Recombinant Shigella boydii serotype 4 Uncharacterized protein ytcA (ytcA)

What is the amino acid sequence of Shigella boydii serotype 4 Uncharacterized protein ytcA?

The amino acid sequence of Shigella boydii serotype 4 Uncharacterized protein ytcA is: CSLSPAIPVIGAYYPSWFFCAIASLILTLITRRIIQLAFNLAIVGIIYTALFAVYAMLFLWLAFF. This sequence corresponds to the expression region 27-91 of the full-length protein . When working with this protein, researchers should note that the hydrophobic nature of this sequence suggests membrane association, which has implications for experimental design, particularly for solubilization and purification protocols.

What are the recommended storage conditions for recombinant ytcA protein?

Recombinant ytcA protein should be stored in Tris-based buffer with 50% glycerol at -20°C for regular storage and at -80°C for extended storage periods . For working with the protein, it is advised to create small aliquots to avoid repeated freeze-thaw cycles, which can lead to protein degradation and loss of structural integrity. Working aliquots can be maintained at 4°C for up to one week . This approach helps maintain protein stability while minimizing structural modifications that could affect experimental outcomes.

What is the UniProt identification number for ytcA and why is this information important for researchers?

The UniProt identification number for Shigella boydii serotype 4 (strain Sb227) Uncharacterized protein ytcA is Q31TR5 . This identification is crucial for researchers as it provides access to standardized annotation information including sequence data, domain structures, post-translational modifications, and evolutionary relationships. For uncharacterized proteins like ytcA, the UniProt entry serves as a central reference point for connecting disparate research findings and can facilitate comparative genomic analyses with related proteins from other bacterial species.

What expression systems are most effective for producing recombinant ytcA protein?

While the search results don't specify the exact expression system for ytcA, research on similar bacterial membrane proteins suggests that Escherichia coli-based expression systems are generally effective for producing recombinant Shigella proteins. When expressing membrane-associated proteins like ytcA (based on its amino acid sequence), researchers should consider:

• Using E. coli strains optimized for membrane protein expression (such as C41(DE3) or C43(DE3))

• Employing lower induction temperatures (16-25°C) to slow expression and facilitate proper folding

• Testing different promoter systems to control expression levels

• Including appropriate fusion tags to aid in purification and solubility

For membrane proteins like ytcA, the inclusion of appropriate detergents during extraction and purification is critical to maintain native protein conformation.

How can inverse transition cycling (ITC) be applied to purify recombinant ytcA protein?

Inverse transition cycling (ITC) represents an alternative purification strategy for recombinant proteins from E. coli that could be applied to ytcA. The method involves:

• Creating a fusion protein of ytcA with a thermally responsive elastin-like polypeptide (ELP)

• Exploiting the environmentally triggered, reversible solubility of the ELP fusion

• Purifying through sequential cycles of aggregation, centrifugation, and resolubilization

This approach offers several advantages over traditional chromatography methods:

• Increased yield and purification efficiency comparable to immobilized metal affinity chromatography (IMAC)

• Scalability for larger production requirements

• Simplified purification workflow that reduces time and resource requirements

• Retention of protein activity after cycling through the inverse phase transition

A typical ITC purification protocol would involve at least two rounds of cycling to achieve high purity, as demonstrated with other recombinant proteins in scientific literature.

What methods are recommended for structural characterization of ytcA protein?

For structural characterization of an uncharacterized membrane protein like ytcA, a multi-technique approach is recommended:

Given the membrane-associated nature of ytcA, structural studies should incorporate appropriate detergents or lipid nanodiscs to maintain native conformation during analysis. Computational prediction methods can also provide valuable structural insights, especially for proteins difficult to characterize experimentally.

What is known about the function of ytcA in Shigella boydii serotype 4?

The function of ytcA in Shigella boydii serotype 4 remains largely uncharacterized, as indicated by its designation as an "uncharacterized protein" . Based on its amino acid sequence, which contains hydrophobic regions typical of membrane proteins, and its localization within the bacterial genome (ordered locus name: SBO_4114) , researchers can make preliminary functional hypotheses:

• The protein likely has a membrane-associated function, possibly involved in transport, signaling, or structural support

• Its conservation within Shigella species suggests functional importance

• Comparative analysis with similar proteins in related bacterial species may provide functional clues

Research approaches to determine ytcA function could include gene knockout studies, protein-protein interaction analyses, and phenotypic characterization of mutant strains under various environmental conditions. The proximity of uncharacterized proteins to DNA replication and transcription machinery, as seen with other uncharacterized bacterial proteins, can provide important functional insights .

How might ytcA contribute to Shigella pathogenesis or antibiotic resistance?

