Recombinant Yersinia pestis bv. Antiqua UPF0266 membrane protein YpAngola_A1654 (YpAngola_A1654)

What is YpAngola_A1654 and what organism does it originate from?

YpAngola_A1654 is a UPF0266 membrane protein found in Yersinia pestis biovar Antiqua, specifically strain Angola. This protein belongs to the UPF0266 family of membrane proteins with currently uncharacterized function (UPF stands for Uncharacterized Protein Family). The protein is encoded in the genome of Y. pestis bv. Antiqua, which is one of the historically significant strains of the plague-causing bacterium .

Yersinia pestis is the causative agent of plague and is known to have caused three major pandemics throughout human history. The Antiqua biovar represents one of the ancestral lineages of this pathogen, which emerged through the evolutionary divergence from Y. pseudotuberculosis at least 2,000 years ago .

What are the available forms of recombinant YpAngola_A1654 for research?

The recombinant forms of YpAngola_A1654 available for research are typically expressed in E. coli expression systems with affinity tags to facilitate purification. The most common form available is:

The His-tagged version is particularly useful for purification using nickel affinity chromatography and for detection using anti-His antibodies in various experimental applications .

How should I optimize the expression and purification of recombinant YpAngola_A1654?

Optimizing the expression and purification of YpAngola_A1654 requires careful consideration of its membrane protein nature. Since membrane proteins are often challenging to express in soluble form, consider these methodological approaches:

• Expression System Selection: While E. coli is commonly used, consider specialized strains designed for membrane protein expression such as C41(DE3) or C43(DE3). These strains are better at accommodating the potential toxicity of membrane protein overexpression .

• Solubilization Strategies: After expression, membrane proteins require proper solubilization. Use mild detergents such as n-dodecyl-β-D-maltoside (DDM) or CHAPS for initial solubilization, and test different detergent concentrations to optimize protein extraction without denaturation.

• Purification Optimization: For His-tagged YpAngola_A1654, use immobilized metal affinity chromatography (IMAC) with increasing imidazole concentrations to distinguish between full-length protein and truncated forms. A stepwise elution can help separate the full-length protein from contaminants .

• Quality Control: Verify the integrity of the purified protein using SDS-PAGE, Western blotting, and mass spectrometry to confirm the full sequence and proper folding.

Remember that transmembrane proteins present unique challenges, and maintaining their native conformation requires careful attention to buffer conditions throughout the purification process .

What are the optimal storage conditions for maintaining YpAngola_A1654 stability and activity?

Based on the available information, the optimal storage conditions for recombinant YpAngola_A1654 are:

• Buffer Composition: The protein should be stored in a Tris-based buffer containing 50% glycerol, optimized specifically for this protein.

• Storage Temperature: Store at -20°C for regular use, or at -80°C for extended storage to maintain stability.

• Handling Recommendations: Avoid repeated freeze-thaw cycles as these can lead to protein degradation and loss of function. Instead, prepare working aliquots and store them at 4°C for up to one week.

• Stability Considerations: As a membrane protein, YpAngola_A1654 may have specific stability requirements. Monitor the protein's integrity over time using analytical techniques like size-exclusion chromatography or dynamic light scattering .

What is the known or predicted function of YpAngola_A1654 in Yersinia pestis?

• Membrane Integration: The amino acid sequence suggests multiple transmembrane domains, indicating that it functions within the bacterial membrane architecture .

• Potential Roles: As a membrane protein in a pathogenic bacterium, it may be involved in:

  • Cell membrane integrity maintenance

  • Transport of molecules across the membrane

  • Sensing environmental conditions

  • Potentially contributing to virulence mechanisms

• Evolutionary Context: In the context of Yersinia evolution, membrane proteins can play crucial roles in adaptation to different environments. The Yersinia genus has evolved through ecological specialization and gene gain-and-loss events that led to different pathogenic capabilities .

Further functional characterization would require targeted studies including gene knockout experiments, localization studies, and interaction analyses to determine its precise biological role.

How can I design experiments to characterize potential interactions between YpAngola_A1654 and host proteins?

