Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ0332.1 (MJ0332.1)
Description
Overview
Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ0332.1 (MJ0332.1) is a protein derived from the archaeon Methanocaldococcus jannaschii. M. jannaschii is a hyperthermophilic methanogen, meaning it thrives in extremely hot environments and produces methane as a metabolic byproduct . MJ0332.1 is referred to as an uncharacterized protein, which indicates that its specific function within the organism is not yet确切的已知 .
Basic Information
Production and Characteristics
MJ0332.1 is produced using recombinant DNA technology, where the gene encoding MJ0332.1 is expressed in E. coli . The recombinant protein is full-length (1-132 amino acids) and includes an N-terminal His tag for purification purposes . The protein is available in lyophilized form . It is recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the option to add 5-50% glycerol for long-term storage at -20°C/-80°C .
Potential Functions and Research Context
As an uncharacterized protein, the exact function of MJ0332.1 remains unknown. Proteins from Methanocaldococcus jannaschii are of interest in various biochemical and genetic studies . Research has focused on understanding the organism's genetic system, including creating strains with modified genes and studying protein activity . For instance, researchers have engineered M. jannaschii strains to overexpress specific proteins with affinity tags, facilitating protein purification and activity testing . Studying uncharacterized proteins like MJ0332.1 may provide insights into unique metabolic pathways or adaptations specific to hyperthermophilic archaea .
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 unless dry ice shipping is requested in advance. Additional fees apply for dry ice shipping.
Note: Tag type is determined during production. If a specific tag type is required, please inform us; we will prioritize development of the specified tag.
Target Background
KEGG: mja:MJ_0332.1
STRING: 243232.MJ_0332.1
Q&A
Advanced Research Questions
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What experimental approaches can determine the function of MJ0332.1?
Given that MJ0332.1 is uncharacterized, a multi-disciplinary approach is necessary to elucidate its function:
The recent development of a genetic system for M. jannaschii (as described by Sarmiento et al., 2019) allows for in vivo studies of MJ0332.1 . This genetic system includes:
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Heat shock-based DNA transformation method
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Mevinolin/simvastatin selection systems
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Homologous recombination for genome modification
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Promoter systems for controlled gene expression
For functional studies, it's crucial to consider the native conditions of M. jannaschii:
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Optimal growth temperature: 85°C
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Anaerobic environment
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Methanogenic metabolism
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How can structural studies of MJ0332.1 be designed and optimized?
Structural characterization of MJ0332.1 presents unique challenges due to its archaeal origin and potential membrane association:
| Structural Method | Sample Requirements | Advantages | Limitations |
|---|---|---|---|
| X-ray Crystallography | Highly pure, homogeneous protein (>95%) | High resolution (potentially <2Å) | Difficult for membrane proteins |
| Nuclear Magnetic Resonance (NMR) | 15N/13C-labeled protein, <30 kDa | Solution structure, dynamics | Size limitation (MJ0332.1 is suitable) |
| Cryo-Electron Microscopy | Pure protein, can work with complexes | No crystallization needed | Typically for larger proteins/complexes |
| Small-Angle X-ray Scattering | Monodisperse samples | Low resolution envelope, flexibility | Limited molecular details |
For MJ0332.1 crystallization, consider:
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Detergent screening for membrane protein solubilization
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Thermostability assays to identify optimal buffer conditions
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Crystallization at elevated temperatures (20-30°C) to accommodate the thermophilic nature
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Lipidic cubic phase crystallization if traditional methods fail
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Addition of stabilizing partners (antibodies, nanobodies) to aid crystallization
NMR studies may be particularly suitable given MJ0332.1's size (132 amino acids), enabling:
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Backbone assignments
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Secondary structure determination
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Dynamics studies
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Binding site identification through chemical shift perturbation
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How can genetic manipulation techniques be applied to study MJ0332.1 in vivo?
The recent development of genetic tools for M. jannaschii enables sophisticated in vivo studies of MJ0332.1 :
For generating a MJ0332.1 knockout in M. jannaschii:
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Construct a suicide plasmid containing 500bp upstream and downstream homology regions
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Include the P<sub>sla-hmgA</sub> cassette for selection
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Linearize the construct before transformation
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Transform M. jannaschii using heat shock (85°C for 5 minutes)
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Select transformants on solid medium containing mevinolin or simvastatin
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Verify the knockout by PCR and sequencing
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Characterize the phenotype under various growth conditions
The transformation efficiency for M. jannaschii is approximately half that observed with laboratory strains, but still sufficient for genetic studies .
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What strategies can be employed to identify potential interacting partners of MJ0332.1?
Identifying protein-protein interactions is crucial for understanding MJ0332.1's function:
For affinity purification-MS studies:
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Generate an M. jannaschii strain expressing MJ0332.1 with a 3xFLAG-twin Strep tag using the genetic system described by Sarmiento et al.
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Culture the strain under various conditions (different growth phases, stress conditions)
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Perform gentle lysis preserving protein complexes
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Purify using tandem affinity purification (Strep-Tactin followed by anti-FLAG)
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Analyze co-purifying proteins by mass spectrometry
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Validate interactions through reciprocal pull-downs and functional assays
The recent demonstration of homologous protein expression with affinity tags in M. jannaschii (shown for Mj-FprA) provides a template for similar studies with MJ0332.1 .
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How can post-translational modifications of MJ0332.1 be analyzed?
Post-translational modifications (PTMs) can significantly impact protein function and may be particularly important in archaea:
| PTM Analysis Method | Application | Sensitivity | Sample Requirements |
|---|---|---|---|
| Mass Spectrometry | Comprehensive PTM identification | High (femtomole) | Purified protein, enzymatic digestion |
| Western Blotting | Detection of specific PTMs | Moderate | Specific antibodies against PTMs |
| Mobility Shift Assays | Detection of large PTMs | Low | Purified protein |
| Chemical Derivatization | Targeting specific PTM types | Variable | Depends on modification |
For comprehensive PTM analysis of MJ0332.1:
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Express and purify the protein from both E. coli and native M. jannaschii
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Perform parallel analysis to identify archaeal-specific modifications
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Use a combination of proteases (trypsin, chymotrypsin, Glu-C) for maximal sequence coverage
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Apply enrichment strategies for specific PTMs (phosphopeptide enrichment, etc.)
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Perform LC-MS/MS analysis with high-resolution instruments
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Use database search algorithms with archaeal-specific PTM options
Based on studies of other M. jannaschii proteins, potential PTMs to investigate include:
