Recombinant Staphylococcus aureus UPF0122 protein SAS1170 (SAS1170)

What is Staphylococcus aureus UPF0122 protein SAS1170?

SAS1170 is an uncharacterized protein from Staphylococcus aureus that belongs to the UPF0122 protein family, a group of proteins whose functions have not been fully elucidated. The designation "UPF" stands for "Uncharacterized Protein Family," indicating that these proteins have been identified through genomic sequencing but have not yet been assigned clear biological functions through experimental validation. SAS1170 specifically refers to the gene locus in certain S. aureus strains, with the protein product being expressed as a recombinant protein for research purposes. The protein is conserved across multiple S. aureus strains, suggesting potential functional importance in bacterial physiology or pathogenesis. Understanding this protein may provide insights into novel aspects of S. aureus biology, potentially revealing new therapeutic targets or virulence mechanisms.

How does SAS1170 compare to other S. aureus proteins?

SAS1170 differs from well-characterized S. aureus virulence factors such as α-hemolysin (Hla), staphylococcal enterotoxin B (SEB), staphylococcal protein A (SpA), iron surface determinant B (IsdB), and manganese transport protein C (MntC) that have established roles in pathogenesis . Unlike these proteins, which have been incorporated into vaccine formulations due to their known virulence functions, SAS1170 belongs to an uncharacterized family with no clearly defined role in pathogenesis. The protein also differs from other S. aureus surface proteins like Clumping factor A, which has a well-established function in promoting bacterial attachment to human fibrinogen and inducing bacterial clumping . Comparative genomic analysis indicates SAS1170 is conserved across multiple S. aureus strains, suggesting it may play a fundamental role in bacterial physiology rather than a strain-specific function in virulence or adaptation.

What expression systems are optimal for recombinant SAS1170 production?

For recombinant expression of SAS1170, both prokaryotic and eukaryotic systems have been utilized, with each offering distinct advantages. Based on commercial protein production methods, yeast expression systems appear viable for SAS1170 expression, as indicated by the available recombinant protein preparations . Escherichia coli remains the most common and cost-effective expression system for bacterial proteins, typically using vectors with strong inducible promoters like T7 or tac. When expressing S. aureus proteins in E. coli, codon optimization may improve yields, particularly for proteins with rare codons. For applications requiring post-translational modifications or improved protein folding, yeast systems like Pichia pastoris or Saccharomyces cerevisiae might be preferable, though they generally result in lower yields than bacterial systems. Researchers should consider implementing a small-scale expression screening with different vectors, host strains, and induction conditions to optimize SAS1170 expression before scaling up production.

What purification strategies provide the highest purity and yield for SAS1170?

Purification strategies for SAS1170 typically involve affinity chromatography using tagged recombinant versions of the protein. Most commercially available SAS1170 preparations utilize histidine tags, allowing for immobilized metal affinity chromatography (IMAC) purification . For optimal purification, a multi-step approach should be implemented, beginning with affinity chromatography, followed by size exclusion chromatography to separate monomeric protein from aggregates and contaminants of different molecular weights. Ion exchange chromatography may serve as an additional purification step, particularly if the protein has a distinctive isoelectric point. Critical buffer optimization during purification should include testing various pH conditions, salt concentrations, and potential stabilizing agents to maintain protein integrity. Researchers should verify purification success through SDS-PAGE, Western blotting, and mass spectrometry to confirm identity, purity, and integrity of the final protein preparation.

How can researchers validate the functionality of purified SAS1170?

Validating the functionality of purified SAS1170 presents unique challenges since its biological function remains uncharacterized. Several approaches can be employed to assess protein quality and potential functionality. Circular dichroism spectroscopy can confirm proper secondary structure formation, while thermal shift assays can assess protein stability. Surface plasmon resonance or pull-down assays may identify potential binding partners by screening against S. aureus lysates or host factors. Activity-based assays should be designed based on bioinformatic predictions of potential functions, possibly including assays for enzymatic activity, DNA/RNA binding, or protein-protein interactions. If antibodies against SAS1170 are available, immunological detection methods can confirm the presence of conformational epitopes that might be lost during denaturation. Additionally, functional complementation studies in SAS1170 knockout strains could reveal phenotypic changes associated with the protein's function.

How can SAS1170 be used to study S. aureus pathogenesis mechanisms?

