Recombinant UPF0213 protein M6_Spy1178 (M6_Spy1178)

What is the predicted structural confidence of recombinant UPF0213 protein M6_Spy1178 based on AlphaFold models?

The structural confidence of recombinant UPF0213 protein M6_Spy1178 can be assessed through AlphaFold prediction metrics, similar to other UPF0213 family proteins. Based on comparable UPF0213 proteins from Streptococcus pyogenes, such as SpyM50708, the predicted structure typically demonstrates a global pLDDT (predicted Local Distance Difference Test) score between 75-95 . For reference, the UPF0213 protein from Vibrio campbellii (VIBHAR_05350) shows a high global pLDDT score of 94.36, indicating very high confidence in the predicted model .

When working with these models, researchers should be aware that:

• Regions with pLDDT > 90 are considered very high confidence

• Regions with 70 < pLDDT ≤ 90 are considered confident

• Regions with 50 < pLDDT ≤ 70 have low confidence

• Regions with pLDDT ≤ 50 have very low confidence and may be unstructured in isolation

Laboratory validation of these computational predictions is highly recommended for critical research applications.

How does the sequence of UPF0213 protein M6_Spy1178 compare with other bacterial UPF0213 proteins?

UPF0213 proteins show variable sequence conservation across bacterial species while maintaining structural similarities. For comparative analysis, consider the following:

When designing experiments involving M6_Spy1178, researchers should conduct multiple sequence alignments to identify conserved domains that may be functionally significant. Particular attention should be paid to structural motifs rather than specific sequences, as UPF0213 proteins often maintain functional similarity despite sequence divergence.

What are the optimal expression systems for producing recombinant UPF0213 protein M6_Spy1178?

The selection of an appropriate expression system for UPF0213 protein M6_Spy1178 should be based on project requirements regarding protein folding, post-translational modifications, and downstream applications. Multiple expression options are available:

• E. coli expression systems: Preferred for high-yield production of UPF0213 proteins, as demonstrated by successful expression of related UPF0213 proteins including YPO3475/y0709/YP_0608 and yhbQ . These systems typically yield >85% purity by SDS-PAGE analysis.

• Yeast expression systems: Provide eukaryotic folding machinery that may benefit certain structural confirmations, though yields are typically lower than bacterial systems .

• Baculovirus expression: Offers advantages for proteins requiring complex folding or those that form inclusion bodies in bacterial systems .

• Mammalian cell expression: Consider for applications requiring mammalian-specific post-translational modifications, though this is rarely necessary for bacterial proteins .

For robust expression of M6_Spy1178, our recommended protocol includes:

• Using codon-optimized sequences for the expression host

• Employing fusion tags (His, GST, or Avi-tag) to facilitate purification

• Expression temperature optimization, typically 18°C for improved folding

• Inducer concentration titration to balance yield and solubility

What purification strategies maximize yield and biological activity of recombinant UPF0213 protein M6_Spy1178?

Purification of recombinant UPF0213 protein M6_Spy1178 requires a strategic approach to maintain structural integrity and functional activity:

• Initial capture: Immobilized metal affinity chromatography (IMAC) is effective for His-tagged constructs, while glutathione affinity chromatography works well for GST-fusion proteins.

• Intermediate purification: Ion exchange chromatography to separate based on charge properties. For UPF0213 proteins, anion exchange columns (e.g., Q Sepharose) typically provide better resolution than cation exchangers.

• Polishing step: Size exclusion chromatography to achieve >90% purity and remove aggregates.

Recommended buffer conditions based on related UPF0213 proteins:

• Lysis buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, protease inhibitors

• Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole

• Storage buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 10% glycerol

For long-term storage, add 5-50% glycerol and store at -20°C/-80°C in small aliquots to avoid freeze-thaw cycles. The general shelf life in liquid form is approximately 6 months at -20°C/-80°C, while lyophilized preparations can maintain stability for up to 12 months .

What experimental approaches can determine the biological function of UPF0213 protein M6_Spy1178?

The biological function of UPF0213 protein M6_Spy1178 remains partially characterized, requiring multiple experimental approaches:

• Comparative genomic analysis: Identify syntenic relationships and gene neighborhood conservation across Streptococcus species to infer potential function.

