Recombinant Escherichia coli O9:H4 UPF0266 membrane protein yobD (yobD)

Which E. coli strains are most suitable for expressing the recombinant YobD protein?

For initial expression screening of recombinant YobD protein, BL21(DE3) and derivatives of the K-12 lineage are recommended as primary host strains. BL21(DE3) offers several advantages including:

• Deficiency in the Lon protease, which reduces degradation of foreign proteins

• Absence of the OmpT outer membrane protease, preventing degradation during cell lysis

• High transformation efficiency with exogenous DNA

For membrane proteins like YobD that may impose metabolic burden on the host, specialized strains such as C41(DE3) and C43(DE3) should be considered, as they were specifically developed to withstand the expression of potentially toxic membrane proteins through mutations in the lacUV5 promoter region that drives T7 RNA polymerase expression .

What expression vectors are optimal for recombinant YobD production?

The optimal expression vectors for YobD protein should be selected based on the following considerations:

Temperature-inducible systems may be particularly valuable for YobD expression, as they allow growth at lower temperatures (15°C with cold-shock promoters) which often improves membrane protein folding and incorporation .

What purification strategy would yield the highest purity of recombinant His-tagged YobD protein?

A multi-step purification strategy optimized for membrane proteins like YobD would include:

• Membrane Isolation: Differential centrifugation following cell lysis to separate membrane fractions

• Detergent Solubilization: Using appropriate detergents (DDM, LDAO, or Triton X-100) to extract membrane proteins

• Immobilized Metal Affinity Chromatography (IMAC): Utilizing the N-terminal His-tag for selective binding

• Size Exclusion Chromatography: Final polishing step to remove aggregates and obtain homogeneous protein

The recommended storage buffer for purified YobD protein is Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein stability. For long-term storage, addition of 5-50% glycerol and storage at -20°C/-80°C in small aliquots is recommended to avoid repeated freeze-thaw cycles .

How can researchers optimize soluble expression of recombinant YobD membrane protein?

Membrane proteins like YobD often present challenges for soluble expression. The following methodological approaches can improve solubility:

• Temperature Modulation: Lower growth temperatures (15-25°C) slow protein production rate, allowing proper folding

• Induction Parameters: Lower IPTG concentrations (0.1-0.5 mM) or alternative inducers for gentler expression

• Host Strain Selection: Use of C41(DE3) or C43(DE3) strains specifically developed for membrane protein expression

• Co-expression Strategies: Addition of chaperones or foldases to assist proper folding

• Fusion Partners: N-terminal fusion with solubility enhancers like MBP (maltose-binding protein) or SUMO

A systematic micro-expression trial approach is recommended to identify optimal conditions before scale-up. High-throughput screening in 2-ml tubes or 96-well plates allows testing of multiple combinations of these variables .

What analytical methods should be employed to confirm the structural integrity of purified YobD protein?

To validate the structural integrity of purified YobD, a combination of complementary analytical techniques is recommended:

For membrane proteins like YobD, additional techniques such as hydrogen-deuterium exchange mass spectrometry may provide insights into solvent-accessible regions and membrane-embedded domains .

How can site-directed mutagenesis be applied to study functional domains within the YobD protein?

Site-directed mutagenesis offers a powerful approach to investigate structure-function relationships in YobD protein. The methodological workflow should include:

• Target Selection: Identify conserved residues within the YobD sequence for mutagenesis based on sequence alignment with other UPF0266 family proteins

• Mutagenesis Strategy: Design primers for:

  • Alanine scanning of charged residues (Arg, Lys, Asp, Glu)

  • Conservative substitutions within putative transmembrane domains

  • Cysteine substitutions for subsequent labeling experiments

• Validation: Confirm mutations by sequencing and expression testing

• Functional Analysis: Compare wild-type and mutant proteins in terms of:

  • Membrane integration efficiency

  • Protein stability

  • Interaction with potential binding partners

This systematic approach can help delineate critical regions for YobD function, particularly the amino acid sequence segments that are essential for membrane insertion and protein-protein interactions .

What experimental approaches can elucidate the physiological role of YobD in E. coli?

Despite limited information on YobD function, several experimental strategies can help determine its physiological role:

• Gene Knockout Studies: CRISPR-Cas9 or recombineering to create yobD knockout strains and assess phenotypic changes

• Transcriptomics: RNA-Seq to identify genes co-regulated with yobD under various growth conditions

• Proteomics:

  • Pull-down assays using His-tagged YobD to identify interaction partners

  • Comparative proteomics between wild-type and yobD knockout strains

• Localization Studies: Fluorescent protein fusions to determine subcellular localization

• Stress Response Assays: Testing sensitivity of yobD knockout strains to various stressors (pH, osmotic stress, antibiotics)

The integration of these approaches provides a comprehensive view of YobD function within the cellular context, potentially revealing its role in membrane biology or specific metabolic pathways .

What are the challenges and solutions in studying membrane topology of the YobD protein?

