Recombinant UPF0758 protein STH371 (STH371)

What is UPF0758 protein and its general characteristics?

UPF0758 protein belongs to a family of proteins with uncharacterized protein function (UPF). The name "UPF0758" indicates it is the 758th family of proteins whose functions have not been fully characterized. These proteins typically have conserved sequences across various bacterial species, suggesting important biological roles. The full-length protein consists of 214 amino acid residues with a molecular weight of approximately 23,793 Da . UPF0758 proteins have been identified in various bacterial species including Rhodobacter capsulatus and Prosthecochloris aestuarii, indicating conservation across photosynthetic bacteria .

What expression systems are available for recombinant production of UPF0758 proteins?

Recombinant UPF0758 proteins can be produced using several expression systems, each with distinct advantages depending on research requirements. The primary expression systems include:

The choice of expression system should align with specific experimental requirements. For structural studies where post-translational modifications are less critical, E. coli systems often provide sufficient yields at lower cost . For functional studies requiring proper protein folding and modifications, insect or mammalian expression systems may be preferable despite their higher cost and technical complexity .

How should recombinant UPF0758 protein be stored and handled?

Optimal storage conditions for recombinant UPF0758 protein are critical for maintaining structural integrity and biological activity. Based on standard protocols for similar recombinant proteins, the following guidelines are recommended:

For long-term storage, the protein should be kept at -20°C or preferably -80°C. Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and activity loss . The protein is typically provided in lyophilized form or as a liquid preparation determined during the manufacturing process. When working with lyophilized protein, gentle reconstitution is essential to prevent denaturation. If small volumes become entrapped in the vial seal during shipment, brief centrifugation in a tabletop centrifuge can help dislodge the material .

What structural characterization methods are most effective for UPF0758 proteins?

Structural characterization of UPF0758 proteins requires a multi-method approach to provide comprehensive information. Based on available data and models for related UPF0758 family proteins, researchers should consider the following methodological approaches:

• Computational Modeling: AlphaFold has successfully generated high-confidence models (pLDDT global score: 89.19) for UPF0758 proteins such as Paes_0735, indicating that AI-based structural prediction can be valuable when experimental structures are unavailable .

• X-ray Crystallography: For definitive atomic-level structural determination, optimization of crystallization conditions is essential. Typical approaches include:

  • Screening with commercial crystallization kits at 4°C and 20°C

  • Protein concentration optimization (typically 5-15 mg/ml)

  • Addition of stabilizing ligands if functional hints are available

• Cryo-EM: For proteins resistant to crystallization, single-particle cryo-EM offers an alternative structural determination method.

• NMR Spectroscopy: For dynamic regions or smaller domains, NMR can provide valuable structural and dynamics information, requiring isotope-labeled protein samples (13C, 15N).

The confidence metrics from computational models suggest structured regions with pLDDT scores above 70, which should be prioritized in experimental structure determination efforts .

What purification strategies yield the highest purity UPF0758 protein for structural studies?

Obtaining high-purity (≥95%) UPF0758 protein is critical for structural studies and reliable functional assays. Based on reported protocols, the following multi-step purification strategy is recommended:

• Initial Capture: Affinity chromatography using the appropriate tag (His-tag is commonly employed for UPF0758 proteins)

• Intermediate Purification: Ion exchange chromatography to remove contaminants with different charge profiles

• Polishing Step: Size exclusion chromatography to achieve final purity ≥95% as assessed by SDS-PAGE

An optimized purification protocol typically includes:

Final product quality should be assessed by SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity . For structural biology applications, additional characterization by dynamic light scattering to confirm monodispersity is advisable.

How can researchers assess the functional activity of recombinant UPF0758 protein?

Despite being classified as an uncharacterized protein family, several approaches can be used to investigate the functional activity of UPF0758 proteins:

• Computational Function Prediction:

  • Sequence similarity networks with characterized proteins

  • Structural similarity to proteins of known function

  • Genomic context analysis to identify potential functional partners

• Binding Assays:

  • Thermal shift assays to identify stabilizing ligands

  • Pull-down experiments to identify interaction partners

  • Surface plasmon resonance to quantify binding kinetics

• Enzymatic Activity Screening:

  • Generic activity assays (phosphatase, ATPase, protease, etc.)