• Protein-protein interaction studies:

  • Pull-down assays using recombinant M6_Spy1178 as bait

  • Yeast two-hybrid screening against Streptococcus pyogenes library

  • Proximity-dependent biotin identification (BioID) using M6_Spy1178-BirA fusion

• Gene knockout/knockdown experiments:

  • CRISPR-Cas9 mediated gene deletion in S. pyogenes

  • Antisense RNA approaches for transient depletion

  • Analysis of resulting phenotypes for growth, morphology, and virulence alterations

• Structural biology approaches:

  • X-ray crystallography or cryo-EM to validate and refine AlphaFold predictions

  • Molecular dynamics simulations to identify potential binding pockets

• Biochemical activity assays:

  • Testing for potential enzymatic activities (hydrolase, transferase, etc.)

  • In vitro RNA binding assays, particularly given the role of other UPF proteins in RNA metabolism

Investigations should consider potential relationships to nonsense-mediated mRNA decay pathways, as other UPF family proteins like UPF1 are involved in these processes .

How can differential scanning fluorimetry be optimized for thermal stability analysis of UPF0213 protein M6_Spy1178?

Differential scanning fluorimetry (DSF) provides critical insights into the thermal stability and buffer optimization for UPF0213 protein M6_Spy1178. For robust implementation:

• Sample preparation optimization:

  • Protein concentration: Test range of 0.1-0.5 mg/mL (typically 0.2 mg/mL is optimal)

  • Dye selection: SYPRO Orange at 5-10X final concentration is recommended

  • Buffer screening: Test multiple buffers (MES, HEPES, Tris) at pH 6.0-8.0

• Experimental parameters:

  • Temperature range: 25-95°C

  • Ramp rate: 1°C/min for highest resolution

  • Filter settings: Excitation ~490 nm, emission ~575 nm (SYPRO Orange)

• Data analysis approach:

  • Calculate Tm using first derivative method (inflection point of melting curve)

  • Assess curve shape for indications of multiple domains or aggregation

• Buffer optimization matrix:

  Buffer Component	Range to Test	Typical Optimal Range
  pH	5.5-9.0 (0.5 increments)	7.0-8.0
  NaCl	0-500 mM (50 mM increments)	100-200 mM
  Glycerol	0-20% (5% increments)	5-10%
  Reducing agents	0-5 mM DTT or TCEP	1 mM

• Ligand screening: Test potential ligands/cofactors at 1-10 mM to identify stabilizing compounds, which may indicate biological binding partners.

When interpreting DSF data for UPF0213 proteins, compare results with known proteins in the same family to identify patterns in stabilizing conditions, providing insights into potential shared functional characteristics.

How do UPF0213 proteins relate evolutionarily to other UPF family proteins, particularly UPF1?

While sharing the UPF designation, UPF0213 proteins and UPF1 represent distinct protein families with different evolutionary origins and functions:

• Evolutionary divergence:

  • UPF0213 proteins are primarily bacterial proteins of unknown function

  • UPF1 is a eukaryotic protein with well-characterized roles in nonsense-mediated mRNA decay (NMD)

• Structural domains:

  • UPF0213 proteins are relatively small (~90-105 amino acids) and possess a single domain

  • UPF1 is larger (~1,100 amino acids) with multiple domains including RecA-like domains (RecA1 and RecA2), a 1B domain with a "regulatory loop," and a helicase domain

• Functional mechanisms:

  • UPF1 functions through alternating conformations between "open" and "closed" states, regulated by an 11-amino acid insertion in the regulatory loop (353-GNEDLVIIWLR-363) that enhances its translocation and ATPase activities

  • UPF0213 protein functions remain largely uncharacterized but appear to be distinct from UPF1's role in RNA surveillance

• Molecular partners:

  • UPF1 interacts with UPF2, UPF3, and components of the translation termination machinery

  • UPF0213 interaction partners remain to be identified

Despite sharing the "UPF" designation, researchers should not assume functional similarity between UPF0213 and UPF1 proteins. The UPF (UP-Frameshift) nomenclature often indicates historical classification rather than functional relationships.

What are the predicted binding interfaces of UPF0213 protein M6_Spy1178 based on structural modeling?

Computational analysis of UPF0213 protein M6_Spy1178's structure reveals potential interaction surfaces that should be experimentally validated:

• Electrostatic surface analysis: Using molecular visualization tools and the AlphaFold predicted structure, researchers can identify charged patches that may serve as protein-protein or protein-nucleic acid interaction sites.

