Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_0148 (AF_0148)

What is Archaeoglobus fulgidus and why is AF_0148 of research interest?

Archaeoglobus fulgidus is a hyperthermophilic archaeon that grows optimally at 83°C and is a strict anaerobe . It belongs to the euryarchaeon group and has been a model organism for studying adaptations to extreme environments. The uncharacterized protein AF_0148 is of research interest because it represents one of many proteins in archaea with unknown function, potentially harboring novel enzymatic activities or structural properties adapted to extreme conditions. The protein consists of 85 amino acids , making it a relatively small protein that could serve as a model for studying protein stability in hyperthermophiles.

What expression systems are recommended for recombinant production of AF_0148?

Based on available data for recombinant Archaeoglobus fulgidus proteins, E. coli is the most commonly used heterologous expression system for AF_0148 . The protein has been successfully expressed as a His-tagged construct in E. coli . For optimal expression, consider the following recommendations:

• Use expression vectors like pET21 that have been successful with other A. fulgidus proteins

• Include a His-tag for purification, which has been demonstrated to be effective with other A. fulgidus proteins

• Optimize codon usage for E. coli if expression levels are low

• Consider using E. coli strains designed for expression of proteins from AT-rich organisms

What are the optimal storage conditions for purified recombinant AF_0148?

While specific data for AF_0148 storage is not available, other A. fulgidus proteins have been successfully stored using the following conditions:

• Storage buffer composition: 20 mM Tris-HCl (pH 8.0 at 25°C), 500 mM NaCl, 1 mM DTT, and 50% v/v glycerol

• Storage temperature: -20°C

• For long-term storage, aliquot the protein to avoid repeated freeze-thaw cycles

• Consider flash-freezing in liquid nitrogen before transferring to -80°C for extended storage

How can I verify the identity and purity of recombinant AF_0148?

To confirm the identity and assess the purity of recombinant AF_0148:

• SDS-PAGE analysis: Aim for >90% homogeneity as commonly achieved with other A. fulgidus proteins

• Mass spectrometry: To confirm the molecular weight and sequence identity

• Western blotting: Using anti-His antibodies if the protein contains a His-tag

• N-terminal sequencing: To verify the correct start of the protein and absence of unexpected processing

What are the predicted structural domains and properties of AF_0148?

AF_0148 is a small protein (85 amino acids) with currently uncharacterized domains. To predict its structural elements:

• Employ bioinformatic tools such as HHpred, Phyre2, and I-TASSER for structure prediction

• Analyze the sequence for conserved motifs using PROSITE or PFAM databases

• Look for structural homology with proteins of known function in archaea

• Assess potential nucleic acid-binding capacity, as many small archaeal proteins interact with DNA or RNA

Given the hyperthermophilic nature of A. fulgidus, AF_0148 likely possesses features that contribute to thermostability:

• Higher proportion of charged residues

• Increased number of salt bridges

• More compact hydrophobic core

• Potentially decreased loop regions

How can I assess potential protein-protein interactions of AF_0148?

Several approaches can help identify potential binding partners of AF_0148:

• Pull-down assays: Use His-tagged AF_0148 as bait with A. fulgidus cell lysate

• Bacterial two-hybrid system: Adapted for archaeal proteins

• Cross-linking studies: To capture transient interactions

• Co-immunoprecipitation: If antibodies against AF_0148 are available

• Proximity-dependent biotin identification (BioID): For in vivo interaction studies

Notably, other A. fulgidus proteins have been found to form functional heterodimeric complexes. For example, the Archaeoglobus fulgidus Argonaute (AfAgo) forms a complex with a protein encoded upstream in the same operon . This suggests examining the genomic context of AF_0148 for potential interaction partners.

What approaches can be used to elucidate the potential function of AF_0148?

When working with an uncharacterized protein like AF_0148, consider these approaches to determine its function:

• Genomic context analysis: Identify nearby genes that might be functionally related

• Transcriptomic studies: Determine under which conditions AF_0148 is expressed

• Deletion/knockout studies: Assess phenotypic changes when AF_0148 is removed

• Heterologous complementation: Test if AF_0148 can rescue mutants of related organisms

• Activity screening: Test the protein against panels of potential substrates

How does the thermostability of AF_0148 compare to mesophilic homologs?

To assess the thermostability of AF_0148 relative to potential mesophilic homologs:

• Conduct differential scanning calorimetry (DSC) to determine melting temperature

• Perform circular dichroism (CD) spectroscopy at increasing temperatures

• Assess activity retention after heat treatment at various temperatures

• Compare with homologous proteins from mesophilic organisms if identified

Archaeoglobus fulgidus proteins typically show remarkable thermostability. For example, studies on A. fulgidus DNA repair enzymes demonstrated activity at 60°C and higher temperatures , reflecting adaptations to the organism's optimal growth temperature of 83°C.

What purification protocols are most effective for His-tagged AF_0148?

Based on purification methods used for other A. fulgidus proteins, the following protocol is recommended for His-tagged AF_0148:

• Cell lysis: Sonication in buffer containing 20 mM Tris-HCl (pH 8.0), 200 mM NaCl, 2 mM PMSF, 5 mM 2-mercaptoethanol

• Heat treatment: 30 minutes at 70°C to precipitate E. coli proteins (taking advantage of the thermostability of A. fulgidus proteins)

• Centrifugation: Remove precipitated E. coli proteins

• Immobilized metal affinity chromatography (IMAC): Using HisTrap HP chelating columns

• Heparin affinity chromatography: Using HiTrap Heparin HP columns (especially if the protein is predicted to bind nucleic acids)

• Size exclusion chromatography: For final polishing and buffer exchange

This multi-step approach has shown >90% homogeneity for other A. fulgidus proteins and should be effective for AF_0148.

