Recombinant Escherichia coli O6:K15:H31 UPF0266 membrane protein yobD (yobD)

What is the UPF0266 membrane protein yobD in E. coli O6:K15:H31?

The yobD protein is an uncharacterized protein family 0266 (UPF0266) membrane protein found in pathogenic Escherichia coli strain O6:K15:H31. This protein is part of a pathogenicity island (PAI) that contributes to the virulence of the organism. The K15 capsule determinant in uropathogenic E. coli strain 536 (O6:K15:H31) is encoded within a 79.6-kb pathogenicity island designated PAI V536, which contains multiple virulence-associated genes . The yobD gene may be involved in membrane-associated functions potentially related to the organism's pathogenicity.

Based on homology with similar proteins, such as the UPF0266 membrane protein yobD in Salmonella enteritidis, the protein likely contains approximately 150-160 amino acids and features multiple transmembrane domains characteristic of integral membrane proteins .

What expression systems are recommended for recombinant yobD production?

• BL21(DE3) derivatives: The standard strain for recombinant protein expression, though membrane proteins often present challenges.

• C41(DE3) and C43(DE3): These Walker strains are specifically engineered for toxic and membrane protein expression. Their mutations in the lacUV5 promoter convert it back to the weaker wild-type promoter, reducing expression levels to more tolerable amounts for the cell .

• Lemo21(DE3): This strain allows tunable expression by modulating T7 RNA polymerase activity, which can be critical for toxic membrane proteins.

Methodologically, expression should begin with small-scale trials using multiple strains to determine the optimal conditions before scaling up production.

How should researchers address protein toxicity when expressing recombinant yobD?

Membrane protein toxicity is a significant challenge when expressing proteins like yobD. Several methodological approaches can mitigate this issue:

• Strain selection: Use specialized strains like C41(DE3) and C43(DE3) that were specifically selected to withstand the expression of toxic proteins. These strains have mutations that revert the lacUV5 promoter back to a weaker wild-type promoter, resulting in lower expression levels that are more tolerable to the cell .

• Tightly controlled expression: Use tightly regulated promoters and lower the temperature (18-25°C) during induction to decrease expression rates and allow proper folding.

• Periplasmic targeting: Consider secretion to the periplasm using appropriate signal peptides such as PelB, OmpA, or DsbA. This approach can reduce cytoplasmic accumulation and toxicity .

• Co-expression with chaperones: Express molecular chaperones like DnaK/DnaJ/GrpE or GroEL/GroES to assist in proper folding.

• Fusion tags: Use solubility-enhancing tags like MBP (maltose-binding protein) or SUMO to improve folding and reduce toxicity.

When toxicity is suspected, monitoring growth rates before induction is essential. If the recombinant strain grows slower than the empty-vector control, basal expression of the toxic protein may be occurring, requiring adjustments to the expression strategy .

What purification strategies are most effective for recombinant yobD?

Purifying membrane proteins like yobD requires specialized approaches:

Step-by-step purification protocol:

• Cell lysis and membrane fraction isolation:

  • Disrupt cells using sonication, high-pressure homogenization, or enzymatic lysis

  • Separate membrane fraction through ultracentrifugation (typically 100,000 × g for 1 hour)

• Membrane solubilization:

  • Screen multiple detergents (DDM, LDAO, OG, Triton X-100) at different concentrations

  • Incubate membranes with selected detergent for 1-2 hours at 4°C with gentle rotation

• Affinity chromatography:

  • If expressing His-tagged yobD (recommended), use immobilized metal affinity chromatography (IMAC)

  • Include the optimal detergent at concentrations above critical micelle concentration (CMC) in all buffers

  • Consider using cobalt-based resins for higher specificity than nickel-based resins

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Include detergent at 2-3× CMC in running buffer

• Quality assessment:

  • SDS-PAGE with Coomassie staining to assess purity

  • Western blotting to confirm identity

  • Dynamic light scattering to evaluate homogeneity

Storage recommendations include maintaining the protein in a detergent buffer with 5-50% glycerol at -20°C or -80°C to prevent freeze-thaw damage .

How can researchers optimize solubilization of yobD from membrane fractions?

