Recombinant Bovine Cytochrome b5 (CYB5A)
Description
Molecular Definition and Production
Recombinant bovine CYB5A is expressed in Escherichia coli systems, typically fused with a N-terminal His-tag for simplified purification. The mature protein spans residues 2–134 of the native sequence (134 amino acids, ~15.3 kDa) and retains the canonical cytochrome b5 heme-binding domain . Key production details include:
| Parameter | Specification |
|---|---|
| Expression Host | E. coli |
| Tag | His-tag |
| Purity | >90% (SDS-PAGE) |
| Storage | Lyophilized powder at -20°C/-80°C; reconstituted in Tris/PBS buffer with 50% glycerol |
2.1. Core Architecture
The protein adopts a conserved cytochrome b5 fold, featuring:
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Two hydrophobic cores separated by a five-stranded β-sheet .
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A heme group ligated by His-89 and His-112 residues, critical for redox activity .
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A solvent-exposed loop containing the conserved HPGG motif, essential for electron transfer .
Comparative studies with bovine Cyb5A and human Ncb5or-b5 reveal distinct electrostatic surface properties, particularly in core 1 (α2–α5 helices), which influence docking with reductase partners .
2.2. Functional Roles
CYB5A serves as an electron carrier for:
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Steroidogenesis: Enhances 17,20-lyase activity of CYP17A1, boosting androgen synthesis .
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Fatty acid desaturation: Supports stearoyl-CoA desaturase (SCD) in lipid metabolism .
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Drug detoxification: Facilitates cytochrome P450-mediated oxidation reactions .
3.1. Analytical Tools
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ELISA Kits: Sensitive detection of bovine CYB5A in serum, plasma, and tissue lysates (e.g., AssayGenie’s kit with a detection range of 0.312–20 ng/mL) .
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Kinetic Studies: Measures electron transfer efficiency between CYB5A and reductases (Table 1) .
Table 1: Electron Transfer Kinetics of CYB5A Variants
| Reductase | Cyb5A (k<sub>cat</sub>/K<sub>m</sub>, M<sup>-1</sup>s<sup>-1</sup>) | Ncb5or-b5 (k<sub>cat</sub>/K<sub>m</sub>, M<sup>-1</sup>s<sup>-1</sup>) |
|---|---|---|
| Cyb5R3 | 1730 ± 63 | 24.60 ± 1.73 |
| Ncb5or-b5R | 22.97 ± 1.67 | 5.69 ± 0.29 |
3.2. Metabolic and Disease Research
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Obesity: The rs548402150 variant in CYB5A correlates with increased BMI and altered energy expenditure in specific populations .
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Methemoglobinemia: CYB5A defects impair methemoglobin reduction, linked to type IV hereditary forms .
Comparative Analysis with Orthologs
Bovine CYB5A shares >90% sequence identity with human and murine variants but exhibits unique functional adaptations:
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Electrostatic Surface: Reduced negative charge density in core 1 compared to human Cyb5A, affecting reductase interactions .
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Thermal Stability: Denaturation midpoint (T<sub>m</sub>) of 73.5°C, comparable to human isoforms .
Future Directions
Ongoing research focuses on:
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 unless dry ice shipping is specifically requested in advance. Additional fees apply for dry ice shipping.
Note: While the tag type is determined during production, we can prioritize the development of a specific tag if requested.
Target Background
Cytochrome b5 Function: Key Research Findings
- The conserved Tyr30 in Cyt b5 is irreplaceable due to its involvement in both hydrogen bonding networks and hydrophobic interactions within the heme active site's secondary sphere. PMID: 27888781
- This study calculated electron transfer rates for the [myoglobin(wt), cytochrome b5] complex. PMID: 22955681
- Determination of the solution structure of the oxidized cytochrome b(5) mutant V61H. PMID: 12893266
- Examination of the effects of Val45 mutations (to Tyr45, His45, and Glu45) on the heme microenvironment of cytochrome b(5). PMID: 14644556
- Three combined experiments demonstrated the involvement of the conserved negatively charged region surrounding the solvent-exposed heme edge of cytb5 in its binding interaction with horse heart cytochrome c. PMID: 14674751
- Site-directed mutagenesis studies on Cyt b5 assessing the contributions of amino acid residues influencing the docking and electron transfer (ET) between Cyt b5 and horse heart Cyt c. PMID: 15035623
- Simulations offering qualitative microscopic explanations of the differences in physical properties between outer mitochondrial membrane CYB5 isoform, microsomal CYB5, and two mutants in terms of localized structural and flexibility changes. PMID: 16807901
Q&A
What expression systems are most effective for producing recombinant bovine cytochrome b5?
