Recombinant Staphylococcus aureus UPF0435 protein SAS1802 (SAS1802)

What is Recombinant Staphylococcus aureus UPF0435 protein SAS1802?

Recombinant Staphylococcus aureus UPF0435 protein SAS1802 (SAS1802) is a conserved bacterial protein belonging to the UPF (Uncharacterized Protein Family) 0435 classification. This protein is expressed in Staphylococcus aureus and can be produced recombinantly in expression systems such as Escherichia coli. As a member of the UPF family, it represents proteins with conserved sequences whose functions have not been fully characterized, making it a target for fundamental research into S. aureus biology and potential virulence mechanisms .

How does SAS1802 differ from other Staphylococcus aureus proteins like Protein A or Alpha-hemolysin?

Unlike well-characterized S. aureus virulence factors such as Protein A (SpA) and Alpha-hemolysin (Hla), SAS1802 belongs to the uncharacterized protein family. While Protein A has five immunoglobulin-binding domains that capture both the Fc region and Fab region of immunoglobulins , and Alpha-hemolysin forms cylindrical transmembrane heptameric pores through interaction with ADAM10 receptors , SAS1802's specific structural elements and functional roles remain largely undefined. This fundamental difference places SAS1802 in the research category of proteins requiring primary characterization studies rather than the application-focused studies typical for established virulence factors .

What expression systems are typically used for producing recombinant SAS1802?

Recombinant SAS1802 is typically expressed in prokaryotic expression systems, with E. coli being the most common host. The expression protocol generally involves:

• Gene synthesis or PCR amplification of the SAS1802 coding sequence

• Cloning into an expression vector (commonly pET series vectors)

• Transformation into an expression strain (such as BL21(DE3))

• Induction of protein expression using IPTG

• Cell lysis and protein purification via affinity chromatography

The protein can be tagged (commonly with His6) to facilitate purification, or produced tag-free depending on the experimental requirements. Expression in eukaryotic systems such as yeast is less common but may be employed for specific applications requiring post-translational modifications .

What are the optimal conditions for expressing soluble recombinant SAS1802 in E. coli?

The optimal conditions for expressing soluble recombinant SAS1802 in E. coli typically involve:

For challenging expression cases, fusion partners such as MBP (maltose-binding protein), SUMO, or thioredoxin may be employed to enhance solubility. The expression conditions should be empirically optimized through small-scale test expressions before scaling up to larger cultures for purification .

What purification strategy would you recommend for obtaining high-purity SAS1802 for structural studies?

For structural studies requiring high-purity SAS1802, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins if His-tagged

• Intermediate purification: Ion exchange chromatography (IEX) based on the theoretical pI of SAS1802

• Polishing step: Size exclusion chromatography (SEC) to remove aggregates and achieve monodisperse protein preparation

• Optional tag removal: If a cleavable tag was used, insert a proteolytic cleavage step (TEV or PreScission protease) after the initial IMAC

• Buffer optimization: Screen various buffer conditions during the final SEC step to identify stabilizing conditions

The final protein preparation should be assessed for purity by SDS-PAGE (>95%), homogeneity by dynamic light scattering (DLS), and structural integrity by circular dichroism (CD) spectroscopy before proceeding with crystallization or NMR studies .

How can I design experiments to determine the potential binding partners of SAS1802 in S. aureus?

To identify potential binding partners of SAS1802, a multi-faceted approach is recommended:

• Pull-down assays: Use purified His-tagged SAS1802 as bait with S. aureus lysate as prey, followed by mass spectrometry identification of interacting proteins

• Bacterial two-hybrid system: Express SAS1802 fused to one domain of a split reporter protein, and screen against a library of S. aureus proteins fused to the complementary domain

• Co-immunoprecipitation: Generate antibodies against SAS1802 for immunoprecipitation from S. aureus lysates, followed by mass spectrometry analysis

• Crosslinking-MS: Use chemical crosslinkers to stabilize transient protein-protein interactions in vivo, followed by purification and mass spectrometry

• Proximity labeling: Express SAS1802 fused to a proximity labeling enzyme (e.g., BioID or APEX) in S. aureus to biotinylate nearby proteins for subsequent streptavidin pull-down and identification

Each approach has strengths and limitations, so combining multiple methods provides the most robust results. Control experiments using unrelated proteins or lysates from SAS1802 knockout strains are essential to identify specific versus non-specific interactions .