• Conservation mapping: By aligning UPF0213 sequences across bacterial species and mapping conservation onto the structural model, evolutionarily conserved surface residues likely indicate functional binding interfaces.

• Molecular docking predictions: Computational docking with potential binding partners including:

  • Small molecules

  • Peptides

  • Nucleic acids (particularly RNA given the relationship of other UPF proteins to RNA processing)

• Cavity detection algorithms: Software tools like CASTp, POCASA, or fpocket can identify potential binding pockets within the UPF0213 structure.

• Molecular dynamics simulations: Analysis of conformational flexibility can reveal transient binding pockets not evident in static structures.

For experimental validation of these predictions, site-directed mutagenesis of predicted interface residues followed by functional assays would provide crucial insights into the protein's biological role. Additionally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can experimentally verify predicted binding interfaces by measuring changes in solvent accessibility upon binding partner interaction.

How can CRISPR-Cas9 techniques be optimized for studying UPF0213 protein M6_Spy1178 function in Streptococcus pyogenes?

Deploying CRISPR-Cas9 for UPF0213 protein M6_Spy1178 functional studies in S. pyogenes requires specific optimization strategies:

• Guide RNA design considerations:

  • Target sequences with NGG PAM sites within the M6_Spy1178 gene

  • Perform off-target analysis specific to S. pyogenes genome

  • Design multiple gRNAs targeting different regions to ensure success

  • Consider targeting regions away from predicted functional domains

• Delivery methods for S. pyogenes:

  • Electroporation of ribonucleoprotein (RNP) complexes

  • Plasmid-based delivery systems using compatible origins of replication

  • Phage-based delivery systems for difficult-to-transform strains

• Editing strategies:

  • Complete gene knockout via non-homologous end joining (NHEJ)

  • Domain-specific mutations via homology-directed repair (HDR)

  • CRISPRi for transient knockdown via dCas9 fused to repressor domains

  • CRISPRa for overexpression studies

• Phenotypic analysis pipeline:

  • Growth curve analysis under various stress conditions

  • Transcriptomic profiling (RNA-seq) of knockout vs. wild-type

  • Metabolomic analysis to identify pathway alterations

  • Virulence assays in appropriate infection models

• Complementation strategies:

  • Trans-complementation with inducible expression vectors

  • Cis-complementation by reintegration at native or ectopic loci

  • Expression of tagged versions for localization studies

For S. pyogenes specifically, consider using modified CRISPR systems that avoid interference with the endogenous CRISPR-Cas9 machinery, as S. pyogenes is the source organism for the most commonly used Cas9 enzyme.

What experimental approaches can resolve discrepancies between computational predictions and observed functions of UPF0213 proteins?

Addressing inconsistencies between computational predictions and experimental observations requires systematic investigation:

When computational predictions conflict with experimental results, consider:

• The possibility of condition-specific functions not captured in standard assays

• Potential moonlighting functions beyond the primary predicted role

• Post-translational modifications affecting function but not primary structure

• Protein-protein interactions that modify the function in vivo

How do different expression hosts affect post-translational modifications of recombinant UPF0213 protein M6_Spy1178?

The choice of expression host significantly impacts post-translational modifications (PTMs) of recombinant UPF0213 protein M6_Spy1178, affecting both structural and functional properties:

• Co-expression strategies with PTM-specific enzymes in E. coli

• Cell-free systems supplemented with PTM enzymes

• Site-specific mutation of PTM sites to either mimic (phosphomimetic) or prevent modification

Mass spectrometry analysis of natively expressed M6_Spy1178 from S. pyogenes would provide definitive insights into the natural PTM landscape to guide expression system selection.

What are the critical quality control parameters for ensuring consistency in recombinant UPF0213 protein M6_Spy1178 preparations?

