Recombinant Rabbit Cytochrome b5 (CYB5A)
Description
Introduction to Recombinant Rabbit Cytochrome b5 (CYB5A)
Recombinant Rabbit Cytochrome b5 (CYB5A) is a genetically engineered protein derived from the cytochrome b5 gene in rabbits. This protein is crucial for various cellular processes, including electron transfer reactions, which are essential in metabolic pathways such as drug metabolism, fatty acid desaturation, and steroid biosynthesis. The recombinant form of this protein is produced using host organisms like E. coli, yeast, baculovirus, or mammalian cells, allowing for high purity and yield.
Production and Purity
The recombinant rabbit cytochrome b5 (CYB5A) is typically produced with a purity of greater than or equal to 85%, as determined by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) . This method ensures that the protein is highly purified, which is essential for research applications where specificity and consistency are critical.
Host Organisms and Reactivity
The recombinant rabbit cytochrome b5 can be expressed in various host organisms, including E. coli, yeast, baculovirus, or mammalian cells. This versatility allows researchers to choose the most suitable expression system based on their specific needs, such as protein yield, folding, and post-translational modifications .
Applications in Research
Recombinant rabbit cytochrome b5 is used in various research applications, primarily due to its role in electron transfer reactions. It is involved in studies related to drug metabolism, fatty acid desaturation, and steroid biosynthesis. Additionally, antibodies against cytochrome b5, such as those produced in rabbits, are used in Western blot, ELISA, and immunohistochemistry to detect and analyze cytochrome b5 expression in different cell types .
Research Findings and Implications
Studies involving cytochrome b5, including its recombinant forms, have shown its importance in metabolic processes and disease states. For instance, dysregulation of cytochrome b5 has been linked to metabolic disorders and cancer . Understanding its function can provide insights into potential therapeutic targets for diseases associated with its dysregulation.
Table 1: Characteristics of Recombinant Rabbit Cytochrome b5 (CYB5A)
| Characteristic | Description |
|---|---|
| Gene Names | CYB5A, CYB5 |
| Host Organisms | E. coli, Yeast, Baculovirus, Mammalian Cells |
| Purity | ≥ 85% by SDS-PAGE |
| Applications | Research in drug metabolism, fatty acid desaturation, steroid biosynthesis |
Table 2: Comparison of Recombinant Cytochrome b5 from Different Species
| Species | Host Organisms | Purity |
|---|---|---|
| Rabbit | E. coli, Yeast, Baculovirus, Mammalian Cells | ≥ 85% |
| Human | E. coli | > 95% |
| Bovine | E. coli, Yeast, Baculovirus, Mammalian Cells | ≥ 85% |
References MyBioSource. Recombinant Rabbit Cytochrome b5 (CYB5A). Assay Genie. CYB5A Rabbit Polyclonal Antibody (CAB5401). PMC. Cloning and preliminary pharmacological characterization of the anaphylatoxin C5a receptor in the rabbit. Origene. Cytochrome b5 (CYB5A) (NM_148923) Human Recombinant Protein. PMC. CYB5A polymorphism increases androgens and reduces risk of rheumatoid arthritis. PubMed. Cloning and preliminary pharmacological characterization.
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please specify them during order placement. We will accommodate your request to the best of our ability.
Note: All proteins are shipped with standard blue ice packs. If dry ice shipping is required, please inform us in advance; additional fees will apply.
The tag type will be determined during the production process. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Cytochrome b5 is a membrane-bound hemoprotein that functions as an electron carrier for several membrane-bound oxygenases.
Q&A
What is the basic structure of rabbit cytochrome b5?
Rabbit cytochrome b5 is a 94-amino acid protein that exists as a heterogeneous mixture in solution due to the presence of two isomers (A and B), which differ in the rotation of the heme plane around the axis defined by the alpha and gamma meso protons. The major isomer (A) accounts for approximately 83% of the total protein, with an A/B ratio of 5:1. The solution structure has been solved using NMR spectroscopy, revealing that the oxidized soluble fragment consists of multiple conformers with an average backbone rmsd (for residues 4-84) of 0.060 ± 0.016 nm . The protein adopts a tertiary structure featuring beta sheets, alpha helices, and a heme moiety, similar to other eukaryotic cytochrome b5 proteins .