Membrane protein solubilization is a critical step that requires careful optimization:

Methodological approach for detergent screening:

• Set up a detergent screening panel including:

  • Mild detergents: DDM, LMNG, digitonin

  • Intermediate detergents: DM, UDM, LDAO

  • Harsh detergents: OG, SDS, Triton X-100

• Test solubilization efficiency using a small-scale protocol:

  • Resuspend membrane pellets in buffer containing different detergents

  • Incubate at 4°C for 1-2 hours with gentle agitation

  • Ultracentrifuge at 100,000 × g for 30 minutes

  • Analyze supernatant for protein content using Western blot

• Optimize detergent concentration:

  • Test detergent concentrations ranging from 0.5-2% for initial solubilization

  • After purification, reduce to 2-3× CMC to minimize free micelle content

• Consider detergent mixtures or additives:

  • Cholesterol hemisuccinate (CHS) can stabilize some membrane proteins

  • Lipids like POPC or E. coli lipid extract may improve stability

  • Glycerol (5-10%) can enhance protein stability

The optimal solubilization conditions should balance efficient extraction with maintaining the protein's native conformation and function.

What strategies can improve the expression of correctly folded yobD with proper disulfide bond formation?

The formation of correct disulfide bonds is crucial for many membrane proteins to attain their proper three-dimensional structure. For yobD, consider these advanced approaches:

• Periplasmic expression: Direct the protein to the periplasm using appropriate signal peptides (like PelB or DsbA), as the periplasm provides an oxidizing environment conducive to disulfide bond formation. The DsbA signal sequence is particularly effective as it utilizes the SRP pathway for co-translational translocation .

• Specialized strains: Use engineered E. coli strains with oxidative cytoplasmic environments:

  • Origami strains (Novagen): Contain trxB- gor- mutations with a suppressor mutation in ahpC

  • SHuffle T7 Express strain: Contains trxB- and gor- mutations and constitutively expresses disulfide bond isomerase DsbC, which helps correct mis-oxidized proteins and acts as a chaperone

  • Rosetta-gami B strain: Combines the advantages of the Origami strain with the pRARE plasmid to address codon bias issues

• Co-expression with disulfide bond-forming enzymes: Co-express proteins like DsbC, which promotes the correction of mis-oxidized proteins into their correct form and assists in protein folding.

• Optimization of redox conditions: Add appropriate ratios of reduced/oxidized glutathione to the lysis buffer to maintain the correct redox environment during purification.

The choice between these strategies depends on the specific properties of yobD, particularly the number and positions of cysteine residues. Experimental validation through activity assays or structural analyses is necessary to confirm proper disulfide bond formation .

How can researchers investigate the potential role of yobD in pathogenicity?

To investigate the potential role of yobD in pathogenicity, particularly in relation to its presence in the PAI V536 pathogenicity island of E. coli O6:K15:H31, consider these methodological approaches:

• Gene knockout studies:

  • Create a precise yobD deletion mutant using CRISPR-Cas9 or lambda Red recombination

  • Perform complementation studies by reintroducing the wild-type or mutated yobD gene

  • Assess virulence using appropriate infection models (e.g., cell invasion assays, murine models)

• Protein localization and interaction studies:

  • Use fluorescent protein fusions or immunofluorescence to determine subcellular localization

  • Perform co-immunoprecipitation or bacterial two-hybrid assays to identify interaction partners

  • Consider proximity-labeling approaches like BioID to identify the protein's interaction network in vivo

• Transcriptomic and proteomic profiling:

  • Compare wild-type and yobD mutant strains under infection-relevant conditions

  • Identify differentially expressed genes/proteins that may indicate affected pathways

  • Perform gene set enrichment analysis to identify affected biological processes

• Structure-function analysis:

  • Identify conserved domains through bioinformatic analysis

  • Create point mutations or truncations in functional domains

  • Assess the impact on virulence-associated phenotypes

• Host response studies:

  • Examine host immune responses to wild-type versus yobD mutant strains

  • Measure cytokine production, inflammasome activation, or autophagy induction

  • Assess bacterial survival in macrophages or neutrophils

The K15 capsule of uropathogenic E. coli strain 536 has been shown to be important for virulence in a murine model of ascending urinary tract infection, though not for serum resistance . Investigating whether yobD influences capsule expression or function could provide valuable insights into its role in pathogenicity.

