Recombinant Yersinia pseudotuberculosis serotype O:1b UPF0208 membrane protein YpsIP31758_1444 (YpsIP31758_1444)

What is the structure and basic characteristics of YpsIP31758_1444 protein?

YpsIP31758_1444 is a full-length membrane protein (151 amino acids) from Yersinia pseudotuberculosis serotype O:1b. The protein's amino acid sequence is:

MTIKPSDSVSWFQVLQRGQHYMKTWPADKRLAPVFPENRVTVVTRFGIRFMPPLAIFTLTWQIALGGQLGPAIATALFACGLPLQGLWWLGKRAITPLPPTLLQWFHEVRHKLSEAGQAVAPIEPIPTYQSLADLLKRAFKQLDKTFLDDL

This protein belongs to the UPF0208 membrane protein family, contains hydrophobic regions consistent with transmembrane domains, and has a UniProt ID of A7FGP5 . The recombinant version is typically produced with an N-terminal His-tag to facilitate purification, and when expressed in E. coli systems, yields proteins with greater than 90% purity as determined by SDS-PAGE analysis .

What cellular mechanisms might YpsIP31758_1444 participate in during Yersinia pathogenesis?

While the specific function of YpsIP31758_1444 has not been fully characterized in the provided literature, its classification as a membrane protein suggests potential roles in maintaining membrane integrity, transport functions, or cell signaling pathways. Yersinia pseudotuberculosis, as a gram-negative enteric pathogen, employs various membrane proteins that contribute to virulence mechanisms .

The protein may function within the context of Yersinia's broader pathogenic strategies, potentially interacting with the type III secretion system (T3SS) that delivers effector Yop proteins directly into host cells to modulate anti-bacterial responses . Research methodologies to elucidate its function could include knockout studies comparing wild-type and YpsIP31758_1444-deficient strains, protein-protein interaction studies, or localization experiments during infection processes.

How does the recombinant protein expression system affect YpsIP31758_1444 folding and functionality?

The recombinant YpsIP31758_1444 protein is typically expressed in E. coli expression systems with an N-terminal His-tag . For membrane proteins, expression system selection is critical for proper folding and function. E. coli-based expression may present challenges for maintaining the native conformation of membrane proteins due to differences in membrane composition between E. coli and Yersinia.

To assess proper folding, researchers should implement circular dichroism (CD) spectroscopy to analyze secondary structure content, limited proteolysis to evaluate structural integrity, and fluorescence spectroscopy to examine tertiary structure. Additionally, functionality assessments might include reconstitution into liposomes and measuring specific activities if transport or signaling functions are suspected.

Alternative expression systems worth considering include cell-free systems supplemented with lipids or eukaryotic expression systems for studies requiring post-translational modifications. Comparative expression profiles between different systems may yield insights into optimal conditions for obtaining functionally relevant protein conformations.

What are the optimal conditions for reconstitution and storage of recombinant YpsIP31758_1444?

For optimal reconstitution of lyophilized YpsIP31758_1444 protein:

• Centrifuge the vial briefly before opening to ensure all material is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage stability

• Aliquot to minimize freeze-thaw cycles

Storage recommendations include:

• Short-term (≤1 week): Store working aliquots at 4°C

• Long-term: Store at -20°C/-80°C in Tris/PBS-based buffer containing 6% trehalose at pH 8.0

For membrane proteins like YpsIP31758_1444, additional stability may be achieved by incorporating mild detergents (e.g., n-dodecyl β-D-maltoside or CHAPS) at concentrations just above critical micelle concentration. Researchers should validate protein stability through time-course activity assays or structural analysis to determine optimal storage conditions for their specific experimental needs.

What expression and purification strategies yield optimal functional YpsIP31758_1444?

For optimal expression of recombinant YpsIP31758_1444:

• Expression system: E. coli is the documented system of choice , with BL21(DE3) strains typically preferred for membrane proteins

• Induction conditions: Lower temperatures (16-25°C) often improve membrane protein folding

• Media supplements: Addition of glycerol (0.5-2%) and specific membrane-enhancing compounds (e.g., betaine)

The purification workflow typically includes:

For functional studies, consider reconstitution into nanodiscs or liposomes to maintain the native membrane environment. Validate purification success through SDS-PAGE analysis, where recombinant YpsIP31758_1444 should demonstrate >90% purity .

