Recombinant Uncharacterized membrane protein ynzK (ynzK)
Description
Introduction to ynzK Protein
The ynzK protein is classified as an uncharacterized membrane protein originally identified in Bacillus subtilis strain 168, a model Gram-positive bacterium extensively used in microbiology research. The protein is annotated as a "putative membrane protein of unknown function" with potential phage origin . This classification suggests it may have been incorporated into the B. subtilis genome through horizontal gene transfer from bacteriophages, raising interesting questions about its evolutionary history and functional significance.
Bacillus subtilis belongs to the order Bacillales within the phylum Firmicutes, which is known to harbor various secondary metabolite gene clusters encoding polyketide synthases and non-ribosomal peptide synthetases. These enzymes are responsible for producing a diverse array of polyketides and lipopeptides that may have medical and agricultural applications . While ynzK itself has not been directly linked to these metabolic pathways, understanding its role within this metabolically versatile organism remains an important research focus.
Genetic Context and Conservation
The ynzK gene is identified in the Bacillus subtilis genome under the locus tag BSU17699 . It is part of the ongoing annotation refinement of the B. subtilis 168 genome, which has been a focus of microbiological research for over 20 years. Several key publications have contributed to improving the annotation of the B. subtilis genome, including works published in 2009, 2013, and 2018 .
Table 3.1: Genetic Identifiers for ynzK
| Identifier Type | Values |
|---|---|
| Gene Name | ynzK |
| Locus Tag | BSU17699 |
| UniProtKB IDs | YNZK_BACSU, A0A6M3ZBS8_BACSU |
| UniProt Accessions | C0H417, A0A6M3ZBS8 |
| UniParc | UPI000195C591 |
| EMBL | AL009126, CP052842, CP053102 |
| EnsemblGenome | BSU17699 |
| EMBL-CDS | CAX52625.1, QJP88273.1, QJR46218.1 |
| Gene_ORFName | HIR77_09590, HIR78_09615 |
| RefSeq | YP_003097731.1 |
The annotation as a membrane protein of "phage origin" is particularly noteworthy, as it suggests that this gene may have been acquired through horizontal gene transfer from bacteriophages that infect Bacillus species . Such horizontally-acquired genes often contribute to bacterial adaptation and evolution, potentially providing new functional capabilities to the host organism.
Recombinant Production of ynzK
For research purposes, ynzK is produced as a recombinant protein using Escherichia coli expression systems. This approach allows for the generation of sufficient quantities of the protein for functional and structural studies. The recombinant form of ynzK is commercially available with product codes such as CSB-CF496583BRI and CSB-EP496583BRI1 .
The recombinant ynzK protein is typically produced with high purity (>85% as determined by SDS-PAGE) . The production process may involve the addition of purification tags, although the specific tag type is typically determined during the manufacturing process based on optimization for this particular protein .
Commercially available recombinant ynzK is typically supplied in quantities of around 50 μg, though other quantities can be made available upon request . This standardized production allows researchers to access consistent preparations of this otherwise difficult-to-isolate membrane protein.
Reconstitution Protocol
For lyophilized preparations, reconstitution should be performed in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . Prior to opening, vials should be briefly centrifuged to ensure all contents settle at the bottom. Addition of glycerol to a final concentration of 5-50% is recommended for preparations intended for long-term storage, with 50% being a common default concentration .
Stability and Shelf Life
The stability of recombinant ynzK depends on several factors, including storage conditions, buffer composition, and temperature. Generally, the liquid form has a shelf life of approximately 6 months when stored at -20°C to -80°C, while the lyophilized form can remain stable for up to 12 months under the same conditions .
Repeated freezing and thawing should be avoided as this can lead to protein denaturation and loss of structural integrity . Instead, the protein solution should be aliquoted before freezing to minimize the number of freeze-thaw cycles.
Potential Biological Functions
While the specific function of ynzK remains uncharacterized, several hypotheses can be formulated based on its structural features and genomic context. As a membrane protein potentially of phage origin, ynzK might play roles in:
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Membrane integrity or organization in Bacillus subtilis
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Cell-cell communication or signaling
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Transport of specific molecules across the membrane
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Defense mechanisms related to phage resistance
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Integration of phage components into host metabolism
Bacillus subtilis is noted for its production of diverse secondary metabolites, including polyketides and lipopeptides with potential applications in medicine and agriculture . These compounds are synthesized by polyketide synthases and non-ribosomal peptide synthetases. While there is no direct evidence linking ynzK to these pathways in the available search results, understanding its role in the broader context of B. subtilis metabolism could provide valuable insights.