  • Substrate screening panels

• In vivo Functional Analysis:

  • Complementation studies in knockout strains

  • Phenotypic analysis of overexpression strains

Given the structural features observed in computational models, attention should be directed to potential nucleotide-binding motifs or metal coordination sites that could indicate enzymatic functions .

How does the structure of UPF0758 proteins inform potential functional roles?

The structural analysis of UPF0758 protein family members provides important clues about potential functions. The computational model of UPF0758 protein Paes_0735 shows high confidence scores (pLDDT global: 89.19), suggesting a well-defined tertiary structure . Structure-based analysis reveals:

• Domain Organization: The protein likely contains a single globular domain with conserved structural motifs.

• Potential Active Sites: Regions with highly conserved residues across species may indicate functional importance, possibly for catalytic activity or ligand binding.

• Structural Homology: Despite low sequence identity with characterized proteins, structural similarity searches using DALI or PDBeFold may reveal functional homologs with similar three-dimensional arrangements.

The confidence metrics from the AlphaFold model indicate that most regions of the protein are well-ordered (pLDDT scores between 70 and 90), suggesting a stable fold conducive to specific molecular interactions rather than a highly flexible protein . These structural insights can guide targeted mutagenesis studies to identify functionally important residues.

What phylogenetic distribution patterns exist for UPF0758 proteins and what do they suggest about evolution?

UPF0758 proteins show interesting phylogenetic distribution patterns that provide insights into their evolutionary history and potential functional importance:

• Taxonomic Distribution: UPF0758 proteins have been identified in diverse bacterial species, including photosynthetic bacteria like Rhodobacter capsulatus and Prosthecochloris aestuarii . This distribution suggests the protein emerged early in bacterial evolution.

• Conservation Patterns: Sequence analysis across bacterial species reveals:

  • Highly conserved motifs likely corresponding to functional sites

  • Variable regions that may represent species-specific adaptations

  • Conservation correlating with ecological niches (e.g., particularly in photosynthetic bacteria)

• Genomic Context: Analysis of neighboring genes across species can provide functional hints through the principle of conserved gene neighborhoods.

This phylogenetic information suggests that UPF0758 proteins likely serve a fundamental biological role that has been maintained throughout bacterial evolution, particularly in photosynthetic species where they may play specialized roles in photosynthesis-related processes or adaptations to phototrophic lifestyles.

What are the recommended experimental controls when working with recombinant UPF0758 proteins?

Rigorous experimental design for studies involving UPF0758 proteins requires appropriate controls to ensure reliable and interpretable results:

For functional assays, titration experiments with varying protein concentrations are essential to establish dose-dependence relationships. When studying potential enzymatic activities, time-course experiments should be conducted to determine initial reaction rates and avoid endpoint measurements that can be misleading .

What integration with systems biology approaches might reveal UPF0758 protein function?

Uncharacterized proteins like UPF0758 present excellent candidates for systems biology approaches to elucidate their functions within cellular networks:

• Interactome Analysis: Techniques like BioID, APEX proximity labeling, or tandem affinity purification can identify protein interaction partners, placing UPF0758 in a functional context. These methods involve expressing the protein of interest fused to an enzyme that modifies neighboring proteins, allowing identification of proximal partners.

• Transcriptomics Integration: RNA-seq analysis comparing wild-type and UPF0758 knockout strains can reveal affected pathways and processes through differential gene expression patterns.

• Metabolomics Profiling: Comparing metabolite profiles between normal and UPF0758-depleted conditions may identify affected metabolic pathways, particularly relevant given the protein's presence in photosynthetic bacteria.

• Network Analysis: Integrating multiple -omics datasets to position UPF0758 within cellular networks can generate testable hypotheses about function. Correlation networks, bayesian approaches, and machine learning methods can identify functional modules containing UPF0758.

These systems approaches are particularly valuable for UPF0758 proteins as they can overcome limitations of isolated biochemical assays when specific substrates or activities remain unknown.

How can CRISPR-based methods advance understanding of UPF0758 protein function?