How can researchers accurately assess the oligomeric state of YpsIP31758_1444 in solution?

Determining the oligomeric state of membrane proteins like YpsIP31758_1444 requires multiple complementary approaches:

• Size Exclusion Chromatography (SEC): Calibrate with membrane protein standards of known molecular weight rather than soluble proteins

  • Account for detergent micelle contribution to apparent molecular weight

  • Use multi-angle light scattering (SEC-MALS) for more accurate mass determination

• Analytical Ultracentrifugation (AUC):

  • Sedimentation velocity experiments detect different oligomeric species

  • Sedimentation equilibrium determines absolute molecular weight

• Chemical Crosslinking:

  • Use membrane-permeable crosslinkers (DSS, glutaraldehyde)

  • Analyze crosslinked products via SDS-PAGE and western blotting

• Native-PAGE:

  • Blue native PAGE preserves protein-protein interactions

  • Compare migration patterns with known standards

• Electron Microscopy:

  • Negative staining for initial structure assessment

  • Cryo-EM for higher resolution details of oligomeric assemblies

When interpreting results, remember that detergent choice significantly impacts oligomerization behavior. Cross-validate findings using multiple methods and detergent conditions to distinguish between physiologically relevant oligomers and artifacts of the experimental system.

How might YpsIP31758_1444 interact with the Yersinia type III secretion system (T3SS) and effector proteins?

The potential interaction between YpsIP31758_1444 and the Yersinia T3SS represents an intriguing research avenue. Yersinia pseudotuberculosis employs a sophisticated T3SS to deliver Yop effectors directly into host cells, modulating anti-bacterial responses . As a membrane protein, YpsIP31758_1444 could participate in this process through several mechanisms:

• Structural support: Providing membrane stabilization for the T3SS apparatus

• Regulatory function: Influencing the expression or assembly of T3SS components

• Chaperone activity: Potentially assisting in the proper folding or delivery of Yop effectors

To investigate these possibilities, researchers should consider:

• Co-immunoprecipitation studies with tagged YpsIP31758_1444 to identify interaction partners

• Bacterial two-hybrid screening to detect protein-protein interactions

• Fluorescence microscopy to visualize co-localization with T3SS components

• Quantitative proteomic analysis comparing wild-type and YpsIP31758_1444 knockout strains

• Transcriptomic profiling to detect changes in T3SS gene expression in the absence of YpsIP31758_1444

Given that YopH and YopE have been identified as critical for Yersinia colonization and persistence in intestinal and lymph tissues , investigating whether YpsIP31758_1444 influences their expression or delivery would be particularly valuable.

What structural determinants of YpsIP31758_1444 contribute to its membrane localization and function?

Understanding the structural determinants of YpsIP31758_1444 requires detailed analysis of its amino acid sequence and predicted structural elements:

The 151-amino acid sequence (MTIKPSDSVSWFQVLQRGQHYMKTWPADKRLAPVFPENRVTVVTRFGIRFMPPLAIFTLTWQIALGGQLGPAIATALFACGLPLQGLWWLGKRAITPLPPTLLQWFHEVRHKLSEAGQAVAPIEPIPTYQSLADLLKRAFKQLDKTFLDDL) contains several hydrophobic regions that likely form transmembrane domains.

To investigate structural determinants:

• Computational Analysis:

  • Hydropathy plot analysis to identify transmembrane regions

  • Secondary structure prediction (α-helices, β-sheets)

  • Homology modeling based on structurally characterized UPF0208 family members

• Experimental Approaches:

  • Site-directed mutagenesis of conserved residues

  • Truncation studies to identify minimal functional domains

  • Cysteine accessibility methods to map topology

  • Limited proteolysis combined with mass spectrometry to identify stable domains

• Advanced Structural Techniques:

  • X-ray crystallography of purified protein in detergent micelles or lipidic cubic phase

  • Cryo-EM analysis of the protein in nanodiscs

  • Solid-state NMR of reconstituted protein in lipid bilayers

Creating a library of domain-swapped chimeric proteins between YpsIP31758_1444 and related proteins could help identify regions responsible for specific functions or localization patterns.

