Recombinant Protein CrcB homolog 1 (crcB1)
Description
Protein Structure and Composition
CrcB homolog 1 (crcB1) is a full-length protein consisting of 132 amino acids in humans. The complete amino acid sequence has been determined as: "MPNHDYRELAAVFAGGALGALARAALSALAIPDPARWPWPTFTVNVVGAFLVGYFTTRLLERLPLSSYRRPLLGTGLCGGLTTFSTMQVETISMIEHGHWGLAAAYSVVSITLGLLAVHLATVLVRRVRIRR" . This sequence reveals a protein structure that is consistent with membrane-associated functions, particularly featuring hydrophobic regions that suggest transmembrane domains. Based on sequence analysis, the protein appears to share homology with fluoride ion channels found in various organisms, suggesting evolutionary conservation of this protein family .
The recombinant form of this protein is typically produced with an N-terminal histidine tag to facilitate purification and experimental manipulation. This His-tagged version is cataloged under identification numbers such as RFL16087HF in commercial protein databases and repositories . The protein has been assigned the UniProt ID P63862, which serves as its standard identifier in protein databases .
Genetic Origin and Expression
The human CrcB homolog 1 is encoded by the CRCB1 gene. While the search results do not provide extensive information on the genomic location and organization of this gene, the protein can be recombinantly expressed in prokaryotic systems such as Escherichia coli for research and commercial purposes . This approach allows for the production of significant quantities of the protein for structural and functional studies.
The recombinant protein is typically expressed in E. coli expression systems, which provide an efficient and cost-effective method for producing human proteins in a bacterial host . The expression in E. coli suggests that the protein does not require complex post-translational modifications for its basic structural integrity, though native modifications may still be relevant for full biological function.
Ion Channel Activity
Evidence suggests that CrcB homolog 1 functions as a fluoride-specific ion channel. Related proteins in this family play important roles in reducing fluoride concentration within cells, thereby mitigating the potential toxicity of this ion . This function is critical for cellular homeostasis, as fluoride can interfere with various enzymatic processes and cellular functions when present at elevated concentrations.
The protein's structure appears to facilitate specific ion transport capabilities, with computational models suggesting a configuration suitable for ion passage across cellular membranes . Although direct experimental verification of the human CrcB1's fluoride transport capabilities is not extensively documented in the provided search results, its homology to known fluoride ion channels in other organisms strongly suggests conservation of this function.
Cellular Localization and Integration
The amino acid sequence of CrcB homolog 1 indicates membrane-spanning domains typical of transmembrane proteins . This structural arrangement allows the protein to integrate into cellular membranes where it can perform its putative ion channel functions. The specific subcellular localization (plasma membrane, organelle membranes, etc.) is not definitively established in the search results, though the function suggests plasma membrane or possibly organelle membrane localization.
Expression Systems
Recombinant CrcB homolog 1 is primarily produced using bacterial expression systems, with E. coli being the predominant host organism . The use of E. coli for expression indicates that the protein can fold properly in prokaryotic systems without requiring the complex post-translational modification machinery present in eukaryotic cells. The expression protocol typically involves transformation of E. coli with an expression vector containing the CRCB1 gene sequence, followed by induction of protein expression under controlled conditions.
Purification Methodology
The recombinant protein is designed with an N-terminal histidine tag, which facilitates purification through affinity chromatography methods . This tag consists of a series of histidine residues that bind with high affinity to metal ions such as nickel, allowing for selective isolation of the tagged protein from the complex mixture of cellular components. Following purification, the product typically achieves greater than 90% purity as determined by SDS-PAGE analysis .
The purified protein is often provided in lyophilized powder form, which enhances stability during storage and shipping. For experimental use, the protein can be reconstituted in deionized sterile water to concentrations ranging from 0.1 to 1.0 mg/mL . Addition of glycerol (typically to a final concentration of 50%) is recommended for long-term storage at -20°C or -80°C to prevent damage from freeze-thaw cycles.
Experimental Utility
Recombinant CrcB homolog 1 has potential applications in various research areas, particularly those focusing on ion transport mechanisms and membrane protein function. The availability of the purified recombinant protein enables structural studies, functional assays, and investigations into interaction partners.
The protein can serve as a valuable tool for:
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Studying fluoride transport mechanisms across membranes
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Investigating membrane protein topology and structure
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Exploring ion channel pharmacology and modulation
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Developing assays for screening compounds that modulate ion channel activity
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Generating antibodies for detection and localization studies
Biomedical Relevance
While the direct clinical significance of CrcB homolog 1 is not extensively documented in the search results, the protein's function in ion homeostasis suggests potential relevance to certain physiological and pathological processes. Fluoride transport mechanisms may have implications for dental health, bone metabolism, and cellular responses to environmental fluoride exposure.
The protein's role in detoxification of fluoride ions may also provide insights into cellular defense mechanisms against potentially harmful environmental compounds. Further research into this protein family could potentially reveal new targets for therapeutic intervention in conditions involving disrupted ion homeostasis.
