Recombinant Inner membrane protein ybbJ (ybbJ)
Description
Introduction to Recombinant Inner Membrane Protein ybbJ
The recombinant inner membrane protein ybbJ is a conserved protein found in bacteria, such as Escherichia coli. It belongs to the NfeD protein family, which often works in tandem with proteins from the SPFH family, including QmcA, to form functional complexes. These complexes play crucial roles in various cellular processes, including membrane organization and protein quality control.
Structure and Function
Recent studies have elucidated the structure of the QmcA-YbbJ complex using cryo-electron microscopy (cryo-EM). This complex forms an intricate cage-like structure composed of 26 copies of QmcA-YbbJ heterodimers . The transmembrane helices of YbbJ act as adhesive elements, bridging adjacent QmcA molecules, while the oligosaccharide-binding domain of YbbJ encapsulates the SPFH domain of QmcA . This structural arrangement suggests a role in stabilizing membrane structures and facilitating interactions between different membrane components.
Biological Significance
The interaction between YbbJ and QmcA highlights the importance of these proteins in maintaining membrane integrity and possibly in protein quality control mechanisms. The SPFH domain, present in QmcA, is evolutionarily conserved and found in proteins localized to lipid rafts, which are crucial for various cellular processes .
Cryo-EM Structure
| Feature | Description |
|---|---|
| Complex Composition | 26 copies of QmcA-YbbJ heterodimers |
| Structural Role of YbbJ | Transmembrane helices act as adhesive elements |
| Interaction with QmcA | Oligosaccharide-binding domain encapsulates SPFH domain of QmcA |
Functional Implications
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Membrane Organization: The complex likely plays a role in organizing and stabilizing membrane structures.
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Protein Quality Control: The interaction between YbbJ and QmcA may facilitate the maintenance of protein homeostasis within the cell.
Potential Applications
While specific applications of recombinant YbbJ are not well-documented, understanding its structure and function can provide insights into bacterial membrane biology. This knowledge could be leveraged in biotechnological applications, such as developing novel membrane-targeting strategies or improving membrane stability in engineered systems.
References Cryo-EM structure of the SPFH-NfeD family protein complex QmcA-YbbJ. UniProtKB entry for YbbJ. Cryo-EM structure of E.coli SPFH-NfeD family protein complex.
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
If you require a specific tag type, please inform us; we will prioritize its development.
Target Background
KEGG: sfl:SF0433
Q&A
What are the optimal expression systems for recombinant ybbJ production?
The selection of an appropriate expression system is critical for successful recombinant membrane protein production. For inner membrane proteins like ybbJ, both prokaryotic and eukaryotic systems offer distinct advantages depending on research goals.
Yeast expression systems have become increasingly popular for eukaryotic membrane proteins due to their ability to perform post-translational modifications while maintaining relatively high yields. Recent years have seen an exponential increase in membrane protein structures being deposited in the Protein Data Bank, indicating improved empirical methods for membrane protein production . For prokaryotic inner membrane proteins like ybbJ, E. coli remains a preferred system due to its simplicity, rapid growth, and genetic tractability.
When expressing ybbJ, consider the following system selection criteria:
| Expression System | Advantages | Limitations | Best For |
|---|---|---|---|
| E. coli | High yields, economical, simple genetics | Limited post-translational modifications | Initial expression screening, functional studies |
| Yeast (S. cerevisiae) | Eukaryotic processing capability, moderate yields | Longer growth time than bacteria | Functional validation, structural studies |
| P. pastoris | High cell density, strong inducible promoters | More complex genetic manipulation | Large-scale production, challenging proteins |
| Mammalian cells | Native folding environment for mammalian proteins | Expensive, lower yields | Functional studies requiring mammalian context |
The choice between these systems should be based on research objectives, required yield, and downstream applications.
How can growth conditions be optimized to maximize ybbJ expression in recombinant systems?
Optimizing growth conditions is crucial for successful membrane protein expression. Research indicates that the fastest growth conditions are not necessarily optimal for membrane protein production . For ybbJ expression, consider:
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Growth temperature: Lower temperatures (20-25°C) often improve membrane protein folding and reduce inclusion body formation.
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Induction timing: Inducing expression during early-mid log phase rather than late log phase often yields better results.
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Harvesting time: It is crucial to harvest cells prior to glucose exhaustion, just before the diauxic shift in yeast systems .
Research has demonstrated that growth phase at harvest significantly impacts membrane protein yields. The differences in yields under various culture conditions are not necessarily reflected in corresponding mRNA levels but rather relate to differential expression of genes involved in membrane protein secretion and cellular physiology .