What cellular functions does cytochrome b5 perform?
Cytochrome b5 serves as a crucial component of the electron transport chain in cells, functioning primarily as an electron transfer protein. It plays key roles in several metabolic pathways, including drug metabolism, fatty acid desaturation, and steroid biosynthesis . The protein interacts with various redox partners, potentially modulating lipid and sterol biosynthetic enzyme activity. Dysregulation of cytochrome b5 has been associated with various pathological conditions, including metabolic disorders and cancer, highlighting its importance in cellular homeostasis .
What is the amino acid sequence of rabbit cytochrome b5?
The amino acid sequence of human CYB5A, which shares high homology with rabbit cytochrome b5, is: MAEQSDEAVKYYTLEEIQKHNHSKSTWLILHHKVYDLTKFLEEHPGGEEVLREQAGGDATENFEDVGHSTDAREMSKTIIGELHPDDRPKLNKPPETLI . Rabbit cytochrome b5 has 94 amino acids in its soluble fragment and exhibits approximately 93% sequence similarity with rat cytochrome b5, despite showing different isomer distributions .
How do the A and B isomers of rabbit cytochrome b5 differ structurally, and what factors determine their distribution?
The A and B isomers of rabbit cytochrome b5 differ in the rotation of the heme plane around the axis defined by the alpha and gamma meso protons. Despite the high sequence similarity (93%) between rabbit and rat cytochrome b5, their A/B isomer ratios differ significantly (5:1 for rabbit versus 1.5:1 for rat). Structural analysis reveals that residues 23 and 74 play crucial roles in determining this distribution through their side chain interactions with the prosthetic group . These interactions appear to be primarily hydrophobic and steric in nature, influencing the relative stability of one isomer compared to the other. Researchers working with recombinant rabbit cytochrome b5 should be aware of this isomeric heterogeneity when analyzing spectroscopic or functional data .
What structural elements are critical for the membrane association of cytochrome b5?
Eukaryotic ER-bound microsomal cytochrome b5 proteins contain a C-terminal transmembrane anchor consisting of 16-18 amino acids that secures the protein to the ER membrane . This anchoring mechanism is distinct from viral cytochrome b5 proteins, which may exist as either soluble proteins (single hydrophilic domain) or membrane-bound through an N-terminal transmembrane anchor . The hydrophobic domain of cytochrome b5 facilitates spontaneous association with plasma membranes, occurring more readily at low pH but proceeding satisfactorily at physiological pH and temperature . The membrane-anchoring properties of cytochrome b5 are robust, as these proteins resist removal from membranes even after extensive washing at high ionic strength .
How do the heme-binding regions of rabbit cytochrome b5 compare with other species?
The heme-binding histidines in cytochrome b5 are spatially conserved across various species, including rabbits, while there is variability in the heme-adjacent residues across different organisms . This conservation reflects the functional importance of the heme coordination for electron transfer activities. Spectroscopic analyses show that recombinant rabbit cytochrome b5 exhibits spectral properties similar to those of human and other mammalian cytochrome b5 proteins, indicating a conserved heme environment . Researchers should consider these conserved elements when designing experiments involving site-directed mutagenesis of recombinant rabbit cytochrome b5 .
What methods are recommended for recombinant expression of rabbit cytochrome b5?
For recombinant expression of rabbit cytochrome b5, researchers should consider the following methodological approach:
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Gene synthesis or cloning of the CYB5A gene into a suitable expression vector with appropriate tags for purification
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Expression in E. coli systems, which have been successfully used for cytochrome b5 proteins
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Induction of protein expression under conditions that promote heme incorporation
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Purification via affinity chromatography, followed by additional chromatographic steps if needed
When working with recombinant rabbit cytochrome b5, researchers should verify proper folding and heme incorporation by comparing the absorption spectrum with published data for the native protein. The oxidized form of properly folded cytochrome b5 should exhibit characteristic peaks in its absorption spectrum similar to those observed for purified native cytochrome b5 .
What techniques are most effective for studying the membrane association properties of recombinant rabbit cytochrome b5?