What buffer conditions optimize AF_0148 stability for structural and functional studies?

While specific optimal conditions for AF_0148 have not been reported, the following buffer compositions have been successful for other A. fulgidus proteins:

• For general storage and handling:

  • 20 mM Tris-HCl (pH 8.0 at 25°C), 500 mM NaCl, 1 mM DTT

• For activity assays:

  • Consider buffers that maintain stability at high temperatures

  • Include divalent cations (Mg²⁺, Mn²⁺) that might be required for activity

  • Test pH range of 6.0-8.5, as archaeal proteins often have broader pH optima

• For structural studies:

  • Buffer screening is recommended (e.g., using thermal shift assays)

  • Consider including osmolytes such as trimethylamine N-oxide that enhance thermostability

  • Maintain reducing conditions with DTT or TCEP to prevent oxidation of cysteine residues

How can I design appropriate experimental controls for functional assays with AF_0148?

When working with an uncharacterized protein like AF_0148, robust controls are essential:

• Negative controls:

  • Heat-denatured AF_0148 (inactive protein)

  • Buffer-only reactions (no protein)

  • Non-related protein of similar size/properties

• Positive controls:

  • If homologous proteins with known function exist, include them in parallel assays

  • If testing enzymatic activity, include commercial enzymes with potentially similar functions

• Specificity controls:

  • Site-directed mutants of conserved residues

  • Truncated versions of the protein

  • Competition assays with excess unlabeled substrates

• Environmental controls:

  • Test activity across temperature range (37-85°C)

  • Vary pH and ionic strength to determine optimal conditions

  • Test dependency on potential cofactors (ATP, metal ions)

What techniques are most appropriate for determining the oligomeric state of AF_0148?

To determine if AF_0148 exists as a monomer or forms higher-order structures:

• Size exclusion chromatography: Compare elution volume to standard proteins

• Dynamic light scattering: To measure hydrodynamic radius

• Analytical ultracentrifugation: For precise determination of molecular weight and shape

• Native PAGE: To visualize oligomeric states under non-denaturing conditions

• Cross-linking studies: To capture transient interactions

• Small-angle X-ray scattering (SAXS): For low-resolution structural information in solution

Other A. fulgidus proteins, such as the Argonaute protein, have been found to form heterodimeric complexes , suggesting the possibility that AF_0148 might also function as part of a multi-protein complex.

How can in silico analyses guide experimental approaches for AF_0148?

In silico analyses can provide valuable insights to focus experimental efforts:

• Sequence homology searches: BLAST against various databases to identify homologs

• Phylogenetic analysis: Determine conservation across archaea and other domains

• Structural prediction: Use AlphaFold or similar tools to predict 3D structure

• Gene neighborhood analysis: Examine genes adjacent to AF_0148 for functional hints

• Promoter analysis: Identify potential regulatory elements controlling expression

These computational approaches can generate testable hypotheses about AF_0148 function, guiding the design of targeted experiments rather than broad screening approaches.

What considerations are important when studying protein-nucleic acid interactions for AF_0148?

If investigating potential nucleic acid binding properties of AF_0148:

• Binding assays:

  • Electrophoretic mobility shift assays (EMSA) with various DNA/RNA substrates

  • Filter binding assays for quantitative determination of binding constants

  • Fluorescence anisotropy with labeled nucleic acids

• Specificity determination:

  • Test binding to single-stranded vs. double-stranded substrates

  • Assess sequence preferences using systematic evolution of ligands by exponential enrichment (SELEX)

  • Examine structure-specific binding (e.g., stem-loops, bulges, G-quadruplexes)

• Thermodynamic characterization:

  • Isothermal titration calorimetry (ITC) to determine binding constants

  • Surface plasmon resonance (SPR) for kinetic analysis of association/dissociation

Note that several A. fulgidus proteins have been found to interact with nucleic acids, including the Argonaute protein that forms a heterodimeric complex and is involved in guide RNA-mediated target DNA binding .

What are the most promising approaches to determine the cellular role of AF_0148?

Based on strategies that have been successful for other uncharacterized archaeal proteins:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map AF_0148 to specific cellular pathways or stress responses

• Interaction mapping:

  • Identify protein interaction networks involving AF_0148

  • Construct guilt-by-association functional predictions

• Comparative genomics:

  • Analyze gene conservation patterns across archaea

  • Identify co-evolution patterns with proteins of known function

• Structural biology:

  • Solve the 3D structure through X-ray crystallography or cryo-EM

  • Identify potential binding pockets or catalytic sites

The multidisciplinary approach combining biochemical, biophysical, and genetic methods offers the best chance of elucidating the function of this uncharacterized protein.

How can AF_0148 research contribute to our understanding of archaeal biology and evolution?

Studying uncharacterized proteins like AF_0148 contributes to:

• Filling knowledge gaps in archaeal biochemistry and metabolism

• Understanding adaptations to extreme environments (high temperature, anaerobic conditions)

• Potentially discovering novel enzymatic activities with biotechnological applications

• Gaining insights into archaeal-specific biological processes

• Expanding our understanding of protein evolution across domains of life