Recombinant UPF0229 protein BA_0551/GBAA_0551/BAS0519 (BA_0551, GBAA_0551, BAS0519)

What is UPF0229 protein BA_0551/GBAA_0551/BAS0519?

UPF0229 protein BA_0551/GBAA_0551/BAS0519 is a recombinant protein from Bacillus anthracis with a full amino acid sequence of 391 residues. The protein belongs to the UPF0229 family of uncharacterized proteins. The sequence is: MGEENQPNYT ISQENWSLHR KGYDDQQRHQ EKVQEAIKNN LPDLVTEESI VMSNGKDVVK IPIRSLDEYK IRYNYDKNKH VGQGNGDSKV GDVVARDGSG GQKQKGPGKG QGAGDAAGED YYEAEVSILE LEQAFFKELE LPNLKRKEMD ENRIEHVEFN DIRKTGLWGN IDKKRTMISA YKRNAMRGKA SFHPIHQEDL KFRTWNEVLK PDSKAVVLAM MDTSGSMGIW EKYMARSFFF WMTRFLRTKY ETVDIEFIAH HTEAKVVPEE EFFSKGESGG TICSSVYKKA LELIDNKYSP DRYNIYPFHF SDGDNLTSDN ARCVKLVEEL MKKCNMFGYG EVNQYNRHST LMSAYKNIKD ENFRYYILKQ KADVFHAMKS FFREESGEKM A .

For research purposes, this protein is produced recombinantly in various expression systems including E. coli, yeast, baculovirus, or mammalian cells, with each system offering different advantages in terms of yield, post-translational modifications, and folding properties .

What expression systems are suitable for recombinant UPF0229 protein production?

Several expression systems can be employed for UPF0229 protein production, each with distinct advantages:

E. coli and yeast offer the best yields and shorter turnaround times for UPF0229 protein production. Expression in insect cells with baculovirus or mammalian cells provides many of the post-translational modifications necessary for correct protein folding or retention of protein activity .

How can I optimize expression of UPF0229 protein in E. coli?

Optimizing expression in E. coli requires careful consideration of several factors:

• Strain selection: For difficult-to-express proteins like UPF0229, consider specialized strains:

  • BL21(DE3) for general high-level expression

  • Origami™ or SHuffle® strains for enhanced disulfide bond formation

  • CodonPlus or Rosetta strains for rare codon optimization

  • C41(DE3) and C43(DE3) for potentially toxic proteins

• Vector selection: pBAD/gIII vectors can provide regulated, secreted recombinant protein expression with tunable induction using L-arabinose at concentrations from 0.00002% to 0.2% .

• Expression temperature: Lower temperatures (16-25°C) often increase solubility for challenging proteins like UPF0229 .

For comprehensive optimization, use the following step-by-step approach:

• Transform the expression vector into the selected E. coli strain

• Grow a small-scale culture (10mL) to OD600 = 0.5

• Split into multiple tubes with different inducer concentrations

• Incubate at different temperatures

• Analyze protein expression at various time points (2h, 4h, overnight)

• Determine soluble vs. insoluble fractions through cell lysis and SDS-PAGE analysis

What strategies can improve translation efficiency for UPF0229 protein?

Recent research has identified specific factors affecting translation efficiency that can be optimized for UPF0229 protein:

• Translation initiation site accessibility: The accessibility of translation initiation sites, modeled using mRNA base-unpairing across Boltzmann's ensemble, outperforms other features in predicting expression success. For UPF0229 protein, ensure the 5' region of the mRNA has minimal secondary structure .

• Codon optimization: Low-abundance tRNA depletion causes ribosome stalling. Analysis of UPF0229 sequence for rare codons is essential, particularly checking for:

  • AGG/AGA (Arg)

  • AUA (Ile)

  • CUA (Leu)

  • CCC (Pro)

  • GGA (Gly)

  If rare codons exceed 5% of total codons, consider using a codon-optimized gene or specialized strains like BL21(DE3)CodonPlus .