Several techniques have proven effective for studying the membrane association properties of recombinant rabbit cytochrome b5:
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Fluorescent labeling followed by microscopy to visualize membrane association
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Photobleaching techniques to measure lateral mobility in membranes
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Quantitative binding assays to determine the number of protein molecules associated per cell
Research has shown that approximately 150,000 cytochrome b5 molecules can associate per erythrocyte . The lateral mobility of fluorescently labeled cytochrome b5 in plasma membranes can be measured by photobleaching techniques, with the apparent lateral diffusion coefficient (D) ranging from 1.0×10⁻⁹ to 8×10⁻⁹ cm²·s⁻¹ and a mobile fraction (M) between 0.4 and 0.6 . These parameters resemble those reported for lipid-anchored proteins rather than membrane-spanning proteins with large cytoplasmic domains .
What analytical methods should be employed to distinguish between the A and B isomers of recombinant rabbit cytochrome b5?
To distinguish between the A and B isomers of recombinant rabbit cytochrome b5, researchers should consider employing the following analytical methods:
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NMR spectroscopy, which can resolve the distinct spectral signatures of the two isomers
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High-performance liquid chromatography (HPLC) with appropriate conditions to separate the isomers
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Circular dichroism (CD) spectroscopy to detect differences in secondary structure
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Molecular dynamics simulations to understand the energetics of the two conformations
NMR spectroscopy has been successfully used to solve the solution structure of the major form (A isomer) of oxidized rabbit cytochrome b5, utilizing NOEs, pseudocontact shifts, and coupling constants . For quantitative analysis of the isomer ratio, researchers should establish calibration standards with known A/B ratios to ensure accurate measurements.
How can recombinant rabbit cytochrome b5 be employed as a membrane anchor for other proteins?
The hydrophobic domain of cytochrome b5 can serve as a universal, laterally mobile membrane anchor for associating various recombinant proteins with cell membranes. A methodological approach includes:
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Design of fusion constructs containing the protein of interest linked to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5
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Expression and purification of the fusion protein
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Introduction to target cells, where spontaneous membrane association will occur
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Verification of membrane localization using fluorescent tags or functional assays
This approach has been demonstrated successfully with LacZ:HP, a recombinant protein consisting of enzymatically active E. coli β-galactosidase coupled to the C-terminal hydrophobic domain of cytochrome b5 . Approximately 100,000 LacZ:HP molecules can associate per erythrocyte, and the lateral mobility of these fusion proteins resembles that of lipid-anchored proteins . This technology has potential applications in the development of diagnostically and therapeutically useful recombinant proteins that require membrane association .
What are the key considerations when using antibodies against rabbit cytochrome b5 in research applications?
When using antibodies against rabbit cytochrome b5 in research applications, several key considerations should be addressed:
| Consideration | Recommended Approach |
|---|---|
| Specificity | Verify cross-reactivity with related proteins in your experimental system |
| Application | Determine optimal dilutions for specific applications (WB: 1:500-1:2000; IHC-P: 1:50-1:200; IF/ICC: 1:50-1:200) |
| Epitope recognition | Consider whether the antibody recognizes the N-terminal domain or C-terminal region |
| Sample preparation | Optimize lysis buffers to preserve protein structure or epitope accessibility |
| Positive controls | Include positive control samples (e.g., MCF7, A-549, HT-29, 293T, M21 cell lines) |
Cytochrome B5 Polyclonal Antibodies produced in rabbits, such as CAB5401, have been validated for various applications including Western blot, immunohistochemistry, immunofluorescence, and ELISA . These antibodies typically recognize human, mouse, and rat cytochrome b5, making them valuable tools for comparative studies across species .
How does the structural comparison between rabbit cytochrome b5 and viral cytochrome b5 proteins inform research on protein evolution?
Structural comparison between rabbit cytochrome b5 and viral cytochrome b5 proteins reveals fascinating insights into protein evolution:
These comparisons provide a framework for understanding the evolution of electron transport proteins and may guide research on the minimal structural requirements for cytochrome b5 function. The viral cytochrome b5 proteins can be viewed as naturally occurring minimalist versions of the protein that retain core functionality .
