Recombinant UPF0374 protein M6_Spy1367 (M6_Spy1367)

What is UPF0374 protein M6_Spy1367 and how is it classified?

UPF0374 protein M6_Spy1367 is a bacterial protein from Streptococcus pyogenes M6 strain, belonging to the UPF (Uncharacterized Protein Family) 0374 class. This protein shares structural similarities with UPF0374 protein MGAS2096_Spy1342 and other proteins in the Streptococcus genus . As an uncharacterized protein family member, its precise biological function remains under investigation, making it a subject of interest for structural and functional genomics research. The protein is typically studied in recombinant form to elucidate its structural properties and potential role in bacterial pathogenesis or cellular processes. Sequence analysis indicates conserved domains that may suggest involvement in cellular signaling or metabolic pathways within S. pyogenes.

What expression systems are most suitable for producing recombinant UPF0374 protein M6_Spy1367?

Multiple expression systems can be employed for producing recombinant UPF0374 protein M6_Spy1367, each offering distinct advantages:

What purification methods are recommended for recombinant UPF0374 protein M6_Spy1367?

Purification of recombinant UPF0374 protein M6_Spy1367 typically employs a multi-step approach similar to other recombinant proteins:

• Initial Capture: Affinity chromatography using polyhistidine tags is common, as seen with other recombinant proteins . The protein can be expressed with a polyhistidine tag at the C-terminus for efficient IMAC (Immobilized Metal Affinity Chromatography) purification.

• Intermediate Purification: Ion exchange chromatography to separate based on charge differences.

• Polishing Step: Size exclusion chromatography to achieve >90% purity, similar to standards for other recombinant proteins .

The specific purification protocol should be optimized based on the expression system used and the desired purity level. For research requiring ultra-high purity, additional chromatography steps may be necessary. Final purity should be assessed using SDS-PAGE analysis, with >90% purity being standard for most research applications .

How should recombinant UPF0374 protein M6_Spy1367 be stored to maintain stability?

Proper storage conditions are critical for maintaining the stability and activity of recombinant UPF0374 protein M6_Spy1367:

Best practices include:

• Store the purified protein at -20°C to -80°C for long-term storage, similar to other recombinant proteins .

• Avoid repeated freeze-thaw cycles, which can cause protein degradation and loss of activity.

• Consider lyophilization for extended stability, as commonly done with recombinant proteins .

• When reconstituting lyophilized protein, use sterile buffers containing at least 0.1% human or bovine serum albumin as a stabilizing agent .

• Divide the reconstituted protein into single-use aliquots to prevent multiple freeze-thaw cycles.

How do post-translational modifications affect the structure and function of UPF0374 protein M6_Spy1367?

Post-translational modifications (PTMs) can significantly impact the structure, stability, and biological activity of UPF0374 protein M6_Spy1367. While bacterial proteins typically undergo fewer PTMs than eukaryotic proteins, they can still experience modifications that affect function.

Potential PTMs for UPF0374 protein M6_Spy1367 include:

• Phosphorylation: May regulate protein-protein interactions or enzymatic activity

• Acetylation: Could affect protein stability and interaction with DNA/RNA

• Glycosylation: Rare in bacterial proteins but possible in recombinant versions expressed in eukaryotic systems

The choice of expression system directly influences PTM profiles:

• E. coli expression typically results in minimal PTMs

• Insect and mammalian expression systems provide more complex PTMs that may be required for proper folding or activity maintenance

For studies requiring PTM analysis, mass spectrometry-based proteomics approaches (including LC-MS/MS) are recommended to identify and quantify modifications. Comparing protein expressed in different systems can help elucidate the functional significance of specific PTMs on UPF0374 protein activity.

What computational methods can be used to predict the structure and function of UPF0374 protein M6_Spy1367?

As an uncharacterized protein, computational methods are valuable for predicting the structure and potential function of UPF0374 protein M6_Spy1367:

• Homology Modeling: When sequence identity with known structures exceeds 30%, homology modeling can provide reliable structural predictions. Tools like SWISS-MODEL, Phyre2, or I-TASSER are applicable.