What expression vector features are critical for successful recombinant yobD production?

Vector design significantly impacts membrane protein expression success. For yobD, consider these critical features:

• Promoter selection:

  • For toxic membrane proteins, PBAD (arabinose-inducible) or Ptac promoters offer tighter regulation than T7

  • If using T7, consider vectors with lacI or lacIq for tighter repression of basal expression

  • The pRha promoter (rhamnose-inducible) provides concentration-dependent induction without all-or-none effects

• Fusion tags and their positioning:

  • N-terminal tags: 6×His, MBP, or SUMO can improve solubility

  • C-terminal tags: May be preferable if the N-terminus is involved in membrane insertion

  • Cleavage sites: Include TEV or PreScission protease sites for tag removal

  • For membrane proteins like yobD, position the tag away from transmembrane domains

• Codon optimization:

  • Optimize codons for E. coli expression while maintaining strategic rare codons that may facilitate proper folding

  • Consider using Rosetta strains that supply tRNAs for rare codons rather than full codon optimization

• Signal sequences:

  • For periplasmic targeting, include efficient signal sequences like PelB or DsbA

  • The DsbA signal sequence is particularly effective for membrane proteins as it utilizes the SRP pathway for co-translational translocation

• Origin of replication and copy number:

  • Low-copy vectors (pACYC or pSC101 origins) often yield better results for toxic membrane proteins

  • pET vectors with ColE1 origin may cause excessive expression leading to toxicity

• Antibiotic resistance:

  • Consider using antibiotics that do not affect membrane integrity (avoid polymyxins)

  • Kanamycin is often preferred over ampicillin for long-term stability

A modular vector system allowing rapid testing of different tags, promoters, and signal sequences can significantly expedite optimization of yobD expression.

How can researchers overcome aggregation and inclusion body formation when expressing yobD?

Membrane proteins like yobD frequently aggregate or form inclusion bodies when overexpressed. Advanced strategies to address this challenge include:

• Controlled expression kinetics:

  • Reduce temperature to 15-20°C during induction

  • Use low inducer concentrations (e.g., 0.1 mM IPTG instead of 1 mM)

  • Implement auto-induction media for gradual protein production

  • Consider continuous exchange cell-free expression systems for direct incorporation into nanodiscs or liposomes

• Specialized solubilization and refolding:

  • If inclusion bodies form, solubilize with 8M urea or 6M guanidine hydrochloride

  • Implement step-wise dialysis with decreasing denaturant concentrations

  • Add detergents during refolding (typically started at concentrations above CMC)

  • Include mixed lipid-detergent micelles during refolding to promote proper insertion

• Co-expression strategies:

  • Co-express with molecular chaperones (DnaK/DnaJ/GrpE or GroEL/GroES)

  • For disulfide-containing proteins, co-express with DsbA or DsbC to assist proper disulfide formation

  • Consider co-expression with FkpA, Skp, or SurA for periplasmic folding assistance

• Fusion partner approach:

  • Use solubility-enhancing fusion partners like MBP, SUMO, or Mistic

  • Consider fusion with fluorescent proteins (GFP) as folding indicators

  • Implement split-GFP systems to monitor and select for properly folded protein

• In vitro folding assessment:

  • Circular dichroism spectroscopy to evaluate secondary structure

  • Fluorescence-detection size exclusion chromatography (FSEC) to assess homogeneity

  • Thermal stability assays (DSF or nanoDSF) to optimize buffer conditions

For membrane proteins like yobD that are particularly prone to aggregation, directing the protein to the periplasm or utilizing specialized strains like SHuffle may be necessary to achieve proper folding .

What structural analysis techniques are most appropriate for characterizing recombinant yobD?