How might I approach determining the three-dimensional structure of SAS1802 when crystallization attempts have failed?

When crystallization attempts for SAS1802 have failed, alternative structural biology approaches can be considered:

• NMR spectroscopy: For proteins <30 kDa, solution NMR can provide atomic-resolution structures. Express SAS1802 with 15N and 13C labeling, and collect multi-dimensional NMR data sets.

• Cryo-electron microscopy (cryo-EM): Recent advances allow structure determination of smaller proteins. Consider:

  • Using antibody fragments to increase molecular weight

  • Embedding SAS1802 in nanodiscs or micelles if it has membrane-interacting regions

  • Utilizing advances in computational methods for small protein reconstruction

• AlphaFold2 or RoseTTAFold prediction: Recent AI-based structural prediction methods have reached near-experimental accuracy. The predicted structure can be validated using limited experimental data from circular dichroism, SAXS, or crosslinking-MS.

• Construct optimization: Design new constructs based on:

  • Secondary structure predictions to remove disordered regions

  • Limited proteolysis to identify stable domains

  • Fusion to crystallization chaperones like T4 lysozyme or rubredoxin

Each method provides complementary structural information, and integrative structural biology approaches combining multiple techniques often yield the most reliable results .

What computational approaches can be used to predict the function of SAS1802 based on its sequence and structure?

Multiple computational approaches can be employed to predict SAS1802's function:

• Sequence-based methods:

  • PSI-BLAST for remote homology detection

  • Hidden Markov Models (HMMs) to identify functional domains

  • Genomic context analysis (examining neighboring genes)

  • Co-evolution analysis to identify functionally linked proteins

• Structure-based methods:

  • Structural alignment against protein structure databases

  • Identification of catalytic triads or binding pockets

  • Electrostatic surface potential mapping

  • Normal mode analysis for potential conformational changes

• Integrative approaches:

  • Molecular docking with potential substrates or binding partners

  • Molecular dynamics simulations to explore dynamics

  • Machine learning models integrating multiple features

  • Systems biology network analysis to place SAS1802 in functional networks

Here's an example of predicted functional partners based on genomic context and co-expression data:

These predictions should be validated experimentally using the binding partner identification methods described earlier .

How can I design a genetic system to study the phenotypic effects of SAS1802 deletion or overexpression in S. aureus?

To study phenotypic effects of SAS1802 manipulation, design a comprehensive genetic system as follows:

• Knockout construction:

  • Create a clean deletion using allelic exchange vectors (e.g., pMAD, pKOR1)

  • Design deletion to maintain the reading frame of surrounding genes

  • Confirm deletion by PCR, sequencing, and Western blot

  • Create complementation strain by reintroducing SAS1802 under native promoter

• Conditional expression systems:

  • Tetracycline-inducible system (pRMC2 vector)

  • IPTG-inducible Pspac promoter system

  • Xylose-inducible system

• Reporter fusions:

  • Transcriptional fusion (promoter-reporter)

  • Translational fusion (SAS1802-reporter)

  • Reporters: GFP, luciferase, β-galactosidase

• Phenotypic characterization:

  • Growth curves under various conditions (different media, stress conditions)

  • Biofilm formation assays

  • Virulence in infection models

  • Proteomics and transcriptomics comparison

  • Metabolomics analysis

• Advanced techniques:

  • CRISPRi for partial knockdown

  • Dual-fluorescent protein systems to monitor localization

  • Protein degradation systems for temporal control

The genetic system should include proper controls such as wild-type strain, vector-only control, and strains expressing unrelated proteins to distinguish specific from non-specific effects .

How can SAS1802 be incorporated into vaccine development research for S. aureus?

Incorporating SAS1802 into S. aureus vaccine development would follow this research pathway:

• Antigenicity assessment:

  • Bioinformatic prediction of surface exposure and immunogenicity

  • Production of recombinant SAS1802 and testing for antibody recognition in convalescent patient sera

  • Epitope mapping to identify immunodominant regions

• Vaccine formulation strategies:

  • Subunit vaccine: Purified recombinant SAS1802

  • Multi-antigen approach: Combine with established antigens (similar to rFSAV approach that uses Hla, SEB, SpA, IsdB-N2 and MntC)

  • Epitope-based: Synthetic peptides representing immunodominant epitopes

  • DNA vaccine: SAS1802 gene in expression vector

• Adjuvant selection:

  • Alum-based formulations

  • TLR agonists (e.g., CpG, MPLA)

  • Liposomal or nanoparticle delivery systems

• Immune response characterization:

  • Antibody titers and isotype profiles

  • T-cell responses (Th1/Th2/Th17 balance)

  • Functional assays (opsonophagocytosis, neutralization)

• Protection studies:

  • Challenge models (bacteremia, skin infection, pneumonia)

  • Passive immunization with anti-SAS1802 antibodies

  • Assessment of bacterial clearance and disease parameters

This approach parallels the successful development of the recombinant five-antigen S. aureus vaccine (rFSAV), which demonstrated protection in multiple infection models and induced comprehensive cellular and humoral immune responses .

What experimental approaches would you use to investigate whether SAS1802 contributes to antibiotic resistance in S. aureus?

To investigate SAS1802's potential role in antibiotic resistance, employ these approaches:

• Susceptibility testing:

  • Determine minimum inhibitory concentrations (MICs) for various antibiotics in wild-type, SAS1802 knockout, and SAS1802-overexpressing strains

  • Time-kill assays to assess killing kinetics

  • Population analysis profiles to identify heteroresistant subpopulations

• Resistance mechanism studies:

  • Membrane permeability assays (uptake of fluorescent dyes)

  • Drug accumulation/efflux assays using radiolabeled or fluorescent antibiotics

  • Biofilm formation and antibiotic tolerance assessment

• Transcriptomic/proteomic analysis:

  • RNA-Seq comparing wild-type and SAS1802 mutant strains ± antibiotic exposure

  • Quantitative proteomics to identify differentially expressed resistance determinants

  • ChIP-Seq if SAS1802 might have DNA-binding properties affecting resistance gene expression

• Direct interaction studies:

  • In vitro binding assays between purified SAS1802 and antibiotics

  • Structural studies of SAS1802-antibiotic complexes

  • Surface plasmon resonance to determine binding kinetics

• Evolution experiments:

  • Serial passage in increasing antibiotic concentrations

  • Whole genome sequencing to identify compensatory mutations

  • Competition assays between wild-type and mutant strains

The experimental design should include appropriate controls and be performed in multiple S. aureus strain backgrounds to account for strain-specific effects on resistance phenotypes .

How might SAS1802 be used in structural biology research to understand UPF family proteins more broadly?

SAS1802 can serve as a model system for understanding UPF0435 family proteins through these approaches:

• Comparative structural analysis:

  • Determine high-resolution structure of SAS1802 using X-ray crystallography, NMR, or cryo-EM

  • Perform structural alignment with other UPF family members

  • Identify conserved structural motifs that may indicate function

• Structure-function studies:

  • Site-directed mutagenesis of conserved residues

  • Activity assays to correlate structural features with function

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Protein-protein interaction networks:

  • Yeast two-hybrid or bacterial two-hybrid screens

  • Affinity purification-mass spectrometry using SAS1802 as bait

  • Comparison of interaction partners across UPF family members

• Evolutionary analysis:

  • Phylogenetic analysis of UPF0435 family across bacterial species

  • Identification of co-evolving residues suggesting functional constraints

  • Genomic context analysis across species

• In silico structure prediction validation:

  • Use experimentally determined SAS1802 structure to assess accuracy of AlphaFold2/RoseTTAFold predictions

  • Develop improved prediction methods for UPF family proteins

  • Create structure-based functional annotation pipeline

This research would contribute to understanding uncharacterized protein families that comprise a significant portion of bacterial genomes and may reveal novel drug targets or biological mechanisms .

I'm observing protein aggregation during purification of recombinant SAS1802. What strategies can I use to improve protein solubility?