What are common challenges in producing active recombinant rabbit cytochrome b5 and how can they be addressed?
Production of active recombinant rabbit cytochrome b5 can present several challenges:
| Challenge | Solution |
|---|---|
| Insufficient heme incorporation | Supplement growth medium with δ-aminolevulinic acid (precursor for heme biosynthesis) |
| Protein misfolding | Optimize expression temperature (typically lower temperatures improve folding) |
| Low solubility | Express as a fusion protein with solubility-enhancing tags or truncate the hydrophobic domain |
| Isomer heterogeneity | Consider site-directed mutagenesis at positions 23 and 74 to favor a specific isomer |
| Oxidation during purification | Include reducing agents in buffers and minimize exposure to air |
To verify the activity of purified recombinant rabbit cytochrome b5, researchers should perform spectroscopic analyses to confirm proper heme incorporation and assess electron transfer capability with known redox partners .
How can researchers optimize experimental conditions to study the lateral mobility of cytochrome b5 in membranes?
To optimize experimental conditions for studying the lateral mobility of cytochrome b5 in membranes, researchers should consider the following methodological approach:
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Fluorescent Labeling: Use fluorophores that minimally perturb protein structure and function. Small organic dyes with appropriate reactive groups for specific labeling are preferred.
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Photobleaching Techniques: Employ fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy (FCS) with optimized parameters for laser power, bleach area, and acquisition settings.
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Membrane Model Systems: Compare results across different membrane systems (erythrocytes, cultured cell lines, artificial lipid bilayers) to understand context-dependent mobility.
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Temperature Control: Maintain precise temperature control during experiments, as lateral mobility is temperature-dependent. Studies typically use physiological temperature (37°C).
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Data Analysis: Apply appropriate mathematical models to extract diffusion coefficients and mobile fractions from raw data.
Previous research has established that the lateral diffusion coefficient for rabbit cytochrome b5 in erythrocyte and 3T3 cell membranes ranges from 1.0×10⁻⁹ to 8×10⁻⁹ cm²·s⁻¹ with a mobile fraction between 0.4 and 0.6 . These baseline values provide useful references for comparative studies.
How might the structure-function relationships in rabbit cytochrome b5 inform the design of novel biomolecular tools?
The structural and functional properties of rabbit cytochrome b5 offer significant potential for the design of novel biomolecular tools:
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Membrane Anchoring Technology: The hydrophobic domain of cytochrome b5 can be exploited as a universal membrane anchor for creating cell surface-associated protein therapeutics or diagnostic tools .
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Electron Transfer Modules: The well-characterized electron transfer properties of cytochrome b5 might be harnessed to design synthetic electron transport chains for biotechnological applications.
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Protein Engineering Platforms: Understanding the structural basis for isomer preference in rabbit versus rat cytochrome b5 (despite 93% sequence identity) provides insights for rational protein engineering approaches .
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Minimal Functional Units: Comparative analysis with viral cytochrome b5 proteins helps identify the minimal structural elements required for electron transfer function, which could inform the design of simplified synthetic proteins .
Researchers interested in these applications should consider the specific properties of rabbit cytochrome b5, including its high A/B isomer ratio (5:1) and the role of residues 23 and 74 in determining isomer stability through hydrophobic and steric interactions with the heme group .
What research questions remain unanswered regarding the structure-function relationship of rabbit cytochrome b5?
Despite substantial progress in understanding rabbit cytochrome b5, several important research questions remain unanswered:
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What is the precise mechanism by which residues 23 and 74 influence the A/B isomer ratio, and can this knowledge be applied to engineer proteins with defined isomer preferences?
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How does the dynamic behavior of rabbit cytochrome b5 in solution relate to its interactions with various redox partners?
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What are the specific contributions of individual amino acids to the redox potential and electron transfer kinetics of rabbit cytochrome b5?
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How do post-translational modifications affect the structure, stability, and function of rabbit cytochrome b5 in various cellular contexts?
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Can the membrane-anchoring properties of rabbit cytochrome b5 be optimized for specific biotechnological applications, such as cell surface engineering or targeted drug delivery?
Addressing these questions will require integrated approaches combining structural biology, biophysical characterization, computational modeling, and functional assays .