Determining membrane topology of YobD presents several methodological challenges:

Additionally, computational prediction tools (TMHMM, MEMSAT, TopPred) should be used as a starting point to guide experimental design. The predicted topology should then be validated through at least two independent experimental approaches to establish confidence in the structural model .

How can researchers address toxic effects during recombinant YobD expression?

Membrane proteins like YobD can exhibit toxicity during expression, manifesting as slower growth rates, low final cell density, or cell death. Methodological approaches to overcome toxicity include:

• Expression Strain Selection: Use C41(DE3) and C43(DE3) strains specifically selected to withstand toxic protein expression. These strains contain mutations in the lacUV5 promoter that reduce T7 RNA polymerase expression levels .

• Promoter Leakiness Control:

  • Add 1% glucose to the medium to suppress basal expression through catabolite repression

  • Use pLysS or pLysE strains that express T7 lysozyme to inhibit basal transcription

  • Consider tight control expression systems like araBAD promoter with glucose repression

• Induction Parameters:

  • Induce at higher cell densities (OD600 > 0.8)

  • Use lower inducer concentrations

  • Reduce post-induction incubation temperature to 15-25°C

• Media Optimization:

  • Use richer media (TB or 2YT) to support cellular stress responses

  • Supplement with glycerol or additional amino acids

These strategies can significantly mitigate toxicity issues and improve viable cell density and protein yield .

What analytical approaches can help diagnose expression problems with recombinant YobD?

When expression yields of YobD are low or undetectable, a systematic diagnostic approach is essential:

• Verification of Construct Integrity:

  • Confirm plasmid sequence

  • Verify presence of intact open reading frame

  • Check for potential recombination events

• Expression Analysis:

  • Small-scale expression trials with samples collected at multiple time points

  • Western blot analysis using anti-His antibodies for sensitive detection

  • qRT-PCR to assess mRNA levels of the target gene

• Fractionation Analysis:

  • Separate soluble, membrane, and inclusion body fractions

  • Analyze each fraction for presence of target protein

  • Optimize lysis conditions to ensure complete membrane protein extraction

• Stability Assessment:

  • Pulse-chase experiments to monitor protein degradation

  • Co-expression with protease inhibitors

This diagnostic workflow helps pinpoint whether issues stem from transcription, translation, protein folding, membrane insertion, or degradation, allowing targeted optimization strategies .

How should researchers design pilot expression studies for YobD protein to maximize success in scale-up?

A systematic approach to pilot expression studies for YobD should include:

• Micro-Expression Screening:

  • Test multiple strains: BL21(DE3), C41(DE3), C43(DE3), and K-12 derivatives

  • Evaluate different vectors with varying promoters and fusion tags

  • Screen induction conditions (temperature, inducer concentration, time)

• Analytical Workflow:

  • SDS-PAGE and Western blot analysis of whole cell lysates

  • Fractionation to determine subcellular localization

  • Detergent screening for optimal solubilization

• Optimization Table:

• Scale-up Considerations:

  • Evaluate oxygen requirements and mixing parameters

  • Determine heat generation and cooling needs

  • Assess potential for high-density cultivation

This systematic approach in 2-ml tubes or 96-well plate format allows rapid screening of up to 1000 conditions within a week, significantly increasing the probability of successful scale-up .

How can structural studies of YobD contribute to understanding membrane protein biogenesis?

YobD represents an excellent model system for studying membrane protein insertion and folding mechanisms. Research approaches include:

• In vitro Translation Studies:

  • Reconstituted systems with purified Sec translocon components

  • Analysis of co-translational vs. post-translational insertion pathways

  • Kinetic studies of membrane insertion using fluorescence techniques

• Crosslinking Approaches:

  • Incorporation of photo-activatable amino acids at specific positions

  • Identification of transient interactions during membrane insertion

  • Mapping of chaperone binding sites

• Comparative Analysis:

  • Expression of YobD in different cellular compartments

  • Cross-species expression to identify conserved insertion mechanisms

  • Chimeric constructs with other membrane proteins

These studies can provide fundamental insights into membrane protein biogenesis, which remains one of the challenging areas in structural biology research .

What are the implications of studying YobD for antimicrobial development?

As a bacterial membrane protein, YobD research has potential implications for antimicrobial development through:

• Target Validation:

  • Determine essentiality of YobD for bacterial viability

  • Assess conservation across pathogenic strains

  • Evaluate role in bacterial stress responses or virulence

• Structural Studies for Drug Design:

  • High-resolution structure determination

  • Identification of potential binding pockets

  • Fragment-based screening for inhibitor development

• Functional Characterization:

  • Metabolomic studies to identify affected pathways

  • Assays for potential transporter or signaling functions

  • Investigation of interactions with existing antibiotics

While limited information exists on YobD's specific function, its study contributes to the broader understanding of bacterial membrane biology, potentially revealing new targets for antimicrobial intervention .

How can advanced biophysical techniques enhance our understanding of YobD structure and dynamics?

Advanced biophysical techniques offer powerful approaches to characterize the structure and dynamics of challenging membrane proteins like YobD:

Integration of these complementary approaches can provide unprecedented insights into membrane protein structure-function relationships that are difficult to obtain through any single technique .