How can researchers develop cell-penetrating variants of YpsIP31758_1444 for potential therapeutic applications?

Recent discoveries of bacterial cell-penetrating effector proteins (CPEs) suggest potential for developing therapeutic variants of bacterial proteins . To develop cell-penetrating variants of YpsIP31758_1444:

• Identify Cell-Penetrating Domains:

  • Analyze the sequence for positively charged regions similar to known CPPs

  • Compare with YopM, the prototype bacterial CPE

  • Screen for membrane-interactive peptide segments

• Engineering Strategies:

  • Fusion with established CPPs (TAT, penetratin, polyarginine)

  • Domain swapping with known cell-penetrating bacterial proteins

  • Directed evolution approaches selecting for membrane penetration

• Functional Modifications:

  • Attenuate virulence-associated activities

  • Enhance immunomodulatory properties

  • Incorporate cargo-carrying capabilities

• Validation Methods:

  • Fluorescence microscopy with labeled protein to track cellular uptake

  • Flow cytometry quantification of internalization

  • Subcellular fractionation to confirm cytoplasmic delivery

  • Functional assays to verify intracellular activity

The development of such variants aligns with the emerging field of bacteria-derived biologics that harness the natural immunomodulatory capabilities of bacterial proteins while minimizing pathogenic effects . These engineered proteins could potentially serve as self-delivering therapeutics for inflammatory conditions.

How can researchers address protein aggregation issues when working with recombinant YpsIP31758_1444?

Membrane proteins like YpsIP31758_1444 are prone to aggregation during expression, purification, and storage. Implement these strategies to minimize aggregation:

Validation of monodispersity can be performed using dynamic light scattering (DLS) or analytical ultracentrifugation. For significantly aggregation-prone preparations, consider reconstitution into nanodiscs or amphipols, which provide a more stable membrane-mimetic environment than detergent micelles.

What are effective strategies for improving yield and purity of recombinant YpsIP31758_1444?

Optimizing the yield and purity of YpsIP31758_1444 requires attention to multiple experimental parameters:

• Expression Optimization:

  • Test multiple E. coli strains (BL21, C41/C43, Rosetta)

  • Evaluate different promoter systems (T7, tac, ara)

  • Compare rich (LB, TB) vs. minimal media with supplements

  • Optimize cell density at induction (typically OD600 0.6-0.8)

• Extraction Efficiency:

  • Screen detergent panel (ionic, non-ionic, zwitterionic)

  • Optimize detergent:protein ratio

  • Test extraction time and temperature

  • Consider sequential extraction methods

• Purification Refinement:

  • Implement two-step IMAC (low imidazole wash followed by gradient)

  • Add secondary purification step (ion exchange, size exclusion)

  • Include scavengers for contaminants (expanded bed adsorption)

• Quality Control Metrics:

  • SDS-PAGE with silver staining (sensitivity to minor contaminants)

  • Western blot for verification

  • Mass spectrometry for identity confirmation

  • Thermal shift assays for stability assessment

• Scale-up Considerations:

  • Maintain consistent parameters when scaling

  • Consider fed-batch strategies for higher cell densities

  • Implement automated purification systems for reproducibility

Expected yields from optimized processes should reach 2-5 mg of pure protein per liter of bacterial culture, with purity exceeding 95% as assessed by densitometric analysis of SDS-PAGE gels.

What analytical methods can validate the structural integrity of purified YpsIP31758_1444?