Establishing rigorous quality control parameters is essential for reproducible research with recombinant UPF0213 protein M6_Spy1178:

• Identity verification:

  • Mass spectrometry (MS/MS) peptide mapping against theoretical sequence

  • N-terminal sequencing for first 5-10 amino acids

  • Western blot with specific antibodies (if available)

• Purity assessment:

  • SDS-PAGE with densitometry analysis (target: >90% purity)

  • Size exclusion chromatography to detect aggregates and fragments

  • Isoelectric focusing to detect charge variants

  • Endotoxin testing for proteins expressed in gram-negative bacteria

• Structural integrity:

  • Circular dichroism to verify secondary structure elements

  • Intrinsic fluorescence for tertiary structure assessment

  • Differential scanning fluorimetry for thermal stability

• Functional activity:

  • Binding assays with identified interaction partners

  • Enzymatic activity assays (if applicable)

  • Cell-based functional assays relevant to biological context

• Stability monitoring:

  • Accelerated stability studies at elevated temperatures

  • Real-time stability testing under storage conditions

  • Freeze-thaw stability over multiple cycles

Critical specifications for research-grade M6_Spy1178 preparations:

• Purity: >85% by SDS-PAGE

• Endotoxin: <1.0 EU per μg protein (for in vitro/in vivo experiments)

• Aggregation: <5% by SEC-HPLC

• Bioactivity: Within 20% of reference standard in applicable assays

Implement acceptance criteria for each parameter and maintain detailed batch records including expression conditions, purification protocols, and quality control results to ensure reproducibility across experiments.

How can recombinant UPF0213 protein M6_Spy1178 be used in structural vaccinology approaches against Streptococcus pyogenes?

Recombinant UPF0213 protein M6_Spy1178 presents potential opportunities for structural vaccinology against Streptococcus pyogenes, requiring a systematic research approach:

• Antigenic potential assessment:

  • Epitope prediction using computational tools (BepiPred, Ellipro)

  • Surface accessibility analysis based on AlphaFold structural models

  • Conservation analysis across S. pyogenes strains to identify invariant regions

  • Cross-reactivity assessment with human proteins to avoid autoimmunity

• Immunogenicity evaluation:

  • Antibody generation in animal models using purified recombinant M6_Spy1178

  • T-cell epitope identification through stimulation assays

  • Cytokine profiling to characterize immune response quality

  • Assessment of antibody functionality (opsonization, neutralization)

• Structure-based design approaches:

  • Generation of structure-guided M6_Spy1178 fragments focusing on antigenic regions

  • Design of chimeric constructs with known immunogenic carrier proteins

  • Surface re-engineering to enhance stability and immunogenicity

  • Multimerization strategies to increase epitope density

• Formulation considerations:

  • Adjuvant selection based on desired immune response profile

  • Stability studies under vaccine formulation conditions

  • Delivery vehicle optimization (liposomes, virosomes, nanoparticles)

Drawing from related coronavirus membrane protein research, 3D model prediction comparison analysis can identify structural features that correlate with immunogenicity . When designing M6_Spy1178-based vaccine components, ensure structural integrity of key epitopes through circular dichroism and epitope-specific antibody recognition assays post-formulation.

How can recombinant UPF0213 protein constructs be optimized for studying protein-protein interaction networks in bacterial systems?

Optimizing recombinant UPF0213 protein constructs for protein-protein interaction (PPI) network analysis requires targeted design strategies:

• Fusion tag selection and positioning:

  • N-terminal vs. C-terminal tag positioning based on structural predictions

  • Tag options for affinity purification (His, GST, MBP)

  • Fluorescent protein fusions (GFP, mCherry) for localization and FRET studies

  • Proximity labeling tags (BirA*, APEX2) for in vivo interaction mapping

  • Consideration for tag removal via protease cleavage sites

• Construct design strategies:

  • Full-length protein with flexible linkers to tags

  • Domain-specific constructs to map interaction interfaces

  • Surface residue mutations to validate computational binding predictions

  • Expression of orthologous UPF0213 proteins for evolutionary comparison

• Bacterial two-hybrid system optimization:

  • BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system adaptation

  • Split-protein complementation assays (split-GFP, split-luciferase)

  • Gateway-compatible construct libraries for high-throughput screening

• In vitro interaction analysis techniques:

  • Bio-layer interferometry with biotinylated constructs

  • Surface plasmon resonance with various immobilization strategies

  • Isothermal titration calorimetry for thermodynamic parameters

  • Microscale thermophoresis for solution-based interaction studies

• In vivo validation approaches:

  • Co-immunoprecipitation with epitope-tagged constructs

  • Fluorescence microscopy to assess co-localization

  • FRET/BRET assays for direct interaction detection

  • Crosslinking mass spectrometry for interaction interface mapping