For E. coli expression of inner membrane proteins like ybbJ, consider this optimization matrix:
| Parameter | Standard Condition | Optimization Range | Effect on Membrane Protein Yield |
|---|---|---|---|
| Temperature | 37°C | 18-30°C | Lower temperatures reduce protein aggregation |
| Inducer concentration | 1.0 mM IPTG | 0.05-0.5 mM IPTG | Lower concentrations reduce toxicity |
| OD₆₀₀ at induction | 0.6-0.8 | 0.4-1.2 | Early-mid log phase balances growth and expression |
| Post-induction time | 3-4 hours | 4-24 hours | Extended time at lower temperatures increases yield |
| Media composition | LB | TB, 2YT, defined media | Rich media or supplemented minimal media enhances yield |
Critically, systematic bioreactor studies have shown that tightly controlled growth conditions with precise harvesting timing significantly improve membrane protein yields .
What are the most effective methods for extracting ybbJ from the inner membrane?
Extraction of inner membrane proteins like ybbJ requires careful consideration of detergent selection and membrane solubilization conditions. The goal is to extract the protein while maintaining its native structure and function.
For inner membrane proteins, a two-step extraction process is often most effective:
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Cell disruption using mechanical methods (sonication, French press, or high-pressure homogenization)
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Selective membrane solubilization using appropriate detergents
The choice of detergent is critical and should be determined empirically for ybbJ. Common detergents for inner membrane protein extraction include:
| Detergent Class | Examples | CMC (mM) | Advantages | Best For |
|---|---|---|---|---|
| Mild non-ionic | DDM, OG, DM | 0.17, 23.4, 1.8 | Maintains protein structure | Initial extraction, functional studies |
| Zwitterionic | LDAO, FC-12 | 1-2, 1.5 | Efficient solubilization | More challenging extractions |
| Harsh ionic | SDS, Sarkosyl | 7-10, 14.4 | Maximum solubilization | When refolding is planned |
| Polymer-based | SMALPs | N/A | Preserves lipid environment | Native-like structural studies |
Recent advances in styrene-maleic acid lipid particles (SMALPs) technology allow extraction of membrane proteins with their surrounding lipid environment, which can be particularly valuable for maintaining ybbJ in a native-like state for functional studies .
How can purification protocols be optimized specifically for ybbJ?
Purification of inner membrane proteins like ybbJ typically employs affinity chromatography followed by size exclusion or ion exchange chromatography. The key considerations include:
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Selection of affinity tag placement: For ybbJ, C-terminal tagging might be preferable if N-terminal sequences are critical for function or membrane insertion.
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Maintaining protein stability during purification: Including appropriate detergents at concentrations above their critical micelle concentration (CMC) throughout purification.
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Removal of contaminating proteins: Washing steps with low concentrations of imidazole for His-tagged proteins can reduce non-specific binding.
When planning ybbJ purification, consider this general workflow:
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Affinity chromatography (IMAC for His-tagged constructs)
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Optional protease cleavage of affinity tag
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Size exclusion chromatography to separate aggregates and oligomeric states
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Optional ion exchange chromatography for further purification
Experimental evidence from studies on other membrane proteins indicates that the integrity of protein domains is critical for proper membrane localization, as demonstrated with HflC and QmcA proteins . This suggests that maintaining domain integrity during purification would be crucial for ybbJ as well.
What techniques are most suitable for determining ybbJ topology and membrane orientation?
Determining the topology and membrane orientation of ybbJ is essential for understanding its structure-function relationship. Multiple complementary approaches should be considered:
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Computational prediction: Use algorithms like TMHMM, SOSUI, and Phobius to predict transmembrane segments and orientation.
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Experimental verification: Several techniques can experimentally validate these predictions:
| Technique | Information Provided | Advantages | Limitations |
|---|---|---|---|
| Cysteine scanning mutagenesis | Accessibility of specific residues | In vivo approach | Labor intensive |
| Protease accessibility | Surface-exposed domains | Simple setup | Limited resolution |
| Fluorescence fusion reporters | Terminal orientation | Direct visualization | Potential interference with function |
| GFP fusion analysis | Topology mapping | Visual confirmation in cells | Tag size may affect localization |
The study of inner membrane protein YqjD revealed that it possesses a transmembrane motif in the C-terminal region (residues 77-98) that is crucial for membrane localization . Similar approaches could be applied to ybbJ to determine its transmembrane regions and orientation.
For inner membrane proteins, it's crucial to determine whether specific domains face the cytoplasm or periplasm. Studies of HflC showed that the proper combination of transmembrane and periplasmic domains is required for correct localization .
How can the function and potential interaction partners of ybbJ be identified?