• Ab Initio Modeling: For regions lacking homologous structures, ab initio approaches like Rosetta or AlphaFold2 can predict structures based solely on physicochemical principles and sequence information.

• Molecular Dynamics Simulations: Can provide insights into protein flexibility, conformational changes, and potential binding sites.

• Function Prediction Tools:

  • Gene Ontology (GO) term prediction

  • Conserved domain analysis using CDD, Pfam, or InterPro

  • Binding site prediction using tools like COACH or COFACTOR

• Integrated Approaches: Combining sequence pattern-driven de novo assembly with pattern recognition algorithms similar to those used in chloroplast genome assembly .

The reliability of computational predictions should be validated experimentally, but they provide valuable starting points for hypothesis generation and experimental design.

What are the challenges in maintaining protein stability during purification of UPF0374 protein M6_Spy1367?

Maintaining protein stability during purification represents a significant challenge for UPF0374 protein M6_Spy1367. Key challenges and strategies include:

• Protein Aggregation:

  • Challenge: The protein may form aggregates during concentration steps

  • Solution: Include low concentrations (1-5%) of glycerol or non-ionic detergents in buffers

• Proteolytic Degradation:

  • Challenge: Endogenous proteases from expression hosts can degrade the target protein

  • Solution: Add protease inhibitor cocktails, maintain low temperatures (4°C) during purification

• Oxidation of Cysteine Residues:

  • Challenge: Formation of non-native disulfide bonds

  • Solution: Include reducing agents like DTT or β-mercaptoethanol in buffers

• Buffer Compatibility:

  • Challenge: Protein stability varies with pH, salt concentration, and buffer composition

  • Solution: Screen multiple buffer conditions using differential scanning fluorimetry (DSF)

• Tag Interference:

  • Challenge: Affinity tags may affect protein folding or activity

  • Solution: Compare tagged and tag-cleaved versions; use smaller tags or place at alternate termini

A systematic approach to optimization is recommended, starting with standard conditions (PBS buffer, pH 7.4, 150 mM NaCl) and iteratively refining based on protein stability assessment via techniques like size exclusion chromatography to verify monodispersity.

How can protein-protein interactions involving UPF0374 protein M6_Spy1367 be studied?

Multiple complementary techniques can be employed to study protein-protein interactions involving UPF0374 protein M6_Spy1367:

For comprehensive characterization, a multi-method approach is recommended. Initial screening with Y2H or pull-down assays can identify potential interaction partners, followed by validation and quantitative analysis using SPR or ITC. Structural characterization of interaction complexes can be achieved through X-ray crystallography or cryo-electron microscopy if sufficient quantities of pure protein complexes can be isolated.

What techniques can be used to assess the expression levels of UPF0374 protein M6_Spy1367 in different host systems?

Multiple analytical techniques can be employed to quantitatively assess expression levels of UPF0374 protein M6_Spy1367:

• SDS-PAGE with Coomassie Staining:

  • Appropriate for abundant proteins (>0.5 μg per band)

  • Semi-quantitative when compared to known standards

  • Limited sensitivity but simple to perform

• Western Blotting:

  • Requires antibodies against the protein or affinity tag (e.g., His-tag)

  • Higher sensitivity than Coomassie staining (10-100 ng range)

  • Semi-quantitative unless specialized protocols are used

• ELISA:

  • Quantitative with proper standard curves

  • High sensitivity (pg-ng range)

  • Requires specific antibodies

• Fluorescence-Based Quantification:

  • Can use GFP/YFP fusion constructs

  • Allows real-time monitoring in living cells

  • May affect protein folding or function

• Mass Spectrometry:

  • Highly specific and sensitive

  • Can be used for absolute quantification with isotope-labeled standards

  • Requires specialized equipment and expertise

When comparing expression levels across different host systems (E. coli, yeast, insect cells, mammalian cells), it's essential to normalize data to account for differences in cell density, lysis efficiency, and sample loading. For accurate quantification, multiple methods should be employed, with mass spectrometry offering the highest specificity and sensitivity for definitive measurements.