Research Applications
The recombinant ynzK protein serves as a valuable tool for various research applications in bacterial physiology, molecular biology, and biotechnology. Potential applications include:
Immunological Applications
The recombinant protein can be used for the development of antibodies specific to ynzK, which can subsequently be employed for localization studies, immunoprecipitation experiments, or Western blot analyses. Commercial preparations are often marketed for ELISA (Enzyme-Linked Immunosorbent Assay) applications , suggesting their utility in immunological detection systems.
Protein-Protein Interaction Studies
Recombinant ynzK can be used in pull-down assays, co-immunoprecipitation experiments, or yeast two-hybrid screens to identify potential interacting partners, which could help elucidate its biological role within the bacterial cell.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order remarks. We will fulfill your request whenever possible.
Note: All proteins are shipped with standard blue ice packs. If you require dry ice shipping, please communicate with us in advance, as additional charges will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: bsu:BSU17699
STRING: 224308.Bsubs1_010100009741
Q&A
What is the uncharacterized membrane protein ynzK from Bacillus subtilis?
The ynzK protein is a putative membrane protein from Bacillus subtilis with 118 amino acids. Its function has been proposed based on the presence of conserved amino acid motifs, structural features, and limited homology analysis. The protein is classified as a putative membrane component with evidence level 3, indicating that direct experimental validation of its function has not been comprehensively performed . The full amino acid sequence is: MYRNIVFYLILLCLFISVVMKSFEVIRTIANVICFIFVLLYFKDEMKTYSRKALAILSICFLFLVGICAFIILQGQTLMKRHLFFTGLETIAGILLIIVSLSICTFILRKISNRLKRM . Current knowledge about ynzK primarily derives from bioinformatic predictions rather than functional characterization.
What predicted functional partners have been associated with ynzK?
According to STRING database analysis, ynzK has several predicted functional partners, with varying confidence scores:
| Predicted Partner | Description | Confidence Score |
|---|---|---|
| yncM | Conserved hypothetical protein; Evidence 4: Homologs of previously reported genes of unknown function | 0.495 |
| cotC | Spore coat protein (outer); Evidence 1a: Function experimentally demonstrated in the studied strain; Product type s: structure | 0.424 |
What expression systems are commonly used for recombinant ynzK production?
For expression of recombinant membrane proteins like ynzK, Escherichia coli is frequently used as a host organism due to its rapid growth, well-established genetic manipulation techniques, and cost-effectiveness. Based on similar membrane protein expression strategies, the protein may be produced with various tags to facilitate purification and analysis . Current commercial preparations of recombinant ynzK are stored in Tris-based buffer with 50% glycerol, optimized for protein stability . When designing expression systems for membrane proteins like ynzK, considerations should include codon optimization, fusion tags selection, and appropriate solubilization methods to maintain native conformation.
What experimental design approaches are most suitable for characterizing the function of ynzK?
A systematic experimental design for characterizing ynzK should follow these key steps:
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Define variables: Establish independent variables (e.g., expression conditions, cell types, stimuli) and dependent variables (e.g., localization patterns, binding partners, phenotypic effects) .
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Formulate testable hypotheses: Based on bioinformatic predictions of ynzK structure and homology to known proteins, develop specific hypotheses about potential functions .
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Design experimental treatments: Create a series of controlled experiments manipulating expression levels, introducing mutations, or altering environmental conditions .
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Subject assignment: Use both within-subject and between-subject designs when appropriate. For cellular studies, ensure proper controls using the same genetic background .
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Measurement methodologies: Employ multiple complementary techniques including fluorescent tagging for localization, co-immunoprecipitation for interaction studies, and phenotypic assays for functional characterization .
A comprehensive approach would integrate computational predictions with wet-lab validation, including subcellular localization studies, protein-protein interaction screens, and phenotypic analysis of knockout/overexpression strains.
How can culture media be optimized for enhanced expression of membrane proteins like ynzK?
Optimizing culture media for membrane protein expression requires systematic manipulation of multiple factors that influence protein production and membrane incorporation:
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Response Surface Methodology (RSM): This statistical approach can identify optimal concentrations of critical permeabilizing factors that enhance protein secretion. For example, in similar experiments with streptokinase, RSM optimization led to a 7-fold increase in secreted recombinant protein (from 802 U/mL to 5824 U/mL) .