While specific information about ytcA's role in pathogenesis is not directly mentioned in the search results, research on Shigella species suggests several possibilities:

Membrane proteins in Shigella often contribute to:

• Antibiotic resistance through efflux pump mechanisms (51% of antibiotic resistance genes in Shigella are involved in efflux pumps)

• Virulence through bacterial invasion of host cells

• Environmental adaptation during infection

Genome-wide investigations of Shigella species have revealed that many previously uncharacterized proteins are now being identified as potential therapeutic targets, particularly those involved in antibiotic resistance mechanisms . Experimental approaches to investigate ytcA's potential role in pathogenesis would include:

• Comparing expression levels between virulent and avirulent strains

• Testing antibiotic susceptibility in ytcA-knockout mutants

• Evaluating bacterial invasion and survival within host cells in the presence and absence of functional ytcA

• Analyzing protein-protein interactions with known virulence factors

What protein-protein interactions have been identified for ytcA?

• Proximity labeling proteomics: This technique has successfully identified interactions for other uncharacterized proteins by tagging proteins in close spatial proximity, allowing researchers to map the interactome .

• Co-immunoprecipitation coupled with mass spectrometry: This approach can identify stable protein complexes containing ytcA.

• Bacterial two-hybrid screening: This method can systematically test for direct interactions between ytcA and other bacterial proteins.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry analysis can capture transient or weak interactions that might be missed by other techniques.

For membrane proteins like ytcA, special considerations include the use of appropriate detergents to maintain protein structure while allowing for interaction studies, and potentially reconstituting the protein in lipid environments that mimic its native membrane context.

How can ytcA be utilized in vaccine development against Shigella boydii?

Uncharacterized proteins like ytcA could potentially serve as novel antigenic targets for Shigella vaccine development. Current Shigella vaccine approaches focus on:

• Serotype-specific lipopolysaccharide (LPS)

• Conserved invasion plasmid antigens (Ipa proteins, particularly IpaB and IpaC)

To evaluate ytcA as a vaccine candidate, researchers would need to:

• Determine protein localization (surface-exposed proteins make better vaccine targets)

• Assess conservation across Shigella strains and serotypes

• Evaluate immunogenicity through animal studies

• Test protective efficacy in appropriate challenge models

The Shigella artificial invasin complex (Invaplex AR) vaccine approach demonstrates how purified recombinant proteins can be combined with LPS to create effective subunit vaccines . Similar methodologies could incorporate ytcA if it proves to be immunogenic and protective. The advantage of utilizing conserved proteins in vaccines is the potential for cross-protection against multiple serotypes, addressing the challenge of serotype diversity in Shigella infections.

What are the implications of ytcA research for understanding bacterial evolution and adaptation?

Research on uncharacterized proteins like ytcA contributes significantly to our understanding of bacterial evolution and adaptation:

• Phylogenetic analysis can reveal the evolutionary history of ytcA across Shigella species and related Enterobacteriaceae

• Comparison of sequence conservation can identify domains under selective pressure, suggesting functional importance

• Analysis of genomic context can provide insights into co-evolution with other genes

Genome-wide studies of Shigella species have revealed distinct clades among circulating strains worldwide, with relatively less genomic diversity compared to other enteric bacteria . Understanding the conservation and variation of proteins like ytcA within these evolutionary patterns can help explain:

• Mechanisms of host adaptation

• Evolution of pathogenicity

• Development of antibiotic resistance

• Niche specialization within the human gut

These insights are particularly relevant given that Shigella species are major contributors to bacterial dysentery worldwide, especially in developing countries with inadequate sanitation and hygiene .

What are the common challenges in expressing and purifying membrane-associated proteins like ytcA?

Membrane-associated proteins like ytcA present several technical challenges:

When working specifically with ytcA, researchers should note that the recommended storage in Tris-based buffer with 50% glycerol suggests potential stability issues that need careful handling throughout the purification process .

How can researchers overcome difficulties in analyzing the structure of uncharacterized proteins like ytcA?

Structural analysis of uncharacterized membrane proteins presents unique challenges. Researchers can employ these strategies:

• Fragment-based approaches: Breaking the protein into domains for easier structural determination

  • Analysis of the ytcA sequence suggests potential domains that could be expressed separately

  • This approach has been successful for other membrane proteins where full-length structures were challenging

• Computational methods:

  • Homology modeling based on structurally characterized proteins with similar sequences

  • Ab initio modeling for novel fold prediction

  • Molecular dynamics simulations to understand flexibility and conformational changes

• Hybrid methods:

  • Combining low-resolution data (e.g., SAXS) with computational models

  • Integrating data from multiple experimental techniques

• Optimized membrane mimetics:

  • Testing various detergents, lipid nanodiscs, or amphipols to find optimal conditions for structural stability

  • Reconstitution in native-like lipid environments

For ytcA specifically, its relatively small size (expression region 27-91) may make it amenable to NMR-based structural studies if sufficient quantities of isotope-labeled protein can be produced.

How can gene editing technologies be applied to study ytcA function in Shigella boydii?