How can one troubleshoot low expression yields of UPF0374 protein M6_Spy1367?

Low expression yields of UPF0374 protein M6_Spy1367 can result from various factors. A systematic troubleshooting approach includes:

• Codon Optimization:

  • Problem: Rare codons in the M6_Spy1367 sequence not optimal for host

  • Solution: Synthesize codon-optimized gene for the specific expression host

• Expression Conditions Optimization:

  • Problem: Suboptimal induction parameters

  • Solution: Systematically vary temperature (15-37°C), inducer concentration, and induction time

• Solubility Enhancement:

  • Problem: Formation of inclusion bodies

  • Solution: Lower expression temperature (16-20°C), co-express with chaperones, use solubility tags (SUMO, MBP, TRX)

• Vector Selection:

  • Problem: Promoter strength or plasmid copy number issues

  • Solution: Test different vectors with varying promoter strengths

• Host Strain Selection:

  • Problem: Host strain incompatibility

  • Solution: Screen multiple strains (BL21(DE3), Rosetta, SHuffle for E. coli)

• Media Optimization:

  • Problem: Insufficient nutrients or toxic metabolites

  • Solution: Try enriched media (TB, 2YT) or fed-batch cultivation

Systematic optimization matrix:

When implementing changes, modify one parameter at a time and assess the impact on both expression level and protein solubility using SDS-PAGE analysis of both soluble and insoluble fractions.

How can the biological activity of recombinant UPF0374 protein M6_Spy1367 be assayed?

Assessing the biological activity of UPF0374 protein M6_Spy1367 presents challenges due to its uncharacterized nature. A multi-faceted approach is recommended:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) spectroscopy to confirm secondary structure

  • Thermal shift assays to evaluate stability

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to verify oligomeric state

• Functional Assays Based on Bioinformatic Predictions:

  • If sequence analysis suggests enzymatic activity, develop specific enzymatic assays

  • For potential DNA/RNA binding proteins, electrophoretic mobility shift assays (EMSA)

  • Protein-protein interaction screening if regulatory functions are predicted

• Cell-Based Assays:

  • Effects on bacterial growth when overexpressed or knocked down

  • Host cell response assays if pathogenesis-related functions are suspected

  • Localization studies using fluorescently tagged versions

• Comparative Activity Assays:

  • Side-by-side testing with known homologs like UPF0374 protein MGAS2096_Spy1342

  • Cross-species complementation studies

Activity assays should be designed based on the most current bioinformatic predictions about the protein's function. As new data emerges about UPF0374 family proteins, assay development should be iteratively refined to better characterize specific activities.

What are the best practices for analyzing the purity and homogeneity of UPF0374 protein M6_Spy1367 preparations?

Comprehensive analysis of protein purity and homogeneity requires multiple complementary techniques:

• Purity Assessment:

  • SDS-PAGE with densitometry (>90% purity standard for most applications)

  • Capillary electrophoresis for higher resolution

  • Reverse-phase HPLC for detecting closely related species

• Homogeneity Analysis:

  • Size-exclusion chromatography (SEC) to detect aggregates and oligomers

  • SEC-MALS for absolute molecular weight determination and oligomeric state analysis (similar to analyses performed for other recombinant proteins)

  • Dynamic light scattering (DLS) for polydispersity assessment

• Contaminant Detection:

  • Mass spectrometry for host cell protein identification and quantification

  • Limulus amebocyte lysate (LAL) assay for endotoxin quantification (<1.0 EU per μg protein is standard)

  • Absorbance ratio (A260/A280) for nucleic acid contamination

• Structural Integrity:

  • Circular dichroism spectroscopy for secondary structure confirmation

  • Differential scanning fluorimetry for thermal stability

  • Limited proteolysis to assess proper folding

Acceptance criteria for research-grade preparations typically include:

• 90% purity by SDS-PAGE

• <10% aggregates by SEC

• <1.0 EU/μg endotoxin content

• Monodisperse population by DLS (polydispersity index <0.2)

For structural biology applications or therapeutic research, more stringent criteria may be necessary, potentially requiring additional purification steps or alternative expression systems.