Designing experiments to characterize potential host-pathogen protein interactions involving YpAngola_A1654 requires a multi-faceted approach:

• Yeast Two-Hybrid Screening:

  • Modified membrane yeast two-hybrid (MYTH) systems are particularly suitable for membrane proteins like YpAngola_A1654

  • Use the bacterial protein as bait against a human cDNA library to identify potential host interacting partners

  • Validate positive interactions with secondary assays

• Co-Immunoprecipitation (Co-IP):

  • Express tagged versions of YpAngola_A1654 in a mammalian cell line

  • Use anti-tag antibodies to pull down the protein complex

  • Identify co-precipitated host proteins through mass spectrometry

  • Confirm specific interactions with Western blotting

• Proximity Labeling Methods:

  • Fuse YpAngola_A1654 with enzymes like BioID or APEX2

  • These enzymes will biotinylate nearby proteins when activated

  • Purify biotinylated proteins and identify them by mass spectrometry

  • This approach is particularly valuable for transient or weak interactions

• Infection Models with Mutant Bacteria:

  • Create Y. pestis strains with modified YpAngola_A1654 (knockout or tagged versions)

  • Infect host cells and analyze differences in host-pathogen interactions

  • Use immunofluorescence microscopy to visualize localization and potential co-localization with host factors

What role might YpAngola_A1654 play in pathogenicity and host immune response?

While specific information about YpAngola_A1654's role in pathogenicity is limited, we can draw insights from our understanding of Yersinia infection mechanisms:

• Potential Immunogenicity:

  • Bacterial membrane proteins can serve as pathogen-associated molecular patterns (PAMPs) recognized by the host immune system

  • YpAngola_A1654 might generate epitopes recognized by the host adaptive immune system, similar to how other Yersinia proteins are presented by MHC molecules

• Comparison with Known Virulence Mechanisms:

  • Yersinia species are known to introduce virulence proteins like Yop51 into host cells

  • While YpAngola_A1654 is not specifically identified as a virulence factor, its membrane localization positions it at the interface between pathogen and host

  • It could potentially be involved in host cell adhesion, invasion, or modulation of host responses

• Experimental Approach to Study Immune Recognition:

  • Express recombinant YpAngola_A1654 and assess its ability to stimulate immune cells

  • Develop an in vitro system to test whether YpAngola_A1654 epitopes are presented by MHC class I molecules, similar to experiments done with Yop51

  • Use proliferation assays with T-cells to determine if YpAngola_A1654 stimulates a cellular immune response

How can I use structural biology approaches to determine the three-dimensional structure of YpAngola_A1654?

Determining the structure of membrane proteins like YpAngola_A1654 presents unique challenges that require specialized approaches:

• X-ray Crystallography:

  • Purify large quantities of homogeneous protein in detergent micelles or lipidic cubic phases

  • Screen multiple crystallization conditions optimized for membrane proteins

  • Consider using antibody fragments (Fab or nanobodies) to stabilize the protein and facilitate crystal contacts

  • Analyze diffraction data to resolve the three-dimensional structure

• Cryo-Electron Microscopy (Cryo-EM):

  • Particularly valuable for membrane proteins that resist crystallization

  • Prepare YpAngola_A1654 in nanodiscs or amphipols to mimic the membrane environment

  • Use direct electron detectors and advanced image processing to achieve high-resolution structures

  • Visualize the protein in different conformational states that may be relevant to its function

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Suitable for smaller membrane proteins or domains (YpAngola_A1654 at 153 amino acids may be amenable)

  • Requires isotopic labeling (15N, 13C) of the recombinant protein

  • Provides dynamic information about protein movements in addition to structure

  • Can detect protein-ligand interactions in solution

• Computational Prediction and Validation:

  • Use AlphaFold2 or similar AI-based tools to predict the structure

  • Validate predictions with experimental data from limited proteolysis, cross-linking mass spectrometry, or hydrogen-deuterium exchange

  • Build refined models incorporating experimental constraints

What approaches can be used to study the evolutionary significance of YpAngola_A1654 in the context of Yersinia pathogen emergence?