Escherichia coli serves as an efficient expression system for bovine microsomal cytochrome b5. The DNA sequence can be amplified from a liver cDNA library using polymerase chain reaction and subsequently cloned into plasmids that support high-level production in E. coli. Successful expression results in bacterial colonies with distinctive red coloration due to the presence of the heme prosthetic group in the properly folded protein. The expression yields can be substantial, with successful purification achieving up to 45% of the theoretical content in some laboratory conditions .
For optimal expression, plasmid selection is critical as it must support the high-level production of cytochrome b5. The full-length protein (Ala1-Asn133) localizes to the bacterial membrane fraction, while truncated versions lacking the membrane-anchoring domain remain in the cytoplasmic phase, which may affect purification strategies and protein yields .
How does protein length affect the cellular localization of recombinant bovine cytochrome b5?
The cellular localization of recombinant bovine cytochrome b5 within prokaryotic cells directly depends on the protein length. Experimental evidence demonstrates a clear pattern:
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Truncated variants Ala1-Lys90 and Ala1-Ser104 localize to the cytoplasmic phase of bacteria
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The complete protein (Ala1-Asn133) is found within the bacterial membrane fraction
This differential localization indicates that the C-terminal region, specifically the last 30 residues of cytochrome b5, contains all necessary information to direct membrane insertion in E. coli. This finding has significant implications for expression strategy selection, as cytoplasmic variants may be easier to purify but lack membrane association properties that might be important for certain functional studies .
What are the spectroscopic characteristics of recombinant bovine cytochrome b5?
Recombinant bovine cytochrome b5 exhibits distinct spectroscopic features that serve as valuable indicators of proper folding and heme incorporation. Electronic spectroscopy reveals:
| Redox State | Soret Peak Maximum | Additional Characteristics |
|---|---|---|
| Reduced form | 423 nm | Sharper, more intense peak |
| Oxidized form | 413 nm | Broader absorption profile |
These characteristic absorbance maxima provide a reliable method for verifying protein integrity and quantifying protein concentration. Additionally, the spectroscopic properties can be used to monitor conformational changes during thermal stability studies. The distinctive spectral signature arises from the heme prosthetic group and its interaction with the protein environment, particularly the coordination of the heme iron by histidine residues within the protein structure .
How does the thermal stability of different recombinant bovine cytochrome b5 variants compare?
Thermal stability studies reveal significant differences between various forms of bovine cytochrome b5, with recombinant variants showing enhanced stability compared to proteolytically derived fragments. The Ala1-Ser104 variant demonstrates remarkable thermal resistance:
| Cytochrome b5 Form | Redox State | Midpoint Temperature (Tm) | Key Thermodynamic Parameters |
|---|---|---|---|
| Ala1-Ser104 variant | Oxidized | 73°C | Single cooperative transition |
| Ala1-Ser104 variant | Reduced | ~78°C | ΔHm = 420 kJ/mol, ΔSm = 1096 J/mol/K, ΔΔG = 12.38 kJ/mol |
| Tryptic fragment | Oxidized | Lower than 73°C | Not specified in source |
The increased stability of recombinant forms including the Ala1-Ser104 variant correlates with the presence of additional residues at both the N- and C-termini compared to the tryptic fragment. This enhanced stability represents a significant advantage for researchers, as it provides greater flexibility in experimental conditions and potentially extends protein shelf-life for laboratory applications .
What structural analyses confirm the integrity of recombinant bovine cytochrome b5?