Evolutionary Conservation
CrcB homolog 1 appears to be part of a conserved family of fluoride ion channels found across various species . This evolutionary conservation suggests a fundamental biological importance of fluoride regulation that has been maintained throughout evolutionary history. The specific functional adaptations of the human CrcB1 compared to homologs in other organisms represents an area for potential further investigation.
It is important to note that CrcB homolog 1 (crcB1) should not be confused with other similarly named proteins such as CRB1 (Crumbs homolog 1), which is involved in eye development and associated with retinitis pigmentosa and Leber congenital amaurosis . Similarly, it is distinct from CREB1 (cAMP responsive element binding protein 1), which functions as a transcription factor involved in various cellular processes including cancer development .
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Crucial for reducing intracellular fluoride concentration, thereby mitigating its toxicity.
KEGG: cdi:DIP1883
Q&A
What is CrcB homolog 1 and what is its primary function?
CrcB homolog 1 (crcB1) is a membrane protein that functions primarily as a fluoride ion transporter. It is part of a genetic system that includes fluoride riboswitches and often co-occurs with crcB2 in an operon structure. The protein consists of relatively short amino acid sequences (ranging from 109-132 amino acids depending on the species) and contains transmembrane domains that facilitate ion transport across cellular membranes . The protein plays a critical role in fluoride homeostasis, likely protecting cells from fluoride toxicity by exporting excess fluoride ions from the cytoplasm.
In which organisms has crcB1 been identified and characterized?
CrcB1 has been identified in multiple bacterial species, with characterized versions including:
The protein sequence and structure show conservation across species, although with notable variations in length and specific amino acid composition that may reflect adaptations to different cellular environments or functional requirements.
How do researchers distinguish between crcB1 and crcB2 proteins?
Researchers distinguish between crcB1 and crcB2 primarily through:
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Sequence analysis and comparison with reference databases
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Positional analysis within the genome (typically adjacent in the same operon)
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Expression pattern analysis
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Functional complementation studies
In Acetobacterium species, crcB1 and crcB2 are typically arranged in an operon, with crcB1 positioned upstream of crcB2. In some species like A. fimetarium, crcB2 appears to be truncated , which provides an additional distinguishing characteristic.
What expression systems are most effective for producing recombinant crcB1 protein?
For recombinant expression of crcB1, E. coli has been the predominant system of choice due to:
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High yield potential for membrane proteins
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Compatibility with various fusion tags (particularly His-tags)
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Well-established protocols for protein induction and harvest
Recombinant crcB1 proteins from both Mycobacterium paratuberculosis and Prochlorococcus marinus have been successfully expressed in E. coli with N-terminal His tags . The expression typically results in proteins with greater than 90% purity as determined by SDS-PAGE analysis.
What methodological challenges exist in purifying functional crcB1 protein?
The purification of functional crcB1 presents several challenges that researchers must address:
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Membrane protein solubilization without denaturing the protein
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Maintaining structural integrity throughout purification steps
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Verifying functional activity post-purification
A recommended methodological approach includes:
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Expression in E. coli followed by cell lysis under non-denaturing conditions
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Solubilization using mild detergents compatible with membrane proteins
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Affinity chromatography using the His-tag for initial purification
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Size exclusion chromatography for further purification if needed
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Storage in buffer containing 6% trehalose at pH 8.0 to maintain stability
Researchers should avoid repeated freeze-thaw cycles by storing working aliquots at 4°C for short-term use (up to one week) and keeping long-term stocks at -20°C/-80°C .
What experimental approaches can determine the structure-function relationship of crcB1?
Several complementary approaches are recommended for elucidating crcB1 structure-function:
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Comparative Sequence Analysis: Align crcB1 sequences from various organisms to identify conserved regions likely critical for function.
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Site-Directed Mutagenesis: Systematically modify specific amino acids to identify residues essential for fluoride transport.
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Fluoride Transport Assays: Measure fluoride transport efficiency using:
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Fluoride-selective electrodes
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Fluorescent reporter systems
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Radioactive tracer methods
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Structural Biology Techniques:
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X-ray crystallography (challenging for membrane proteins)
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Cryo-electron microscopy
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Nuclear magnetic resonance spectroscopy for specific domains
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These approaches, used together, can provide comprehensive insights into how the protein structure enables fluoride transport function.
How does the amino acid sequence of crcB1 relate to its fluoride transport function?
The amino acid sequences of crcB1 from different organisms show patterns related to function:
Figure 1: Cell growth and normalized fluoride concentration in A. bakii
| Time (days) | A. bakii + PFMeUPA (16S rRNA gene copies/mL) | Normalized F⁻ formation per cell (M/(copies*mL⁻¹)) |
|---|---|---|
| 0 | ~1.0×10⁸ | ~0.0×10⁻⁸ |
| 4 | ~2.5×10⁸ | ~2.0×10⁻⁸ |
| 8 | ~5.0×10⁸ | ~4.0×10⁻⁸ |
| 12 | ~7.5×10⁸ | ~6.0×10⁻⁸ |
Data approximated from Figure S8 in source
What is the relationship between fluoride riboswitches and crcB1 gene regulation?