Studying the evolutionary significance of YpAngola_A1654 requires comparative genomic and functional approaches:

• Phylogenetic Analysis:

  • Compare YpAngola_A1654 sequences across Yersinia species and strains

  • Determine whether the gene has undergone positive selection, suggesting functional importance

  • Identify conserved domains that may indicate critical functional regions

  • Map the presence/absence of the gene in relation to the evolutionary emergence of pathogenic Yersinia species

• Comparative Genomic Context:

  • Analyze the genomic neighborhood of ypangola_a1654 across Yersinia species

  • Identify potential operons or gene clusters that may suggest functional relationships

  • Determine if the gene was acquired through horizontal gene transfer or ancient in the Yersinia lineage

• Gene Gain-Loss Analysis:

  • In the context of Yersinia evolution, gene gain-and-loss events have been critical in the emergence of pathogenic species

  • Determine if YpAngola_A1654 was part of the core genome during ecological specialization

  • Assess whether the gene has undergone modifications that correlate with changes in ecological niche or host range

• Functional Conservation Testing:

  • Test whether orthologs from different Yersinia species can complement mutants lacking YpAngola_A1654

  • Compare protein-protein interaction networks across species

  • Evaluate whether sequence variations correspond to functional differences

How can I develop an ELISA-based detection system for YpAngola_A1654 in research or diagnostic applications?

Developing an ELISA system for YpAngola_A1654 detection requires careful consideration of several factors:

• Antibody Development:

  • Generate high-affinity antibodies against YpAngola_A1654 by immunizing animals with the recombinant protein

  • Consider developing antibodies against multiple epitopes to increase specificity

  • Test monoclonal antibodies for their ability to recognize native and denatured forms of the protein

  • Validate antibody specificity against closely related proteins from other Yersinia strains

• ELISA Format Selection:

  • Sandwich ELISA: Use capture antibodies specific to one epitope and detection antibodies to another

  • Direct ELISA: Immobilize samples containing the protein directly on plates

  • Competitive ELISA: Particularly useful for samples containing potential interfering substances

• Optimization Protocol:

  • Determine optimal coating buffer, blocking conditions, and antibody concentrations

  • Establish standard curves using purified recombinant YpAngola_A1654

  • Validate the assay by spiking known quantities of protein into negative samples

  • Test sensitivity and specificity using samples containing other Yersinia proteins

• Application-Specific Considerations:

  • For research applications: Focus on quantitative accuracy and reproducibility

  • For potential diagnostic use: Optimize for sensitivity, specificity, and robustness with clinical samples

  • Incorporate appropriate controls to account for sample matrix effects

What are the most common issues encountered when working with YpAngola_A1654 and how can they be addressed?

Working with membrane proteins like YpAngola_A1654 presents several challenges:

• Low Expression Yields:

  • Problem: Membrane proteins often express poorly in standard systems

  • Solution: Optimize codon usage for the expression host, reduce expression temperature (16-20°C), and use specialized E. coli strains designed for membrane protein expression

  • Alternative: Consider cell-free expression systems that can directly incorporate the protein into liposomes or nanodiscs

• Protein Aggregation:

  • Problem: Hydrophobic regions of membrane proteins tend to cause aggregation

  • Solution: Screen multiple detergents and lipid-like molecules (amphipols, nanodiscs) to identify optimal solubilization conditions

  • Approach: Use dynamic light scattering or size-exclusion chromatography to monitor protein aggregation state

• Loss of Native Conformation:

  • Problem: Membrane proteins may lose their native structure when extracted from the membrane

  • Solution: Use mild detergents and consider adding lipids that mimic the native membrane environment

  • Validation: Develop functional assays to confirm that the purified protein retains its native activity

• Difficult Purification:

  • Problem: Challenging to separate the protein of interest from other membrane components

  • Solution: Use a two-step purification approach combining affinity chromatography with size exclusion or ion exchange

  • Strategy: Increase imidazole concentration gradually during elution to distinguish full-length His-tagged protein from truncated products

How can I validate the structural integrity and functionality of purified YpAngola_A1654?