Multiple complementary structural analysis techniques confirm that recombinant bovine cytochrome b5 maintains proper folding and structural integrity comparable to native protein:
1D NMR studies demonstrate that the chemical shifts of heme and heme ligand resonances in the Ala1-Ser104 variant exhibit only minor perturbations compared to the tryptic fragment of ferricytochrome b5. This indicates a very similar arrangement of residues in the heme pocket between the recombinant and proteolytically derived forms .
2D NMR analysis further extends this observation, confirming that the structural similarity encompasses not just the heme environment but also the conformations of the polypeptide backbone and side chains throughout the protein structure. This comprehensive structural validation is essential for researchers utilizing recombinant proteins as models for the native cytochrome b5 .
How can researchers effectively measure the thermal stability of recombinant bovine cytochrome b5?
Researchers can employ absorbance spectroscopy to precisely measure the thermal stability of recombinant bovine cytochrome b5 through the following methodology:
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Prepare both oxidized and reduced forms of the protein in appropriate buffers
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Monitor absorbance changes at the Soret peak maxima (413 nm for oxidized, 423 nm for reduced forms) while gradually increasing temperature
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Plot the fraction of denatured protein versus temperature to identify cooperative transitions
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Calculate the midpoint temperature (Tm) where 50% of the protein is denatured
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Determine thermodynamic parameters (ΔHm, ΔSm, ΔΔG) from the denaturation curves
This approach allows quantitative comparison between different variants or experimental conditions. The observation that the reduced form typically displays higher thermal stability than the oxidized form (Tm approximately 78°C versus 73°C) provides important insights into the relationship between redox state and protein stability .
What is the biological role of cytochrome b5 in cellular metabolism?
Cytochrome b5 plays critical roles in multiple cellular pathways, particularly in lipid metabolism and electron transfer processes:
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Fatty acid desaturation: Cytochrome b5 is directly involved in the desaturation of fatty acids, serving as an electron transfer component in these essential biochemical pathways. This function impacts membrane fluidity and lipid composition .
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Electron transfer: As a heme-containing protein, cytochrome b5 participates in various redox reactions, functioning as an electron carrier in multiple metabolic processes.
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Potential disease implications: Recent research suggests cytochrome b5 type A (CYB5A) may have significant roles in disease progression. For instance, in hepatocellular carcinoma (HCC), CYB5A appears to regulate metastasis via the JAK1/STAT3 pathway by binding to STOML2, with decreased expression in HCC correlating with poorer patient outcomes .
These diverse functions highlight the importance of properly folded and functional recombinant cytochrome b5 for metabolic studies and potential therapeutic investigations.
How does recombinant bovine cytochrome b5 interact with biological partners?
Understanding protein-protein interactions involving cytochrome b5 requires sophisticated methodological approaches:
Co-immunoprecipitation (co-IP) coupled with mass spectrometry analysis has successfully identified protein binding partners of cytochrome b5. For example, research has demonstrated that cytochrome b5 type A (CYB5A) binds directly to Stomatin Like 2 (STOML2), affecting downstream signaling cascades .
Verification of interactions can be achieved through multiple complementary techniques:
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Western blotting analysis of co-precipitated complexes
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Endogenous co-IP to rule out artifacts from exogenous protein expression
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Immunofluorescence microscopy to demonstrate cytoplasmic colocalization
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Proximity ligation assays (PLA) providing direct visual evidence of protein-protein interactions within 40 nm proximity
Understanding these interactions is crucial for elucidating the molecular mechanisms underlying cytochrome b5's diverse cellular functions and potential disease implications.
What methodologies are effective for studying the effect of cytochrome b5 on cellular pathways?
Researchers investigating cytochrome b5's impact on cellular pathways can employ several powerful methodological approaches:
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Gene expression modulation:
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Lentiviral-mediated overexpression or knockdown to establish stable cell lines
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CRISPR/Cas9 gene editing for permanent genetic modifications
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Transient transfection for short-term expression studies
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Functional readouts:
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In vivo models:
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Molecular mechanism elucidation:
These methodologies provide comprehensive insights into cytochrome b5's functional roles in normal and pathological states.
What mutagenesis approaches are most effective for studying structure-function relationships in bovine cytochrome b5?