Structural characterization of membrane proteins like yobD presents unique challenges that require specialized approaches:

• Crystallography preparation:

  • Screen multiple detergents to identify those that maintain stability while promoting crystal contacts

  • Consider lipidic cubic phase (LCP) crystallization for membrane proteins

  • Implement surface entropy reduction through site-directed mutagenesis of surface residues

  • Use antibody fragments (Fab or nanobodies) to create additional crystal contacts

• Cryo-electron microscopy (cryo-EM):

  • Prepare protein in detergent micelles, nanodiscs, or amphipols

  • Consider using Volta phase plates to enhance contrast

  • Implement focused refinement techniques for flexible regions

  • Use symmetry-restrained refinement if applicable

• NMR approaches:

  • For full-length structural studies, consider solid-state NMR

  • Solution NMR with selective isotope labeling for specific domains

  • Use detergent micelles or bicelles as membrane mimetics

  • Implement TROSY techniques to improve spectral quality

• Small-angle X-ray/neutron scattering (SAXS/SANS):

  • Provide low-resolution envelope information in native-like environments

  • Use contrast matching in SANS to distinguish protein from detergent contributions

  • Combine with computational modeling for hybrid structure determination

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map solvent-accessible regions and conformational dynamics

  • Identify potential ligand binding sites

  • Determine membrane-embedded regions through differential exchange rates

• Cross-linking mass spectrometry (XL-MS):

  • Use membrane-permeable cross-linkers to capture native interactions

  • Identify distance constraints to validate computational models

  • Map protein-protein interfaces for complexes

• Molecular dynamics simulations:

  • All-atom simulations in explicit membrane environments

  • Coarse-grained approaches for larger-scale dynamics

  • Homology modeling and threading for initial structure prediction

The methodology selection should be guided by the specific research questions regarding yobD structure and the available protein quantity and quality.

How can researchers develop functional assays for yobD to characterize its membrane-associated activities?

Developing functional assays for poorly characterized membrane proteins like yobD requires a systematic approach:

• Membrane integration and topology mapping:

  • Construct fusions with reporter enzymes (PhoA, LacZ) at various positions

  • Use accessibility studies with cysteine-reactive reagents

  • Implement protease protection assays to identify cytoplasmic/periplasmic domains

  • Design a topology map based on combined results

• Transport activity assessment:

  • Reconstitute purified yobD into liposomes with encapsulated fluorescent dyes

  • Monitor potential ion transport using voltage-sensitive dyes

  • Implement patch-clamp techniques if channel activity is suspected

  • Assess substrate transport using radiolabeled compounds based on bioinformatic predictions

• Protein-protein interaction studies:

  • Bacterial two-hybrid or BACTH system for membrane protein interactions

  • Split-ubiquitin yeast two-hybrid systems optimized for membrane proteins

  • Co-immunoprecipitation with carefully selected detergents

  • Biolayer interferometry or surface plasmon resonance with detergent-solubilized protein

• Lipid interaction analysis:

  • Liposome binding assays with various lipid compositions

  • Measure changes in intrinsic tryptophan fluorescence upon lipid binding

  • Assess lipid specificity using lipid overlay assays

  • Monitor membrane perturbation using dye-leakage assays

• Functional complementation:

  • Express yobD in knockout strains of related proteins

  • Test complementation of phenotypes in heterologous systems

  • Perform cross-species complementation to identify conserved functions

Since yobD is found in pathogenic E. coli O6:K15:H31 within a pathogenicity island , assays measuring adherence to epithelial cells, biofilm formation, or resistance to host defense mechanisms may reveal its functional role in virulence.

What bioinformatic approaches can predict yobD function and guide experimental design?

Advanced bioinformatic analyses can provide crucial insights for guiding experimental characterization of yobD:

• Structural predictions and modeling:

  • Use AlphaFold2 or RoseTTAFold for ab initio structure prediction

  • Apply membrane protein-specific threading algorithms (e.g., MEMOIR)

  • Validate models through molecular dynamics simulations in membrane environments

  • Identify potential binding pockets or active sites for functional testing

• Evolutionary analysis:

  • Perform sensitive homology searches using HHpred or HMMER

  • Construct phylogenetic profiles to identify co-evolving gene families

  • Analyze syntenic relationships across bacterial genomes

  • Implement evolutionary coupling analysis to predict residue contacts

• Functional annotation transfer:

  • Search for distant homologs with known functions using PSI-BLAST or DELTA-BLAST

  • Analyze domain architecture using InterProScan

  • Look for conserved motifs associated with specific functions

  • Examine Gene Ontology terms of homologs with Bayesian probability assessment

• Genomic context analysis:

  • Analyze operon structure and co-transcribed genes

  • Look for proximity to genes with known functions within pathogenicity islands

  • Examine regulation patterns through promoter analysis

  • Study the distribution across bacterial species and correlation with pathogenicity

• Computational docking and virtual screening:

  • Identify potential ligands through virtual screening against predicted structure

  • Perform molecular docking studies with candidate substrates

  • Validate interactions through molecular dynamics simulations

  • Design mutations to test computational predictions experimentally

• Transcriptomic data mining:

  • Analyze expression patterns across different conditions

  • Identify co-expression networks to suggest functional associations

  • Look for differential expression during infection processes

Since yobD is found within the PAI V536 pathogenicity island in E. coli O6:K15:H31 , analyzing its genomic context relative to virulence factors and comparing its sequence with proteins in other pathogenicity islands may provide valuable functional insights.

How can researchers systematically troubleshoot low expression yields of recombinant yobD?

Low expression yields of membrane proteins like yobD require systematic troubleshooting:

Comprehensive troubleshooting strategy:

• Assess expression at the mRNA level:

  • Perform RT-qPCR to determine if transcription is occurring

  • Analyze mRNA stability using actinomycin D chase experiments

  • Optimize mRNA secondary structure around the start codon

  • Consider codon optimization focusing on the first 15-20 codons

• Evaluate protein stability and degradation:

  • Add protease inhibitors during cell lysis

  • Test expression in protease-deficient strains

  • Perform pulse-chase experiments to measure half-life

  • Consider fusion to stabilizing domains or partners

• Optimize induction parameters:

  • Test induction at various growth phases (early, mid, late log)

  • Titrate inducer concentration (0.01-1 mM IPTG)

  • Vary post-induction temperature (15°C, 20°C, 25°C, 30°C, 37°C)

  • Test different induction durations (2h, 4h, overnight)

• Strain optimization:

  • Try specialized strains like C41(DE3), C43(DE3) designed for toxic proteins

  • Test Lemo21(DE3) with variable lysozyme expression

  • Consider Rosetta strains if rare codons are present

  • For disulfide bonds, use Origami or SHuffle strains

• Media optimization:

  • Compare rich media (LB, TB, 2YT) versus minimal media

  • Test auto-induction media for gradual protein production

  • Supplement with membrane components (phospholipids)

  • Add osmolytes like betaine or sucrose to stabilize protein

• Detection optimization:

  • Ensure antibodies can access the epitope in membrane context

  • Try multiple extraction methods (various detergents)

  • Use more sensitive detection methods (fluorescent Western blot)

  • Check for protein in different cellular fractions

For toxic membrane proteins, expression often improves dramatically when using specialized strains like C41(DE3) and C43(DE3), which contain mutations that weaken the promoter driving T7 RNA polymerase expression .

What analytical techniques can assess the quality and homogeneity of purified yobD protein?

Ensuring the quality and homogeneity of membrane proteins like yobD requires specialized analytical approaches:

• Size and homogeneity assessment:

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine protein-detergent complex size

  • Analytical ultracentrifugation (AUC) to assess oligomeric state and homogeneity

  • Native PAGE with Coomassie or silver staining

  • Dynamic light scattering (DLS) to evaluate polydispersity

• Structural integrity evaluation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Intrinsic tryptophan fluorescence to assess tertiary structure

  • Thermal shift assays to determine stability and optimal buffer conditions

  • Limited proteolysis followed by mass spectrometry to identify flexible regions

• Detergent and lipid content analysis:

  • Thin-layer chromatography (TLC) to analyze co-purified lipids

  • Colorimetric assays to quantify detergent content

  • Mass spectrometry to identify bound lipids

  • 31P-NMR to analyze phospholipid content

• Ligand binding assessment:

  • Microscale thermophoresis (MST) to measure binding affinity

  • Isothermal titration calorimetry (ITC) for complete binding thermodynamics

  • Surface plasmon resonance (SPR) for binding kinetics

  • Fluorescence-based ligand binding assays

• Activity measurements:

  • ATPase activity assays if homology suggests ATP binding

  • Transport assays in proteoliposomes

  • Electrophysiology for channel activity

  • Functional complementation in knockout strains

• Long-term stability monitoring:

  • Accelerated stability testing at different temperatures

  • Freeze-thaw stability with various cryoprotectants

  • Time-course activity measurements

  • Regular SEC analysis to detect aggregation

The choice of techniques should be guided by the research questions and the predicted function of yobD. A combination of these methods provides a comprehensive assessment of protein quality.

How does yobD compare with homologous proteins in other bacterial species?

A comprehensive comparative analysis of yobD with homologs in other bacteria provides evolutionary and functional insights:

Comparative analysis framework:

• Sequence-based comparison:

  • Sequence identity and similarity metrics across bacterial species

  • Multiple sequence alignment to identify conserved motifs and residues

  • Conservation mapping onto predicted structural models

  • Analysis of species-specific insertions or deletions

• Structural comparison:

  • Superposition of predicted or experimentally determined structures

  • Analysis of conserved structural features versus variable regions

  • Electrostatic surface potential comparison

  • Membrane topology conservation assessment

• Genomic context analysis:

  • Operon structure comparison across species

  • Analysis of conserved gene neighborhoods

  • Correlation with pathogenicity islands or virulence clusters

  • Horizontal gene transfer evidence through GC content and codon usage analysis

• Functional comparison:

  • Known functions of homologs in other species

  • Association with specific pathogenic mechanisms

  • Differences between pathogenic and non-pathogenic strains

  • Expression patterns during infection or stress conditions

• Evolutionary analysis:

  • Phylogenetic tree construction

  • Selection pressure analysis (dN/dS ratios)

  • Evolutionary rate comparison with housekeeping genes

  • Identification of recent duplications or pseudogenization events

The yobD gene is found within a pathogenicity island (PAI V536) in uropathogenic E. coli strain 536 (O6:K15:H31) but is absent from non-pathogenic E. coli K-12 strain MG1655 . This suggests a potential role in virulence, possibly related to the K15 capsule determinant, which has been shown to be important for virulence in urinary tract infections .

What experimental approaches can validate computational predictions about yobD function?

To bridge the gap between computational predictions and experimental validation for yobD function:

• Site-directed mutagenesis targeting:

  • Conserved residues identified through multiple sequence alignments

  • Predicted active sites or binding pockets from structural models

  • Residues under positive selection from evolutionary analysis

  • Potential membrane-interacting regions

• Domain deletion and chimeric proteins:

  • Create truncation series to identify functional domains

  • Swap domains with homologs to determine specificity determinants

  • Design chimeric proteins to test function transfer

  • Express isolated domains to assess independent functions

• Ligand and interaction validation:

  • Screen predicted ligands using binding assays

  • Validate protein-protein interactions through pull-downs or crosslinking

  • Perform competition assays with predicted substrates

  • Use photoaffinity labeling with predicted binding partners

• Functional complementation studies:

  • Express yobD in knockout strains of homologous genes

  • Create conditional knockdowns to assess phenotype

  • Perform cross-species complementation to test functional conservation

  • Use heterologous expression systems to isolate specific functions

• Structural validation:

  • Confirm predicted topology through cysteine accessibility methods

  • Validate structural predictions through distance measurements (FRET, crosslinking)

  • Test membrane insertion and orientation predictions

  • Use hydrogen-deuterium exchange to validate predicted solvent-exposed regions

• Phenotypic assays based on predictions:

  • If transport function is predicted, test substrate specificity

  • For predicted roles in virulence, assess pathogenicity in relevant models

  • If stress response functions are suggested, test survival under stress conditions

  • For predicted signaling roles, examine downstream pathway activation

Since yobD is located within a pathogenicity island containing the K15 capsule determinant in uropathogenic E. coli , testing its role in capsule formation, adherence to epithelial cells, and virulence in animal models would be particularly relevant validation approaches.