• N-terminal sequence engineering: The "translation rheostat" approach can tune expression levels by modifying codons 3-5. For UPF0229 protein, consider implementing one of these high-scoring motifs at positions 3-5:

  • AAD UAU (D = A, G, or U; encoding KY or NY)

  • AAV AUU (V = A, C, or G; encoding KI or NI)

• Local G+C content: The G+C content in regions -24:24 and -30:30 affects opening energy and MFE (Minimum Free Energy), respectively. Lower G+C content in these regions often correlates with higher expression success .

What is the most effective purification strategy for UPF0229 protein?

UPF0229 protein BA_0551 can be effectively purified using the following approach:

• Detection methods: For UPF0229 protein detection during purification, antibodies against the appropriate epitope can be used:

  • Anti-Myc or Anti-Myc-HRP for c-myc tagged protein

  • Anti-His(C-term) or Anti-His(C-term)-HRP for C-terminal polyhistidine tagged protein

• Additional purification steps: For higher purity, consider:

  • Ion exchange chromatography based on the theoretical pI of UPF0229

  • Size exclusion chromatography to separate aggregates or oligomeric forms

  • Hydrophobic interaction chromatography if the protein has hydrophobic patches

How can I assess the quality and folding of purified UPF0229 protein?

To assess the quality and proper folding of UPF0229 protein:

• SDS-PAGE analysis: Evaluate purity and approximate molecular weight (expected ~43 kDa based on the 391 amino acid sequence)

• Western blot: Confirm identity using antibodies against the protein or tag

• Size exclusion chromatography: Determine oligomeric state and detect aggregation

• Circular dichroism (CD): Assess secondary structure content

• Dynamic light scattering (DLS): Evaluate homogeneity and detect aggregation

• Thermal shift assay: Determine thermal stability and optimum buffer conditions

• Mass spectrometry: Confirm molecular weight and post-translational modifications

• Limited proteolysis: Evaluate domain organization and stability

For UPF0229 specifically, which belongs to an uncharacterized protein family, structural characterization may be particularly important to gain insights into its function.

Why is my UPF0229 protein expression low or forming inclusion bodies?

Low expression or inclusion body formation are common challenges with UPF0229 protein. Here's a systematic approach to troubleshooting:

For UPF0229 protein specifically, if you observe no expression on a Coomassie-stained gel:

• Re-run samples on SDS-PAGE and perform western blot

• Use antibody to your protein or Anti-Myc/Anti-His antibodies

• Include a negative control (empty vector) and positive control

How can I improve the solubility of UPF0229 protein?

For improving UPF0229 protein solubility:

• Expression conditions:

  • Lower temperature (16-20°C)

  • Reduced inducer concentration

  • Slower induction rate using lactose instead of IPTG

  • Extended expression time (overnight)

• Fusion partners:

  • MBP (Maltose-Binding Protein) - highly effective for enhancing solubility

  • SUMO (Small Ubiquitin-like Modifier) - promotes correct folding

  • Thioredoxin (Trx) - enhances disulfide bond formation

  • GST (Glutathione S-Transferase) - improves solubility but may form dimers

• Co-expression strategies:

  • Molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Folding modulators (Trigger factor, FkpA)

  • For UPF0229, which may contain disulfide bonds, co-express with DsbC

• Protein engineering:

  • Identify and remove aggregation-prone regions

  • Add solubilizing residues (charged residues on surface)

  • Modify translation rate using the "translation rheostat" approach for slower, more accurate folding

How does UPF0229 protein expression compare across different host systems?

A comparative analysis of UPF0229 protein expression across different host systems reveals important considerations for researchers:

For UPF0229 protein specifically, E. coli and yeast offer the best yields and shorter turnaround times, while insect cells and mammalian cells provide better post-translational modifications that may be necessary for correct folding or activity retention .

Recent studies indicate that P. pastoris might replace mammalian cell cultures for many applications, potentially offering a good balance between yield and proper folding for UPF0229 protein .