SAS1170 may serve as a valuable tool in studying novel aspects of S. aureus pathogenesis, particularly for investigating uncharacterized bacterial processes. Researchers can generate SAS1170-specific antibodies to track the protein's expression and localization during different stages of infection or under various stress conditions. Gene knockout or knockdown studies targeting SAS1170 can reveal potential roles in bacterial survival, virulence, or antibiotic resistance through phenotypic analysis. Protein interaction studies using techniques such as bacterial two-hybrid systems, co-immunoprecipitation, or crosslinking mass spectrometry may identify SAS1170 binding partners, potentially placing it within known pathogenesis pathways. Comparative studies across multiple S. aureus strains with different virulence profiles can establish correlations between SAS1170 sequence variations and strain-specific pathogenic properties. Additionally, heterologous expression of SAS1170 in non-pathogenic bacterial models may reveal gain-of-function phenotypes that provide clues to its role in pathogenesis.

What approaches can determine the function of uncharacterized proteins like SAS1170?

Determining the function of uncharacterized proteins like SAS1170 requires an integrated multi-omics approach combining genomic, transcriptomic, proteomic, and phenotypic data. Comparative genomics analysis across bacterial species can identify conserved genomic contexts or co-evolution patterns that suggest functional relationships. Transcriptomic profiling using RNA-seq to compare SAS1170 knockout mutants with wild-type strains may reveal dysregulated pathways indicating the protein's functional role. Proteomic approaches like proximity labeling (BioID or APEX) can identify proteins physically close to SAS1170 in vivo, suggesting potential interaction networks. Metabolomic profiling of knockout mutants may reveal altered metabolic pathways associated with SAS1170 function. Phenotypic screening using diverse growth conditions, stress factors, or infection models can identify conditions where SAS1170 provides a fitness advantage to the bacterium. Integration of these multi-omics datasets through computational approaches may reveal functional patterns not evident from any single approach.

What storage and handling protocols ensure optimal stability of recombinant SAS1170?

Optimal storage and handling of recombinant SAS1170 is critical for maintaining protein integrity and activity across experiments. Based on standard protocols for similar bacterial recombinant proteins, SAS1170 should be stored in a stabilizing buffer containing 50% glycerol at -20°C for routine storage, with -80°C recommended for long-term preservation . Researchers should avoid repeated freeze-thaw cycles, which can lead to protein denaturation and aggregation; instead, preparing single-use aliquots upon receipt is advised. Working aliquots may be stored at 4°C for up to one week, but extended storage at this temperature is not recommended . Buffer composition significantly impacts protein stability, with Tris-based buffers adjusted to appropriate pH (typically 7.5-8.0) being commonly used. The addition of reducing agents like DTT or β-mercaptoethanol (1-5 mM) can prevent disulfide bond formation if the protein contains cysteine residues. Researchers should validate protein integrity after extended storage periods using methods such as SDS-PAGE, size exclusion chromatography, or activity assays before proceeding with critical experiments.

How can structural biology approaches advance understanding of SAS1170?

Advanced structural biology approaches can significantly enhance our understanding of SAS1170 function despite its current uncharacterized status. X-ray crystallography remains the gold standard for obtaining high-resolution structural information, though crystallization of bacterial membrane or membrane-associated proteins often presents challenges requiring extensive condition screening. Cryo-electron microscopy (cryo-EM) offers an alternative approach that doesn't require protein crystallization and can reveal structural details of SAS1170 in different conformational states or in complex with potential binding partners. Nuclear magnetic resonance (NMR) spectroscopy is particularly valuable for studying protein dynamics and identifying binding interfaces with potential interaction partners. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map solvent-accessible regions and conformational changes upon ligand binding, potentially revealing functional domains. Computational approaches including molecular dynamics simulations can predict structural flexibility and potential binding pockets that might suggest functional roles. Integration of multiple structural techniques typically provides the most comprehensive understanding of protein structure-function relationships.

What role might SAS1170 play in S. aureus infection and antibiotic resistance?

While the specific function of SAS1170 remains undetermined, several experimental approaches can investigate its potential role in S. aureus infection and antibiotic resistance. Gene expression analysis comparing SAS1170 transcript levels across different growth phases, infection conditions, and antibiotic exposure may reveal regulation patterns suggesting functional contexts. Construction of SAS1170 gene deletion mutants followed by comprehensive phenotypic characterization in infection models could identify altered virulence, host adhesion, immune evasion, or antibiotic susceptibility. Complementation studies reintroducing wild-type or modified SAS1170 into knockout strains can confirm phenotypic observations and identify critical functional domains. Assessing protein-protein interactions between SAS1170 and known virulence factors or resistance determinants may reveal functional associations with established pathogenesis pathways. Heterologous expression of SAS1170 in model organisms can determine if the protein alone confers phenotypic changes related to virulence or antibiotic resistance, potentially identifying a direct functional role rather than regulatory effects.