Cassette mutagenesis has proven particularly effective for studying structure-function relationships in bovine cytochrome b5. This approach has successfully generated truncated versions of cytochrome b5 cDNA encoding different protein lengths:
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The first 90 amino acid residues (Ala1-Lys90)
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The first 104 amino acids (Ala1-Ser104)
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The complete protein (Ala1-Asn133)
These truncated variants have revealed critical insights about protein localization and domain functions. For instance, the discovery that the last 30 residues contain all necessary information for membrane insertion would not have been possible without these systematic truncation studies .
For researchers interested in specific residue functions rather than domain analysis, site-directed mutagenesis offers a complementary approach. This technique allows precise alteration of individual amino acids to evaluate their contribution to:
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Heme binding and orientation
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Protein stability
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Membrane insertion
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Protein-protein interactions
What purification strategies are most effective for recombinant bovine cytochrome b5?
Purification strategies must be tailored to the specific form of recombinant bovine cytochrome b5 being expressed, with consideration for localization differences:
For cytoplasmic variants (Ala1-Lys90, Ala1-Ser104):
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Cell lysis using sonication or mechanical disruption
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Clarification by centrifugation to remove cell debris
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Initial purification using ion exchange chromatography
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Further purification via size exclusion chromatography
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Assessment of purity through SDS-PAGE and spectroscopic analysis
For membrane-bound variants (Ala1-Asn133):
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Membrane fraction isolation through differential centrifugation
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Detergent solubilization of membrane proteins
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Affinity chromatography if tagged constructs are used
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Final polishing steps to achieve high purity
The distinctive red color of cytochrome b5 provides a visual indicator during purification, allowing for easier monitoring of protein elution profiles. Specific content can be assessed by comparing the absorbance ratio between the Soret peak and 280 nm, with higher ratios indicating greater purity .
How can researchers overcome challenges in expressing full-length recombinant bovine cytochrome b5?
Expressing full-length recombinant bovine cytochrome b5 presents unique challenges compared to truncated variants due to its membrane localization. Researchers can implement several strategies to overcome these challenges:
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Optimize codon usage for the expression host to enhance translation efficiency
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Employ specialized E. coli strains designed for membrane protein expression
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Modulate growth temperature (typically lowering to 18-25°C) to slow expression and improve folding
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Add supplements to the growth medium:
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δ-aminolevulinic acid as a heme precursor
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Trace metal solutions containing iron
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Specialized induction agents for controlled expression
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Consider fusion partners that can:
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Enhance solubility
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Facilitate detection and purification
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Be removed by specific proteases after purification
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For functional studies requiring membrane association, explore:
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Nanodisc incorporation
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Liposome reconstitution
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Detergent micelle stabilization
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These approaches can significantly improve expression yields and protein quality for the challenging full-length variant .
How does bovine cytochrome b5 compare with cytochrome b5 proteins from other species?
Comparative analysis reveals important similarities and differences between bovine cytochrome b5 and other mammalian homologs:
Understanding these comparative properties is essential for researchers choosing appropriate model systems and interpreting results in the context of human or other mammalian applications .
What are the most promising future research directions for recombinant bovine cytochrome b5?
Several promising research directions emerge from current understanding of recombinant bovine cytochrome b5:
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Disease implications and therapeutic potential:
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Structure-function relationship refinement:
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High-resolution structural analysis using cryo-electron microscopy
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Molecular dynamics simulations to understand conformational changes during function
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Systematic mutation studies to map functional epitopes
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Biotechnological applications:
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Development of cytochrome b5 as an electron transfer component in engineered metabolic pathways
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Exploration of potential biocatalytic applications
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Design of stable cytochrome b5 variants with enhanced properties for industrial applications
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Interaction network elucidation:
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Comprehensive proteomics approaches to map the complete interactome
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Investigation of context-dependent interaction partners in different cellular environments
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Exploration of potential regulatory roles beyond currently known functions
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These research directions leverage the solid foundation of knowledge about recombinant bovine cytochrome b5 while extending into new territories with significant scientific and potentially therapeutic implications.