CRISPR technologies offer powerful approaches to investigate the biological roles of UPF0758 proteins in their native contexts:

• CRISPR Knockout/Knockdown Studies:

  • Generation of clean deletion mutants in model organisms

  • CRISPRi for inducible, reversible gene repression

  • Phenotypic characterization under various growth conditions

• CRISPR Activation (CRISPRa):

  • Overexpression studies to identify gain-of-function phenotypes

  • Counterscreens to knockout studies to validate specificity

• CRISPR Base/Prime Editing:

  • Introduction of specific point mutations at conserved residues

  • Structure-guided mutagenesis based on computational models

• CRISPR Screens:

  • Genetic interaction mapping with genome-wide CRISPR libraries

  • Synthetic lethal/sick interactions to identify functional networks

For bacterial systems, optimized CRISPR methods adapted to particular photosynthetic bacterial species will be necessary, as efficiency can vary significantly between organisms. When designing guide RNAs, conservation analysis can identify optimal target sites that minimize off-target effects.

What is the potential relevance of UPF0758 proteins in understanding bacterial adaptation and evolution?

The conservation of UPF0758 proteins across diverse bacterial species suggests they may play roles in fundamental biological processes or specialized adaptations:

• Environmental Adaptation: The presence of UPF0758 in photosynthetic bacteria like Rhodobacter capsulatus and Prosthecochloris aestuarii suggests possible roles in:

  • Photosynthesis-related processes

  • Stress responses to light or oxygen

  • Energy metabolism under varying environmental conditions

• Evolutionary Significance:

  • Comparative genomics across species can reveal selection pressures

  • Analysis of synonymous vs. non-synonymous mutations can identify functionally constrained regions

  • Horizontal gene transfer patterns may indicate adaptive advantages

• Biotechnological Applications:

  • Understanding UPF0758 function could inform engineering of photosynthetic bacteria

  • Potential applications in bioenergy production or environmental remediation

Investigations focusing on expression patterns under different environmental conditions (light/dark cycles, nutrient limitation, oxidative stress) could provide valuable insights into the ecological roles of these proteins in bacterial adaptation strategies.

How can researchers overcome solubility issues with recombinant UPF0758 proteins?

Solubility challenges commonly arise during recombinant protein production. For UPF0758 proteins, several strategies can be employed:

• Expression Optimization:

  • Temperature reduction during induction (typically 15-25°C)

  • Induction at higher cell densities (OD600 0.8-1.0)

  • Lower inducer concentrations for slower, more controlled expression

  • Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

• Fusion Partners:

  • Solubility-enhancing tags (MBP, SUMO, Trx, GST)

  • C-terminal versus N-terminal tag positioning based on structural considerations

• Buffer Optimization:

  • Screening additives (glycerol, arginine, proline, non-detergent sulfobetaines)

  • Ionic strength variations (100-500 mM NaCl)

  • pH screening based on theoretical isoelectric point

• Refolding Strategies (if necessary):

  • Gradual dialysis from denaturant

  • On-column refolding during affinity purification

  • Pulse dilution methods

The availability of UPF0758 protein in various expression systems (E. coli, yeast, baculovirus, and mammalian cells) suggests that system selection should be guided by specific experimental requirements and solubility considerations .

What are the best approaches for studying protein-protein interactions involving UPF0758 proteins?

Investigating the interacting partners of UPF0758 proteins requires a multi-technique approach to identify both stable and transient interactions:

• In vitro Methods:

  • Pull-down assays using tagged recombinant protein

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Microscale thermophoresis (MST) for interactions in solution

• Structural Methods:

  • X-ray crystallography of co-crystals with binding partners

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

  • Crosslinking mass spectrometry (XL-MS) to identify proximity relationships

• Cellular Methods:

  • Co-immunoprecipitation from native sources

  • FRET/BRET for real-time interaction monitoring

  • Yeast two-hybrid or bacterial two-hybrid screening

• Computational Predictions:

  • Interface prediction from structural models

  • Co-evolution analysis to identify potential partners

  • Molecular docking with candidate interactors

When designing these experiments, it's important to consider the native cellular environment of UPF0758 proteins from photosynthetic bacteria, which may involve specialized compartments or membrane associations.