How can computational approaches predict potential functions of SAS1170?

Computational approaches offer powerful tools for predicting potential functions of uncharacterized proteins like SAS1170 when experimental data is limited. Sequence-based homology analysis using tools like BLAST, HHpred, or HMMER can identify remote homologs with known functions, potentially revealing evolutionary relationships not apparent through standard alignment methods. Structural prediction using AlphaFold2 or RoseTTAFold can generate high-confidence structural models even in the absence of close homologs with known structures, revealing potential functional domains or binding sites. Protein-protein interaction prediction tools such as STRING or InterPreTS can suggest potential binding partners based on co-evolution, genomic context, or structural complementarity. Pathway enrichment analysis of proteins predicted to interact with SAS1170 may reveal biological processes in which the protein participates. Machine learning approaches trained on known protein functions can identify patterns in sequence, structure, or genomic context that correlate with specific functional categories. Integrating predictions from multiple computational methods typically provides more reliable functional hypotheses that can direct focused experimental validation.

What emerging technologies could accelerate functional characterization of SAS1170?

Emerging technologies across multiple disciplines could significantly accelerate the functional characterization of uncharacterized proteins like SAS1170. CRISPR-Cas9 genome editing in S. aureus now enables precise genetic manipulation, allowing for targeted mutations, domain deletions, or regulatory element modifications to assess SAS1170 function in its native context. Single-cell technologies including single-cell RNA-seq and CyTOF can reveal cell-to-cell variation in SAS1170 expression and identify subpopulations of bacteria with distinct functional states during infection or stress responses. Advanced imaging techniques such as super-resolution microscopy and correlative light and electron microscopy (CLEM) can track SAS1170 localization with unprecedented spatial resolution, revealing associations with specific cellular structures. Protein engineering approaches including non-canonical amino acid incorporation can enable site-specific protein labeling for tracking or crosslinking studies. Microfluidic systems allow for high-throughput phenotypic screening of bacterial strains under precisely controlled environmental conditions, potentially identifying specific conditions where SAS1170 function becomes essential. Integration of these technologies with computational approaches will likely provide the most comprehensive functional characterization.

How might understanding SAS1170 contribute to novel therapeutic approaches?

The functional characterization of SAS1170 could potentially contribute to novel therapeutic approaches against S. aureus infections, particularly given the urgent need for alternatives to conventional antibiotics. If SAS1170 proves essential for bacterial survival or virulence, it could represent a novel target for antimicrobial development, especially valuable if the protein has no human homologs that could lead to off-target effects. High-resolution structural data could enable structure-based drug design targeting specific functional domains or binding interfaces of SAS1170. Antibody-based approaches targeting SAS1170 might be developed if the protein is surface-exposed, potentially facilitating bacterial clearance through opsonization or neutralizing critical functions. Vaccine development incorporating SAS1170 as an antigen could be explored, particularly if the protein proves immunogenic and protective in animal models, potentially contributing to multi-antigen vaccine formulations similar to the five-antigen vaccine approach described in the literature . Therapeutic strategies targeting SAS1170 regulation rather than the protein itself might be effective if the protein proves challenging to target directly, potentially involving antisense RNA approaches or small molecules affecting transcriptional or translational control.

What experimental challenges must be addressed in SAS1170 research?

Several significant experimental challenges must be addressed to advance SAS1170 research effectively. Developing specific and sensitive detection methods for SAS1170 represents a primary challenge, requiring generation of high-quality antibodies or tagged versions that don't disrupt protein function. Establishing reliable SAS1170 knockout or knockdown systems in S. aureus strains faces technical hurdles related to transformation efficiency and genetic manipulation in this pathogen. Determining appropriate experimental conditions where SAS1170 function becomes evident remains difficult without functional clues, potentially requiring screening across diverse environmental stresses, growth conditions, and infection models. Distinguishing direct effects of SAS1170 from indirect consequences of its manipulation presents analytical challenges, requiring thorough controls and complementation studies. Translating in vitro findings to in vivo relevance necessitates appropriate animal models that recapitulate human S. aureus infections. Integrating disparate data types from various experimental approaches into coherent functional models requires sophisticated computational tools and interdisciplinary expertise. Addressing these challenges will require collaborative approaches combining expertise in microbiology, structural biology, genetics, immunology, and computational biology.