Recombinant Yersinia pseudotuberculosis serotype IB UPF0266 membrane protein YPTS_1754 (YPTS_1754)

What is YPTS_1754 and what is its fundamental structure?

YPTS_1754 is a membrane protein from Yersinia pseudotuberculosis serotype IB with a full protein length of 153 amino acids. It belongs to the UPF0266 protein family, a group of uncharacterized proteins with predicted membrane localization. The recombinant version is typically expressed with a histidine tag to facilitate purification and downstream applications . The protein's membrane localization suggests it likely contains hydrophobic domains that span the bacterial cell membrane, though detailed structural studies are still needed to confirm its topology and three-dimensional conformation.

What expression systems are most effective for producing recombinant YPTS_1754?

Based on available data, E. coli expression systems have been successfully used to produce recombinant YPTS_1754 with histidine tags . When working with membrane proteins like YPTS_1754, researchers should consider several factors that influence successful expression:

• Selection of an appropriate E. coli strain optimized for membrane protein expression

• Codon optimization of the construct to avoid rare codon usage issues

• Temperature modulation during induction phase (typically lower temperatures of 16-25°C)

• Inclusion of specific detergents during cell lysis and purification steps

For challenging membrane proteins, alternative expression systems like yeast (P. pastoris) or insect cells may be considered if E. coli expression yields are insufficient .

How conserved is YPTS_1754 across Yersinia species?

While the search results don't provide specific information about the conservation of YPTS_1754 across Yersinia species, genome sequence analyses of different Yersinia strains have revealed considerable heterogeneity. Unlike the evolutionary young Y. pestis lineage that shows limited genetic diversity, Y. pseudotuberculosis demonstrates greater heterogeneity in its genome . Researchers interested in YPTS_1754 conservation should conduct comparative genomic analyses across different Yersinia strains and related enterobacteria to determine the uniqueness of this protein and its potential role in serotype-specific functions.

What bioinformatic resources are most useful for analyzing YPTS_1754?

For comprehensive analysis of YPTS_1754, researchers should utilize multiple bioinformatic tools:

• Protein structure prediction tools such as AlphaFold2 for three-dimensional structure modeling

• Transmembrane topology prediction servers (TMHMM, Phobius, TOPCONS) to identify membrane-spanning regions

• Protein family databases (Pfam, InterPro) to identify conserved domains

• Comparative genomics platforms to assess conservation across bacterial species

• Protein-protein interaction prediction tools to hypothesize potential binding partners

These approaches can generate testable hypotheses about YPTS_1754 function even before experimental validation begins .

What role might YPTS_1754 play in Yersinia pseudotuberculosis pathogenicity?

While the specific function of YPTS_1754 in pathogenicity has not been explicitly described in the search results, its nature as a membrane protein in Y. pseudotuberculosis suggests several potential roles:

• Cell surface interactions with host tissues

• Potential involvement in secretion systems

• Possible role in nutrient acquisition during infection

• Contribution to bacterial survival under host-specific stress conditions

Y. pseudotuberculosis contains multiple virulence factors, including the type IVB secretion system found on plasmids that may contribute to scarlatinoid fever symptoms through immunomodulatory effects . Further research is needed to determine if YPTS_1754 interacts with any known virulence mechanisms or pathogenicity islands in Yersinia.

How does the genomic context of YPTS_1754 inform its potential function?

The genomic context analysis of YPTS_1754—examining neighboring genes and potential operons—could provide crucial insights into its function. Y. pseudotuberculosis contains specialized genomic regions like the Yersinia adhesion pathogenicity island (YAPI) that houses virulence-associated genes . Determining whether YPTS_1754 is located within or near such pathogenicity islands, or if it's part of a conserved operon with functionally characterized genes, would provide valuable functional clues. Researchers should perform detailed genomic context analysis to identify potential functional associations.

What experimental approaches are most suitable for elucidating YPTS_1754 function?