Advanced gene editing technologies offer powerful approaches to study ytcA function:

• CRISPR-Cas9 system:

  • Precise knockout of ytcA to study loss-of-function phenotypes

  • Introduction of point mutations to identify critical residues

  • Creation of tagged versions for localization studies

  • Conditional expression systems to study essentiality

• Transposon mutagenesis:

  • High-throughput screening for conditions where ytcA is important

  • Identification of genetic interactions through synthetic lethality screens

• Site-directed mutagenesis:

  • Systematic mutation of conserved residues to assess functional importance

  • Creation of dominant-negative variants

• Allelic exchange:

  • Replacement of native ytcA with variants from other species to study species-specific functions

  • Introduction of reporter fusions for expression studies

For Shigella boydii specifically, these genetic approaches would need to be optimized for transformation efficiency, considering the inherent challenges in genetic manipulation of clinical isolates compared to laboratory strains.

What bioinformatic approaches can predict functional domains and interactions of ytcA?

Advanced bioinformatic approaches can provide valuable insights into the potential functions of uncharacterized proteins like ytcA:

• Sequence-based analysis:

  • Profile Hidden Markov Models to identify distant homologs

  • Conservation analysis across bacterial species

  • Identification of functional motifs and domains

  • Transmembrane topology prediction (particularly relevant for ytcA given its sequence characteristics)

• Structure-based prediction:

  • AlphaFold2 or RoseTTAFold for ab initio structural prediction

  • Structure-based function prediction using fold recognition

  • Binding site prediction and virtual screening for potential ligands

• Systems biology approaches:

  • Genomic context analysis to identify functionally related genes

  • Co-expression network analysis

  • Protein-protein interaction network prediction

• Evolutionary analysis:

  • Selection pressure analysis to identify functionally important residues

  • Phylogenetic profiling to associate ytcA with specific biological processes

These computational approaches can generate testable hypotheses about ytcA function that can guide targeted experimental investigations, particularly valuable for proteins like ytcA that lack obvious homology to well-characterized proteins.

How can multi-omics approaches be integrated to elucidate ytcA function in different environmental conditions?

A comprehensive multi-omics strategy can provide a systems-level understanding of ytcA function:

Integration of these multi-omics datasets would require advanced computational methods:

• Network analysis to identify relationships between different data types

• Machine learning approaches to predict function from complex data patterns

• Visualization tools to make integrated datasets interpretable

For ytcA specifically, comparing multi-omics profiles between wild-type and ytcA-knockout strains under conditions relevant to Shigella pathogenesis (e.g., acid stress, oxidative stress, nutrient limitation) could reveal its functional role in bacterial adaptation and survival during infection.

What information should be included in NIH grant applications for ytcA-focused research?

NIH grant applications focused on ytcA research should strategically include:

• Significance and Innovation:

  • Frame the study of ytcA within broader contexts of antibiotic resistance and Shigella pathogenesis

  • Highlight the novelty of studying uncharacterized proteins as potential therapeutic targets

  • Emphasize how understanding ytcA contributes to basic knowledge of bacterial membrane protein function

• Preliminary Data:

  • Include initial characterization data of ytcA expression and purification

  • Present any preliminary functional or structural insights

  • Demonstrate feasibility of proposed methods using pilot studies

• Research Design and Methods:

  • Clearly describe expression and purification protocols specific to membrane proteins

  • Detail structural and functional characterization approaches

  • Include appropriate controls and alternative approaches

• Data Tables and Organization:

  • Follow the current FORMS-I data tables format required by NIH

  • Present preliminary data in well-organized tables and figures

  • Include a timeline of proposed research activities

Researchers should also emphasize interdisciplinary approaches and the potential translational impact of their findings, particularly in addressing the global health burden of Shigella infections in developing countries with inadequate sanitation and hygiene .

How can researchers establish effective collaborations for comprehensive ytcA research?

Effective collaborations for ytcA research should strategically integrate complementary expertise:

• Identifying Collaboration Partners:

  • Structural biologists with expertise in membrane protein characterization

  • Microbiologists specializing in Shigella pathogenesis

  • Computational biologists for sequence and structure analysis

  • Immunologists for vaccine-related applications

  • Clinical researchers with access to Shigella isolates

• Establishing Collaborative Frameworks:

  • Clearly define research questions and objectives

  • Establish material transfer agreements for sharing protein samples and bacterial strains

  • Develop data sharing protocols that respect intellectual property concerns

  • Create regular communication channels for progress updates

• Leveraging Institutional Resources:

  • Core facilities for specialized techniques (cryo-EM, mass spectrometry, etc.)

  • Bioinformatics support for data analysis

  • Grant writing assistance for multi-investigator proposals

• International Collaborations:

  • Partner with researchers in regions with high Shigella prevalence

  • Engage with global health initiatives focused on enteric diseases

  • Collaborate with vaccine development organizations if ytcA shows immunogenic potential

Successful collaborations should balance diverse scientific perspectives while maintaining focus on concrete research objectives related to ytcA characterization and functional understanding.