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Critical permeabilizing factors: Based on research with other recombinant proteins, the following components can be optimized:
| Factor | Function | Optimal Range |
|---|---|---|
| Glycine | Increases membrane permeability | 0.1-1.0% |
| Triton X-100 | Membrane permeabilizing agent | 0.1-0.5% |
| Ca²⁺ | Stabilizes membrane structure | 0.1-10 mM |
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Induction conditions: Careful control of inducer concentration, induction timing, and post-induction temperature significantly impacts membrane protein expression .
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Growth phase considerations: For membrane proteins, induction during mid-log phase often provides better results than early or late growth phases due to optimal membrane synthesis rates.
When implementing these optimizations, researchers should conduct parallel measurements of both total and extracellular protein to assess the efficiency of translocation across the bacterial membrane.
What analytical approaches can resolve structure-function relationships in uncharacterized membrane proteins like ynzK?
Elucidating structure-function relationships for uncharacterized membrane proteins requires an integrated analytical approach:
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Computational prediction: Start with bioinformatic tools to predict transmembrane domains, secondary structure, and potential functional domains based on the known amino acid sequence (MYRNIVFYLILLCLFISVVMKSFEVIRTIANVICFIFVLLYFKDEMKTYSRKALAILSICFLFLVGICAFIILQGQTLMKRHLFFTGLETIAGILLIIVSLSICTFILRKISNRLKRM) .
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Site-directed mutagenesis: Based on computational predictions, design mutations targeting conserved residues or predicted functional domains to assess their impact on protein activity or localization.
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Structural analysis techniques:
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Circular dichroism spectroscopy for secondary structure assessment
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Limited proteolysis to identify domain boundaries
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Cryo-electron microscopy or X-ray crystallography for detailed structural information if sufficient quantities of purified protein can be obtained
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Functional assays: Develop specific assays based on predicted functions or interactions with known partners (such as yncM or cotC) .
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Cross-linking studies: Use chemical cross-linking followed by mass spectrometry to identify spatial relationships between protein domains and interaction partners.
This multifaceted approach can generate testable hypotheses about structure-function relationships even in the absence of a priori functional knowledge.
How can researchers leverage People Also Ask data to guide ynzK research directions?
Utilizing Google's People Also Ask (PAA) data represents an innovative approach to identifying research gaps and priorities for poorly characterized proteins like ynzK:
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Identify knowledge gaps: PAA data appears in over 80% of English searches and reveals patterns in searcher behavior and information needs . For scientific topics, these patterns often highlight unresolved questions that researchers are actively seeking answers to.
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Track evolving research interests: Regular monitoring of PAA data can reveal shifts in research focus over time, potentially identifying emerging areas of interest in membrane protein research .
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Framework for prioritizing experiments:
| PAA Data Analysis Approach | Research Application |
|---|---|
| Frequency analysis of questions | Identify most common knowledge gaps |
| Complexity assessment of PAA questions | Distinguish between solved and unsolved problems |
| Temporal tracking of PAA changes | Detect emerging research trends |
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Content development strategy: Using PAA insights can help researchers position their work to address high-interest questions, potentially increasing citation rates and research impact .
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Cross-disciplinary connections: PAA data often reveals unexpected connections between research areas, potentially identifying novel applications or experimental approaches from adjacent fields.
This data-driven approach to research planning complements traditional literature reviews by capturing real-time information needs within the scientific community.
What controls and validation steps are essential when studying protein-protein interactions involving ynzK?
Given the predicted interactions between ynzK and proteins like yncM and cotC , robust controls and validation steps are crucial for reliable protein-protein interaction studies:
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Multiple methodological approaches: Employ at least two independent methods to validate interactions:
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Co-immunoprecipitation followed by western blotting
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Bacterial/yeast two-hybrid systems
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Fluorescence resonance energy transfer (FRET)
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Bimolecular fluorescence complementation (BiFC)
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Essential controls:
| Control Type | Purpose | Implementation |
|---|---|---|
| Negative control | Eliminate false positives | Use unrelated membrane protein with similar properties |
| Specificity control | Verify direct interaction | Test interaction with truncated versions of ynzK |
| Affinity tag control | Exclude tag-mediated artifacts | Compare results with differently tagged versions |
| Expression level control | Prevent overexpression artifacts | Use native promoter constructs or titrated expression |
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Validation in native context: Confirm interactions observed in heterologous systems within Bacillus subtilis under physiologically relevant conditions.
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Functional validation: Demonstrate biological significance of the interaction through phenotypic analysis of interaction-deficient mutants.
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Quantitative assessment: Determine binding affinities and kinetics using surface plasmon resonance or isothermal titration calorimetry where feasible.
This comprehensive validation framework ensures that reported protein-protein interactions reflect biologically relevant phenomena rather than experimental artifacts.