Validating the structural integrity of purified YpsIP31758_1444 is essential before functional studies. A comprehensive validation approach includes:

• Spectroscopic Methods:

  • Circular Dichroism (CD): Confirms secondary structure content and stability

  • Fluorescence Spectroscopy: Assesses tertiary structure through intrinsic tryptophan fluorescence

  • Fourier Transform Infrared Spectroscopy (FTIR): Provides information on secondary structure in membrane environments

• Hydrodynamic Techniques:

  • Size Exclusion Chromatography: Evaluates homogeneity and detects aggregation

  • Dynamic Light Scattering: Measures particle size distribution

  • Analytical Ultracentrifugation: Determines molecular weight and oligomeric state

• Thermal and Chemical Stability:

  • Differential Scanning Calorimetry: Measures thermal transitions

  • Thermal Shift Assays: Monitors unfolding using fluorescent dyes

  • Chemical Denaturation: Assesses stability against chaotropic agents

• Structural Probing:

  • Limited Proteolysis: Identifies flexible vs. structured regions

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Maps solvent accessibility

  • Crosslinking Mass Spectrometry: Identifies spatial relationships between domains

• Functional Verification:

  • Liposome Binding Assays: Confirms membrane association capability

  • Protein-Protein Interaction Studies: Verifies ability to engage with binding partners

  • Activity Assays: If enzymatic function is known

For membrane proteins like YpsIP31758_1444, additional consideration must be given to the detergent environment, as different detergents can significantly alter structural properties. Cross-validation using multiple techniques provides the most reliable assessment of structural integrity.

How might comparative genomics enhance our understanding of YpsIP31758_1444 evolutionary conservation and function?

Comparative genomics approaches offer powerful insights into YpsIP31758_1444's evolutionary significance and functional predictions:

• Phylogenetic Analysis:

  • Construct phylogenetic trees using UPF0208 family proteins across bacterial species

  • Identify conserved vs. variable regions within the protein sequence

  • Map evolutionary relationships between Yersinia species and other enterobacteriaceae

• Synteny Analysis:

  • Examine gene neighborhood conservation across Yersinia strains

  • Identify co-evolved gene clusters that may function together

  • Compare genomic context in pathogenic vs. non-pathogenic strains

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify regions under positive or purifying selection

  • Correlate selection patterns with predicted functional domains

  • Compare selection signatures between strains with different host specificities

• Structure-Based Phylogeny:

  • Integrate predicted structural information with sequence divergence

  • Identify structurally conserved regions despite sequence variation

  • Predict functional sites based on evolutionary conservation patterns

• Horizontal Gene Transfer Assessment:

  • Evaluate evidence for horizontal acquisition of YpsIP31758_1444

  • Compare GC content and codon usage with genomic averages

  • Investigate potential mobile genetic element associations

This approach could reveal whether YpsIP31758_1444 represents a core component of Yersinia biology or a specialized adaptation in certain lineages, potentially correlating with virulence potential or host range. The findings would guide more targeted functional studies and potentially identify critical regions for therapeutic intervention.

What potential applications might arise from engineering YpsIP31758_1444 as a bacteria-derived biologic?

The emerging field of bacteria-derived biologics offers intriguing possibilities for engineered YpsIP31758_1444 applications:

• Therapeutic Delivery Systems:

  • Engineer cell-penetrating variants similar to YopM

  • Develop fusion proteins that deliver therapeutic cargo to specific cell types

  • Create attenuated immunomodulatory derivatives for anti-inflammatory applications

• Diagnostic Applications:

  • Design biosensors using YpsIP31758_1444 membrane-integration capabilities

  • Develop detection systems for Yersinia-specific antibodies in clinical samples

  • Create imaging probes for visualizing bacterial infections in vivo

• Biotechnological Applications:

  • Utilize as a membrane protein expression and display platform

  • Engineer synthetic membrane systems with controlled permeability

  • Develop protein-based nanomaterials with self-assembling properties

• Research Tools:

  • Create tagged variants for tracking bacterial localization during infection

  • Develop protein-based probes of membrane dynamics

  • Engineer reporter systems for studying bacterial-host interactions

Recent work with recombinant Yersinia pseudotuberculosis as delivery systems demonstrates the feasibility of such approaches. For example, studies have created a recombinant Yptb strain (PB1+) designed to synthesize an adjuvant form of lipid A , suggesting bacterial components can be engineered for beneficial applications rather than virulence.

The development of these applications would require detailed understanding of YpsIP31758_1444 structure-function relationships and extensive safety testing to ensure therapeutic variants lack pathogenic potential.