Circular dichroism (CD) spectroscopy at increasing temperatures (25-100°C)

  • Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

  • Expected Tm values for T. thermophilus proteins typically exceed 70°C

• Activity Assays at Various Temperatures:

  • Measure enzymatic activity or binding capacity at temperature ranges (37-95°C)

  • Compare with mesophilic homologs to assess relative thermostability

• Structural Stability Assessment:

  • Trypsin resistance assays with and without reducing agents

  • For proteins containing disulfide bonds, compare stability before and after treatment with reducing agents like TCEP

Potential applications based on thermostability:

• Use as scaffold for designing thermostable mini-proteins for therapeutic applications

• Development of thermostable reagents for molecular biology applications

• Structure-guided engineering of mesophilic proteins to enhance thermostability

What computational approaches can predict the function of TT_C1352?

A comprehensive bioinformatics workflow includes:

• Sequence-based Analysis:

  • PSI-BLAST against non-redundant protein database

  • Multiple sequence alignment of homologs

  • Identification of conserved residues

  • Analysis of gene neighborhood and operons

• Structure-based Prediction:

  • Homology modeling if crystal structure unavailable

  • Structural alignment with characterized proteins

  • Active site prediction and comparison

  • Molecular docking with potential substrates

• Integrated Analysis:

  • Gene co-expression networks

  • Phylogenetic profiling

  • Protein-protein interaction prediction

Methodological example from literature:
The function of TTHA0281 (UPF0150 protein) was predicted by observing structural similarity to the partially degraded RNase H fold of HicB-family proteins, suggesting involvement in RNA metabolism .

How can researchers design experiments to investigate potential RNA/DNA interactions of TT_C1352?

Based on findings with other T. thermophilus proteins like TtAgo and Hera that interact with nucleic acids :

Experimental Design Strategy:

• In vivo nucleic acid binding characterization:

  • Express tagged TT_C1352 in T. thermophilus

  • Immunoprecipitate the protein

  • Extract and sequence bound nucleic acids

  • Map binding sites to genome

• In vitro binding assays:

  • Electrophoretic mobility shift assays (EMSA)

  • Filter binding assays

  • Surface plasmon resonance (SPR)

  • Test binding to different RNA/DNA structures (single-stranded, double-stranded, specific secondary structures)

• Nucleic acid preference analysis:

  • Study nucleotide distribution in bound sequences

  • Analyze GC-content of bound RNAs/DNAs

  • Examine preference for specific sequence motifs

  • Assess thermodynamic stability of bound nucleic acids

• Functional validation:

  • Mutagenesis of predicted nucleic acid binding residues

  • In vitro and in vivo assessment of mutant binding capacity

  • Phenotypic analysis of binding-deficient mutants

What are the key considerations in designing controls for experiments with uncharacterized proteins like TT_C1352?

Control Design Framework:

• Protein-level controls:

  • Inactive mutants (mutated catalytic residues) while maintaining folding

  • Tagged vs. untagged protein comparisons to assess tag interference

  • Heat-denatured protein as negative control

  • Well-characterized proteins from the same family as reference points

• Experimental design controls:

  • Between-subjects vs. within-subjects design considerations

  • Randomization protocols to minimize bias

  • Appropriate sample sizes based on statistical power analysis

• Structural and functional validation controls:

  • Scrambled core residues or loop replacement with Gly-Ser linkers to test structural determinants

  • Competition assays with unlabeled ligands to validate binding specificity

Example methodology from literature:
In studies of the DNA-guided Argonaute TtAgo, researchers used catalytically inactive double mutants (DM) as controls to distinguish between binding and catalytic activities .

How can researchers design experiments to determine if TT_C1352 functions in stress response or DNA replication?

Based on findings with other T. thermophilus proteins involved in stress response or DNA replication :

Experimental Design Strategy:

• Stress response assessment:

  • Expose T. thermophilus cultures to various stressors (heat shock, cold shock, oxidative stress)

  • Monitor TT_C1352 expression levels using RT-qPCR

  • Analyze protein levels under stress conditions via Western blotting

  • Compare wild-type and TT_C1352 knockout strains for stress tolerance

• DNA replication involvement:

  • Synchronize cell cultures and analyze protein expression throughout cell cycle

  • Co-immunoprecipitation with known replication proteins

  • Fluorescence microscopy to determine subcellular localization

  • Growth curve analysis with and without replication inhibitors (like ciprofloxacin)

• Competition experiments:

  • Grow wild-type and knockout strains in mixed culture

  • Monitor population dynamics over time using strain-specific markers

  • Analyze competition outcomes under different growth conditions

Data analysis approach:
Use statistical methods appropriate for experimental design, such as two-way ANOVA for experiments with multiple variables, ensuring proper reporting of effect sizes and confidence intervals .

What methodologies can identify potential binding partners or interactomes of TT_C1352?

For comprehensive interactome analysis:

Experimental Approaches:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged TT_C1352 in T. thermophilus

  • Perform pull-down under native conditions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by reciprocal pull-downs

• Yeast Two-Hybrid (Y2H) Screening:

  • Construct bait plasmid with TT_C1352

  • Screen against T. thermophilus genomic library

  • Confirm interactions by secondary assays

• Protein Microarray Analysis:

  • Synthesize protein arrays containing T. thermophilus proteome

  • Probe with labeled TT_C1352

  • Identify binding partners through fluorescence detection

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse TT_C1352 to a promiscuous biotin ligase

  • Express in T. thermophilus

  • Identify biotinylated proteins by pull-down and mass spectrometry

Data Analysis and Validation:

• Apply appropriate statistical thresholds for significance

• Eliminate common contaminants and non-specific binders

• Validate key interactions through independent techniques

• Perform functional assays to determine biological relevance of interactions