A multi-pronged experimental strategy is recommended for investigating YPTS_1754 function:

• Genetic manipulation studies:

  • Construction of knockout mutants in Y. pseudotuberculosis

  • Complementation studies with wild-type and mutated versions

  • Conditional expression systems to study essentiality

• Protein interaction studies:

  • Pull-down assays using tagged recombinant YPTS_1754

  • Bacterial two-hybrid screens

  • Cross-linking followed by mass spectrometry

• Localization studies:

  • Immunofluorescence microscopy with anti-YPTS_1754 antibodies

  • Fractionation of bacterial membranes

  • GFP fusion proteins to track localization during infection

• Functional assays:

  • Assessing virulence of wild-type vs. YPTS_1754 mutants in cellular and animal models

  • Testing sensitivity to environmental stressors

  • Evaluating adherence to host cells with and without YPTS_1754

What challenges exist in structural characterization of YPTS_1754?

As a membrane protein, YPTS_1754 presents several challenges for structural studies:

• Expression and purification obstacles:

  • Potential toxicity when overexpressed

  • Proper folding in heterologous systems

  • Maintaining stability during detergent extraction

• Crystallization difficulties:

  • Finding appropriate detergents that maintain native conformation

  • Limited hydrophilic surfaces for crystal contacts

  • Potential conformational heterogeneity

• NMR spectroscopy limitations:

  • Size constraints for solution NMR

  • Detergent micelle effects on spectral quality

  • Isotopic labeling challenges

Researchers might consider newer approaches like cryo-electron microscopy, which has revolutionized membrane protein structural biology in recent years .

What purification strategies are optimal for obtaining functional YPTS_1754?

For membrane proteins like YPTS_1754, purification requires specialized approaches:

To distinguish full-length YPTS_1754 from truncated products, researchers should consider using dual-tagged constructs (e.g., N-terminal His-tag and C-terminal FLAG-tag) and increasing imidazole concentration during elution to ensure specificity .

How can protein-protein interactions of YPTS_1754 be reliably studied?

To identify and validate protein-protein interactions involving YPTS_1754, researchers should employ multiple complementary techniques:

• Co-immunoprecipitation studies:

  • Using anti-His antibodies to pull down YPTS_1754 and associated proteins

  • Followed by mass spectrometry identification of binding partners

  • Confirmation with reverse co-IP experiments similar to methods described for dynamin-NME interactions

• Crosslinking approaches:

  • Chemical crosslinkers with varying spacer lengths

  • Photo-activatable crosslinkers for capturing transient interactions

  • Mass spectrometry analysis of crosslinked peptides

• Bacterial two-hybrid screening:

  • Library screening to identify potential interactors

  • Validation with deletion constructs to map interaction domains

  • Controls to exclude false positives

• Surface plasmon resonance:

  • Kinetic analysis of interactions with predicted partners

  • Competition assays to determine binding sites

  • Effect of environmental conditions on binding strength

What are the best approaches for studying YPTS_1754 in the context of host-pathogen interactions?

To investigate YPTS_1754's role in host-pathogen interactions, researchers should consider:

• Infection models:

  • Comparing wild-type and YPTS_1754 mutant strains in cellular infection models

  • Quantifying bacterial adherence, invasion, and intracellular survival

  • Measuring host inflammatory responses using cytokine assays

• Transcriptomic approaches:

  • RNA-seq of host cells infected with wild-type vs. YPTS_1754 mutants

  • Bacterial transcriptomics to identify co-regulated genes

  • Dual RNA-seq to capture both host and pathogen responses simultaneously

• Microscopy techniques:

  • Immunofluorescence to track YPTS_1754 localization during infection

  • Live-cell imaging with fluorescently tagged YPTS_1754

  • Super-resolution microscopy to visualize membrane distribution

• Functional assays:

  • Evaluating the impact of YPTS_1754 on Yersinia's ability to suppress host immune responses

  • Testing for potential roles in type III or type IVB secretion systems, which are important virulence mechanisms in Yersinia

  • Examining possible involvement in bacterial stress responses during host infection

How should researchers approach YPTS_1754 functional annotation?