What are the latest advances in proteomics methodologies applicable to studying UPF0229 protein?

Recent advances in proteomics methodologies that can be applied to UPF0229 protein research include:

• Improved protein separation strategies:
  Research shows that the success rate of proteome analysis depends significantly on the degree of protein separation. For UPF0229 protein characterization, extensive protein separation improves the relative dynamic range (RDR) and extends the success rate of the experiment .

• Optimized loading amounts:
  Increasing the amount of peptides loaded on reversed-phase chromatography (RPC) columns significantly improves detection success rates. For UPF0229 protein analysis, loading 10 µg of peptides (compared to 0.1 µg) can lead to substantial gains in relative dynamic range and success rate .

• Enhanced peptide separation:
  The effect of improved peptide separation varies depending on other experimental parameters. For UPF0229 protein analysis, enhancing peptide separation from 100 to 1,000 fractions can substantially improve detection, but only when combined with increased loading amounts .

• Targeted proteomics approaches:
  Using targeted approaches like selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can improve sensitivity for detecting UPF0229 protein in complex samples.

The simulations of proteome analysis indicate that when analyzing a protein like UPF0229, researchers should first optimize protein separation, then maximize sample loading, and finally improve peptide separation to achieve the best results .

How can adaptive evolution approaches improve UPF0229 protein expression?

Adaptive evolution has emerged as a powerful approach for enhancing recombinant protein expression, including proteins like UPF0229:

• Coupling growth to product formation:
  Recent research demonstrates that synthetic pathways can be evolved from theoretical yields of 7-20% to near quantitative yield by coupling cell growth with product titers. For UPF0229 protein, this approach could potentially increase yields by identifying mutations that favor its production .

• Genome-wide mutations affecting expression:
  Genome sequencing of evolved strains has identified global RNA processors (rpoB/rpoC, pcnB, and rne) as key targets for mutation in successful producer cells. For UPF0229 protein expression, specific mutations in these genes could potentially increase yields .

• Transcriptional remodeling:
  Adaptive evolution can lead to transcriptional remodeling that significantly increases the availability of central building blocks like acetyl-CoA (up to 25-fold increase). This metabolic shift could benefit UPF0229 protein production by improving cellular resources allocation .

This approach has been successfully applied to evolve strains for production of industrially-relevant compounds and could be adapted for enhancing UPF0229 protein yields .

What computational approaches can predict the structure and function of UPF0229 protein?

As an uncharacterized protein family member, computational approaches are valuable for predicting UPF0229 structure and function:

• Homology modeling:

  • Use tools like SWISS-MODEL, Phyre2, or AlphaFold2 to predict 3D structure

  • For UPF0229 family proteins, compare with related UPF0229 proteins from E. coli (YeaH) and other organisms that may have more annotation

• Domain prediction:

  • Use InterProScan, SMART, or Pfam to identify conserved domains

  • UPF0229 proteins may contain domains that provide clues to their function

• Structural classification:

  • Use CATH or SCOP to classify predicted structures

  • Compare with structurally similar proteins of known function

• Functional prediction:

  • Use tools like GOblet, ProFunc, or ConSurf for functional annotation

  • Analyze conservation patterns to identify functionally important residues

  • Predict binding sites using tools like SiteHound or FTSite

• Protein-protein interaction prediction:

  • Use STRING or PrePPI to predict interaction partners

  • These predictions may provide insights into cellular pathways involving UPF0229

• Molecular dynamics simulations:

  • Explore conformational flexibility and stability

  • Identify potential ligand binding sites through water/small molecule mapping

For UPF0229 protein BA_0551 specifically, comparing with related proteins like YeaH from E. coli, which is associated with stress response in some organisms like B. subtilis (YhbH), could provide functional insights .

How can I determine the biological function of UPF0229 protein?