Identifying the function and interaction partners of inner membrane proteins like ybbJ requires multiple complementary approaches:
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Co-immunoprecipitation: Using tagged ybbJ to pull down interaction partners
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Bacterial two-hybrid assays: Modified for membrane protein interactions
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Chemical cross-linking: To capture transient interactions
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Proteomic analysis: To identify proteins co-purifying with ybbJ
Research on inner membrane protein YqjD revealed its association with ribosomes through specific protein domains . Similarly, for ybbJ, domain-specific interactions should be investigated to understand its functional role.
A systematic approach to identifying ybbJ function might include:
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Growth phenotype analysis of ybbJ deletion/overexpression strains
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Stress response testing under various conditions
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Localization studies using fluorescence microscopy
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Proteomic analysis to identify changes in protein expression profiles
Studies of SPFH membrane proteins showed that HflKC proteins contribute to aminoglycoside and oxidative stress resistance . Similar phenotypic analyses could reveal the functional role of ybbJ.
What are the common challenges in expressing ybbJ and how can they be addressed?
Membrane protein expression often encounters specific challenges that require systematic troubleshooting. For ybbJ, common issues include:
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Low expression levels: Often related to toxicity or protein instability
| Challenge | Potential Solution | Implementation |
|---|---|---|
| Toxicity to host cells | Use tightly controlled inducible promoters | T7-lac or arabinose-inducible systems with glucose repression |
| Protein instability | Lower expression temperature | Reduce to 18-25°C post-induction |
| Inefficient membrane insertion | Co-express with chaperones or foldases | Include plasmids expressing DnaK-DnaJ-GrpE or GroEL-GroES |
| Host cell stress responses | Optimize strain selection | C41(DE3), C43(DE3) or SHuffle strains for difficult proteins |
Research indicates that successful membrane protein overproduction is linked to avoidance of stress responses in the host cell . Monitoring and mitigating these stress responses can significantly improve expression yields.
How can the quality and functionality of purified ybbJ be assessed?
Quality assessment of purified ybbJ should include both biophysical characterization and functional validation:
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Biophysical characterization:
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SDS-PAGE and Western blotting for purity and identity
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Size exclusion chromatography for aggregation and oligomeric state
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Circular dichroism for secondary structure assessment
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Thermal stability assays to assess protein folding
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Functional validation:
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Reconstitution into liposomes or nanodiscs
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Activity assays based on predicted function
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Binding assays for potential ligands or interaction partners
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For inner membrane proteins, functionality is often tied to proper membrane insertion and orientation. Immunodetection in fractionated cell components (cytoplasmic, inner membrane, and outer membrane) can verify the localization of ybbJ, as demonstrated for HflC-mCherry and QmcA-GFP fusion proteins .
How does lipid composition affect ybbJ localization and function?
Membrane lipid composition significantly impacts the localization and function of inner membrane proteins. Research shows that specific membrane proteins localize to microdomains enriched in particular lipids.
Studies on HflC and QmcA demonstrated that the lack of cardiolipin and isoprenoid lipids altered their membrane localization . Similarly, for ybbJ:
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Consider the impact of lipid composition on:
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Protein localization within the membrane
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Functional activity and interactions
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Stability and oligomerization state
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Experimental approaches to assess lipid dependence:
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Expression in lipid biosynthesis mutants
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Reconstitution in liposomes of varying composition
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Detergent resistance membrane fractionation
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The presence of membrane microdomains, also known as functional membrane microdomains (FMMs) or "lipid rafts," may influence ybbJ localization. These domains are enriched in polyisoprenoid lipids and scaffolding proteins .
What considerations are important for structural studies of ybbJ?
Structural characterization of membrane proteins like ybbJ presents unique challenges. Several approaches can be considered:
| Structural Method | Resolution | Sample Requirements | Advantages | Challenges |
|---|---|---|---|---|
| X-ray crystallography | Atomic | Highly pure, stable crystals | Highest resolution | Difficult crystallization |
| Cryo-EM | Near-atomic | Purified protein (>50 kDa preferred) | No crystals needed | Size limitations, sample homogeneity |
| NMR spectroscopy | Atomic | Isotope-labeled protein | Dynamic information | Size limitations |
| Small-angle X-ray scattering | Low | Monodisperse samples | Solution state measurements | Limited resolution |
For successful structural studies, optimization of the expression plasmid, host cell, and culture conditions is critical . The extraction and purification of functional protein for crystallization trials requires careful consideration of detergent choice and stability conditions.
Recent advances have led to an almost exponential increase in membrane protein structures being deposited in the Protein Data Bank, suggesting that empirical methods have improved to ensure adequate protein supply for these challenging targets .