Validating the structural integrity and functionality of purified YpAngola_A1654 requires multiple complementary approaches:

• Biophysical Characterization:

  • Circular Dichroism (CD) Spectroscopy: Assess secondary structure content and thermal stability

  • Fluorescence Spectroscopy: Monitor tertiary structure through intrinsic tryptophan fluorescence

  • Dynamic Light Scattering: Confirm monodispersity and absence of aggregation

  • Size-Exclusion Chromatography: Verify the oligomeric state of the purified protein

• Structural Validation:

  • Limited Proteolysis: Properly folded proteins show distinct digestion patterns

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Map solvent-accessible regions

  • Cross-linking Studies: Identify spatial relationships between different protein regions

• Functional Assays:

  • Membrane Incorporation: Test the ability of the purified protein to insert into model membranes

  • Liposome Binding Assays: Measure interaction with lipid bilayers

  • If specific function is unknown, develop surrogate assays based on predicted functions (e.g., binding to potential ligands, interactions with other proteins)

• Comparative Analysis:

  • Compare properties with related proteins of known function

  • Use computational predictions to guide experimental validation of specific functional domains

What emerging technologies could advance our understanding of YpAngola_A1654 function?

Several cutting-edge technologies hold promise for elucidating the function of YpAngola_A1654:

• CRISPR-Cas9 Genome Editing:

  • Create precise knockouts or tagged versions of YpAngola_A1654 in Yersinia

  • Perform high-throughput phenotypic screens under various conditions

  • Introduce specific mutations to test structure-function hypotheses

• Single-Cell Techniques:

  • Single-cell RNA-seq to examine host transcriptional responses to bacteria expressing or lacking YpAngola_A1654

  • Mass cytometry (CyTOF) to analyze protein expression and signaling at single-cell resolution

  • Live-cell imaging with fluorescently tagged YpAngola_A1654 to track localization during infection

• Proteomics Approaches:

  • Thermal proteome profiling to identify proteins whose stability is affected by YpAngola_A1654

  • Secretome analysis to determine if YpAngola_A1654 influences protein secretion

  • Phosphoproteomics to identify signaling pathways affected by the protein

• Structural Biology Innovations:

  • Cryo-electron tomography to visualize YpAngola_A1654 in its native membrane environment

  • Integrative structural biology combining multiple data sources (NMR, SAXS, crosslinking) for comprehensive models

  • AI-augmented structure prediction with experimental validation

How might understanding YpAngola_A1654 contribute to broader knowledge about bacterial pathogenesis and host-pathogen interactions?

Research on YpAngola_A1654 has potential to contribute to our understanding of bacterial pathogenesis in several ways:

• Evolutionary Insights:

  • Comparison across Yersinia species may reveal how membrane proteins contribute to adaptation to different ecological niches

  • Understanding whether YpAngola_A1654 was gained or lost during the evolution of pathogenic Yersinia could provide insights into essential virulence determinants

  • Analysis of selection pressure on the gene could indicate its importance in host adaptation

• Novel Pathogenesis Mechanisms:

  • If YpAngola_A1654 is found to interact with host proteins, this could reveal new mechanisms of host manipulation

  • Investigation of its role in membrane integrity might uncover strategies used by bacteria to survive host defenses

  • Studying its expression regulation during infection could identify environmental cues important for virulence

• Immune Recognition Pathways:

  • Research could determine whether YpAngola_A1654 generates epitopes recognized by the host immune system

  • This would contribute to our understanding of how bacterial membrane proteins are processed and presented to T cells

  • Findings could parallel discoveries about other Yersinia proteins that are recognized by the adaptive immune system

• Therapeutic Implications:

  • If YpAngola_A1654 proves essential for Yersinia survival or virulence, it could represent a novel therapeutic target

  • Understanding its structure and function might facilitate the design of inhibitors or vaccines

  • Knowledge of its role in host-pathogen interactions could inform broader antimicrobial strategies