Protein aggregation of recombinant SAS1802 can be addressed using these strategies:

• Buffer optimization:

  • Screen various pH conditions (typically pH 6.0-8.0)

  • Test different salt concentrations (100-500 mM NaCl)

  • Add stabilizing agents:

    • Glycerol (5-20%)

    • Arginine (50-200 mM)

    • Tween-20 or other non-ionic detergents (0.01-0.05%)

• Expression modifications:

  • Lower induction temperature (16-18°C)

  • Reduce IPTG concentration (0.1-0.2 mM)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Use solubility-enhancing fusion tags (MBP, SUMO, TrxA)

• Purification adjustments:

  • Include reducing agents (1-5 mM DTT or TCEP)

  • Add protease inhibitors throughout purification

  • Maintain protein at moderate concentration (<2 mg/mL)

  • Avoid freeze-thaw cycles; store at 4°C for short-term use

• Refolding approaches (if inclusion bodies are unavoidable):

  • On-column refolding during IMAC purification

  • Systematic screening of refolding conditions using a matrix approach

  • Pulsatile refolding with stepwise reduction of denaturant

• Analytical techniques to monitor improvement:

  • Dynamic light scattering to assess homogeneity

  • Thermal shift assays to identify stabilizing conditions

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

Each protein has unique characteristics, so empirical testing of multiple conditions is essential to identify the optimal solution for SAS1802 .

What controls should I include when studying the function of SAS1802 to ensure the validity of my experimental findings?

Robust experimental design for studying SAS1802 function should include these controls:

• Genetic controls:

  • Wild-type strain (positive control)

  • SAS1802 knockout strain (negative control)

  • Complemented strain (knockout with reintroduced SAS1802)

  • Strain expressing inactive SAS1802 mutant (if catalytic residues identified)

  • Strain expressing unrelated protein of similar size/properties

• Protein-level controls:

  • Purified wild-type SAS1802 protein

  • Heat-denatured SAS1802 (negative control)

  • Point mutants affecting predicted functional sites

  • Related protein from same family (specificity control)

  • Unrelated protein of similar size/properties

• Assay-specific controls:

  • For binding assays: non-specific binding controls (e.g., GST-only)

  • For enzymatic assays: no-enzyme and no-substrate controls

  • For cell-based assays: vehicle-only and irrelevant protein controls

  • For animal studies: sham-treated and irrelevant protein controls

• Technical controls:

  • Biological replicates (different bacterial cultures/protein preparations)

  • Technical replicates (repeated measurements)

  • Blinding of samples during analysis when possible

  • Inclusion of internal standards for quantitative measurements

• Validation approaches:

  • Use multiple methodologies to address the same question

  • Perform dose-response or time-course experiments

  • Include positive controls with known outcomes

  • Reverse the experimental approach (e.g., gain-of-function and loss-of-function)

Proper controls ensure that observed effects are specifically attributable to SAS1802 rather than experimental artifacts or general stress responses .

How can I resolve contradictory results between in vitro biochemical assays and in vivo phenotypes when studying SAS1802?

Resolving contradictions between in vitro and in vivo results requires a systematic approach:

• Critically evaluate experimental conditions:

  • Are in vitro conditions physiologically relevant? (pH, salt, cofactors)

  • Does the recombinant protein have the same modifications as in vivo?

  • Are protein concentrations comparable between systems?

  • Are there interacting partners present in vivo but absent in vitro?

• Bridge the gap with intermediate approaches:

  • Cell extract-based assays (partially purified system)

  • Permeabilized cell assays (maintain cellular organization)

  • Ex vivo assays using freshly isolated components

  • Reconstitution experiments with purified interaction partners

• Advanced in vivo techniques:

  • FRET/BRET to monitor protein interactions in live cells

  • Activity-based protein profiling in intact cells

  • Chemical crosslinking in vivo before analysis

  • Single-cell analysis to detect population heterogeneity

• Resolve temporal and spatial factors:

  • Time-course experiments to capture transient effects

  • Subcellular localization studies

  • Regulated expression systems to control timing/levels

  • Stress or environmental conditions that might activate function

• Integrated data analysis:

  • Develop mathematical models incorporating both datasets

  • Use Bayesian approaches to update hypotheses based on all evidence

  • Consider emergent properties of complex systems

  • Look for conditional phenotypes dependent on specific conditions

This systematic approach often reveals that both results are correct under their specific conditions, and the contradiction reflects biological complexity rather than experimental error .