Given the limited information available about YPTS_1754 function, researchers should implement a systematic approach to functional annotation:

• Comprehensive bioinformatic analysis:

  • Detailed sequence analysis to identify conserved motifs

  • Structural prediction with current tools like AlphaFold2

  • Comparative genomics with well-characterized bacterial species

• High-throughput phenotypic screening:

  • Testing YPTS_1754 mutants against diverse environmental conditions

  • Antibiotic susceptibility profiling

  • Nutrient utilization screening

• Interaction network mapping:

  • Identification of protein-protein interactions

  • Genetic interaction mapping through synthetic lethality screens

  • Integration with existing bacterial interactome data

• Targeted functional hypotheses testing:

  • Based on bioinformatic predictions and preliminary data

  • Site-directed mutagenesis of predicted functional residues

  • Complementation with homologs from related species

What are the common pitfalls in recombinant YPTS_1754 expression and purification?

Several challenges might arise when working with recombinant YPTS_1754:

When expressing full-length proteins like YPTS_1754, researchers should be vigilant about truncated products that may result from proteolysis or improper translation initiation. Using dual-tagged constructs and optimizing elution conditions can help ensure the isolation of full-length protein .

How can researchers validate that recombinant YPTS_1754 maintains native conformation?

Validating the native conformation of recombinant membrane proteins like YPTS_1754 requires multiple approaches:

• Circular dichroism spectroscopy:

  • To assess secondary structure content

  • Compare with predicted structural elements

• Tryptophan fluorescence spectroscopy:

  • To evaluate tertiary structure integrity

  • Monitor changes under different conditions

• Limited proteolysis:

  • Compare digestion patterns between recombinant and native protein

  • Identify accessible versus protected regions

• Functional assays:

  • Develop activity assays based on predicted function

  • Compare with native protein when possible

• Antibody recognition:

  • Generate conformation-specific antibodies

  • Use for validation of proper folding

How might advanced structural biology methods enhance our understanding of YPTS_1754?

As structural biology techniques continue to advance, several approaches could provide critical insights into YPTS_1754:

• Cryo-electron microscopy:

  • Single-particle analysis for high-resolution structure determination

  • Visualization of YPTS_1754 in membrane environments

  • Potential for capturing different conformational states

• Integrative structural biology:

  • Combining data from multiple experimental sources

  • Cross-validation between computational predictions and experimental data

  • Modeling of protein dynamics in membrane environments

• In-cell structural studies:

  • Emerging techniques for studying protein structure in cellular context

  • Potential to reveal native interactions and conformations

With AI-based protein structure prediction tools like AlphaFold2 showing remarkable accuracy, researchers can now generate high-confidence structural models of proteins like YPTS_1754 that can guide experimental design and functional hypotheses .

What is the potential of YPTS_1754 as a therapeutic target?

While commercial applications were not to be emphasized, from a research perspective, understanding the potential of YPTS_1754 as a therapeutic target is academically relevant:

• Target validation studies:

  • Determining essentiality of YPTS_1754 for bacterial viability

  • Assessing contribution to virulence in infection models

  • Evaluating conservation across clinically relevant strains

• Structure-based drug design approaches:

  • Using structural information to identify potential binding pockets

  • Virtual screening of compound libraries

  • Fragment-based drug discovery

• Epitope mapping for vaccine development:

  • Identifying surface-exposed regions of YPTS_1754

  • Evaluating immunogenicity of these regions

  • Assessing protective potential in animal models

The involvement of Y. pseudotuberculosis in scarlatinoid fever through immunomodulatory capabilities underscores the importance of studying its membrane proteins as potential therapeutic targets.