To determine the biological function of this uncharacterized protein:

• Expression profiling:

  • Analyze when and where UPF0229 is expressed in B. anthracis

  • Compare expression under different stress conditions (heat, pH, oxidative stress)

  • Look for co-expressed genes that may indicate function

• Genetic approaches:

  • Create gene knockout and analyze phenotypes

  • Perform complementation studies

  • Use CRISPR-Cas9 for precise genome editing

• Protein interaction studies:

  • Perform pull-down assays followed by mass spectrometry

  • Use yeast two-hybrid or bacterial two-hybrid systems

  • Employ proximity labeling approaches (BioID, APEX)

• Biochemical characterization:

  • Test for enzymatic activities based on structural predictions

  • Perform substrate screening

  • Analyze post-translational modifications

• Localization studies:

  • Determine subcellular localization using fluorescent protein fusions

  • Perform fractionation studies

  • Immunolabeling with electron microscopy for high-resolution localization

• Structural studies:

  • X-ray crystallography or cryo-EM to determine high-resolution structure

  • NMR spectroscopy for dynamic regions and ligand binding

Based on related UPF0229 family members, consider testing for stress response functions, as the B. subtilis homolog YhbH is classified as a stress response protein .

What are the emerging technologies for improving UPF0229 protein production?

Emerging technologies with potential application to UPF0229 protein production include:

• Cell-free protein synthesis systems:

  • Eliminates constraints of cell viability

  • Allows direct manipulation of the translation environment

  • Enables production of proteins toxic to host cells

  • Can be optimized specifically for UPF0229 production with precise control of reaction components

• Machine learning for expression optimization:

  • Predicts optimal expression conditions based on protein sequence

  • Models trained on successful expression data for similar proteins

  • Can design optimal coding sequences for UPF0229 expression

  • Recent studies show that machine learning models can accurately predict expression success based on mRNA features

• Synthetic minimal cells:

  • Reduced genome complexity

  • Fewer competing pathways for resources

  • May provide higher yields for UPF0229 protein

  • Eliminates unnecessary cellular processes

• Continuous evolution systems:

  • PACE (Phage-Assisted Continuous Evolution) or similar approaches

  • Allows rapid evolution of expression systems for UPF0229

  • Can develop strains with optimized expression capabilities

  • Continuous selection for high expression over many generations

• Genome editing technologies:

  • CRISPR-Cas9 for precise genetic modifications

  • Creation of customized host strains specifically for UPF0229 expression

  • Introduction of beneficial mutations identified through adaptive evolution

  • Generation of synthetic hosts with optimized metabolic pathways

These emerging technologies hold promise for overcoming current limitations in recombinant UPF0229 protein production and may enable more efficient, cost-effective, and higher-yielding processes in the future.

How might the study of UPF0229 protein contribute to our understanding of bacterial physiology?

As an uncharacterized protein family, research on UPF0229 proteins has potential to reveal important aspects of bacterial physiology:

• Stress response mechanisms:

  • Related UPF0229 family member YhbH in B. subtilis is classified as a stress response protein

  • Understanding UPF0229 function may reveal novel stress adaptation pathways in B. anthracis

  • May uncover previously unknown mechanisms for bacterial survival under adverse conditions

• Regulatory networks:

  • Identification of interaction partners could reveal regulatory pathways

  • May function in transcriptional or post-transcriptional regulation

  • Could be involved in signaling cascades important for virulence or adaptation

• Metabolic functions:

  • May participate in currently unknown metabolic pathways

  • Could be involved in specialized metabolism related to B. anthracis lifestyle

  • May function in metabolic adaptation during host infection

• Structural insights:

  • Novel protein folds or domains could expand our understanding of protein structure-function relationships

  • May reveal new classes of protein interactions or enzymatic mechanisms

  • Could serve as a target for antimicrobial development

• Evolutionary relationships:

  • Comparing UPF0229 proteins across species may reveal evolutionary adaptations

  • Could provide insights into bacterial speciation and adaptation

  • May identify conserved functions important across bacterial phyla