Fluoride riboswitches function as genetic regulatory elements that specifically bind fluoride ions, leading to conformational changes that affect transcription of downstream genes. For crcB1:
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The riboswitch acts as a sensor of intracellular fluoride concentration
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When fluoride levels increase, the riboswitch undergoes structural changes
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These changes promote transcription of the crcB operon, including crcB1
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Increased crcB1 expression enhances fluoride export, reducing cellular toxicity
Notably, the genomic arrangement of this system varies between bacterial species. While most Acetobacterium species contain fluoride riboswitches upstream of their crcB operons, A. woodii lacks an identifiable fluoride riboswitch in this position . This suggests evolutionary divergence in regulatory mechanisms for fluoride response.
How can completely randomized design (CRD) be applied to study crcB1 function?
Completely Randomized Design (CRD) provides a robust methodological framework for studying crcB1 function by:
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Eliminating systematic bias in experimental conditions
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Allowing for rigorous statistical analysis of multiple variables
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Enabling isolation of specific effects from confounding factors
For crcB1 research, a CRD approach might involve:
Experimental Setup:
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Random assignment of bacterial cultures to different fluoride exposure conditions
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Systematic variation of factors such as fluoride concentration, exposure time, or environmental conditions
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Multiple biological and technical replicates to ensure statistical power
Analysis Approach:
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ANOVA for evaluating statistical significance of observed differences
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Post-hoc tests for multiple comparisons between specific conditions
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Regression analysis for dose-response relationships
This approach is particularly valuable when studying how crcB1 expression and function respond to varying environmental conditions, genetic modifications, or pharmacological interventions .
What role might crcB1 play in CREB1-mediated transcriptional responses?
While direct evidence linking crcB1 to CREB1-mediated transcription is limited, research on transcription factor-mediated responses suggests potential relationships:
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CREB1 (cAMP response element-binding protein 1) functions as a transcription factor that responds to various cellular signals
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CREB1 target genes include cytokines and chemokines that mediate immune responses
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Cellular stress responses, including those triggered by ion imbalances, can activate CREB1-mediated transcription
In immunological research, CREB1 activation has been shown to drive expression of cytokines/chemokines including Fractalkine (CX3CL1), which influences monocyte migration . While speculative, it is possible that fluoride stress detection systems (including crcB1-mediated responses) could intersect with CREB1 pathways in some organisms, particularly if fluoride homeostasis disruption triggers broader cellular stress responses.
This represents an area for future research that could connect ion homeostasis mechanisms with broader cellular signaling networks.
How can secretion systems be optimized for extracellular production of recombinant crcB1?
For researchers seeking to optimize extracellular production of recombinant crcB1, several secretion systems can be considered:
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Type I Secretion System (T1SS):
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Optimization Strategies:
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Considerations for crcB1:
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As a membrane protein, secretion may require redesign of hydrophobic domains
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Fusion to soluble carrier proteins might improve secretion efficiency
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Potential need for chaperones to maintain proper folding during secretion
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While challenging due to crcB1's natural membrane localization, these approaches could enable production of soluble, secreted forms of the protein for research applications requiring larger quantities of purified material.
What are the most promising future research directions for crcB1?
Future research on crcB1 could productively focus on:
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Structural Biology: Determining high-resolution structures of crcB1 to understand the molecular basis of fluoride transport.
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Comparative Genomics: Analyzing crcB1 variants across diverse bacterial species to understand evolutionary adaptations.
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Regulatory Networks: Elucidating the complete regulatory mechanisms controlling crcB1 expression, including potential interactions beyond fluoride riboswitches.
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Biotechnological Applications: Exploring potential applications in fluoride bioremediation or as biosensors for environmental fluoride detection.
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Medical Relevance: Investigating whether crcB1 or similar proteins in pathogenic bacteria might serve as potential drug targets.
These research directions would address significant knowledge gaps while potentially yielding practical applications in environmental science and medicine.
What methodological advances would most benefit crcB1 research?
Methodological advances that would significantly benefit crcB1 research include:
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Improved Membrane Protein Expression Systems: Development of specialized expression systems optimized for membrane proteins like crcB1.
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Advanced Fluoride Transport Assays: More sensitive and high-throughput methods to quantify fluoride transport activity in vitro and in vivo.
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Cryo-EM Techniques: Refinements in cryo-electron microscopy methods for smaller membrane proteins would facilitate structural studies.
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Computational Modeling: Enhanced computational approaches for predicting protein-ion interactions and transport mechanisms.
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Single-Cell Analysis: Methods to monitor fluoride transport at the single-cell level to capture heterogeneity in transport activity.