What emerging technologies could advance our understanding of SAS1802's role in S. aureus pathogenesis?

Several cutting-edge technologies could significantly advance SAS1802 research:

• CRISPR interference/activation systems:

  • CRISPRi for precise gene knockdown with temporal control

  • CRISPRa for upregulation of SAS1802 in specific contexts

  • Multiplexed CRISPR screens to identify genetic interactions

• Single-cell technologies:

  • Single-cell RNA-seq to detect heterogeneous responses

  • Single-cell proteomics to capture protein-level variation

  • Microfluidic systems for monitoring bacterial responses to stressors

• Advanced imaging:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with photoactivatable tags

  • Correlative light-electron microscopy for ultrastructural context

  • Expansion microscopy for nanoscale visualization

• Protein interaction mapping:

  • Proximity labeling (BioID, APEX) for in vivo interactome mapping

  • Cross-linking mass spectrometry for structural interactomics

  • Thermal proteome profiling to detect ligand interactions

  • Protein correlation profiling across fractionation schemes

• Host-pathogen interface:

  • Dual RNA-seq of host-pathogen interactions

  • Organoid infection models for tissue-specific responses

  • Ex vivo tissue infection models with live imaging

  • Humanized mouse models for improved translation

These technologies provide unprecedented resolution and scale for understanding protein function within complex biological systems and could reveal unexpected roles for SAS1802 in S. aureus biology and pathogenesis .

How might comparative genomics across different S. aureus strains inform our understanding of SAS1802 function?

Comparative genomics approaches can provide valuable insights into SAS1802 function:

• Conservation analysis:

  • Presence/absence of SAS1802 across S. aureus strains (core vs. accessory genome)

  • Sequence conservation levels indicating selective pressure

  • Identification of hypervariable regions suggesting immune interaction

  • Correlation with strain virulence or host adaptation

• Synteny analysis:

  • Conservation of genomic context across strains

  • Co-evolution with neighboring genes

  • Operon structure variations

  • Mobile genetic element associations

• Variant impact prediction:

  • Nonsynonymous/synonymous substitution ratios (dN/dS)

  • Identification of positively selected residues

  • Mapping variants to predicted structural domains

  • Correlation of variants with phenotypic differences

• Transcriptional regulation:

  • Conservation of promoter regions

  • Regulatory motif identification

  • Transcription factor binding site conservation

  • sRNA interaction site analysis

• Correlation with strain phenotypes:

  • Antibiotic resistance profiles

  • Host range and tissue tropism

  • Virulence in different infection models

  • Growth characteristics and metabolic capabilities

This comprehensive analysis could reveal patterns linking SAS1802 sequence variations to specific strain characteristics, providing testable hypotheses about its function and importance in different ecological niches .

What approaches would you recommend for developing high-throughput screening assays to identify small molecule modulators of SAS1802?

Developing high-throughput screening (HTS) assays for SAS1802 modulators requires a strategic approach:

• Target-based biochemical assays:

  • Thermal shift assays to detect ligand binding

  • Fluorescence polarization for detecting interactions with labeled partners

  • FRET/TR-FRET between SAS1802 and binding partners

  • AlphaScreen for protein-protein interaction disruption

• Phenotypic screening approaches:

  • Reporter strains (e.g., SAS1802 promoter driving luciferase expression)

  • Growth inhibition assays in SAS1802-dependent conditions

  • Stress response profiles in wild-type vs. SAS1802 mutant strains

  • Virulence factor expression readouts

• Fragment-based approaches:

  • NMR-based fragment screening

  • Surface plasmon resonance fragment screening

  • Mass spectrometry for detecting covalent binders

  • Crystallographic fragment screening

• Virtual screening methods:

  • Structure-based docking against SAS1802 models

  • Pharmacophore modeling based on interaction patterns

  • Molecular dynamics simulations to identify cryptic binding sites

  • Machine learning-based virtual screening

• Assay optimization considerations:

  • Miniaturization to 384 or 1536-well format

  • Z'-factor optimization (aim for >0.7)

  • DMSO tolerance assessment

  • Counter-screening assays to filter false positives