Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1249.1 (MJ1249.1)
Description
Introduction to Methanocaldococcus jannaschii and MJ1249.1
Methanocaldococcus jannaschii is an archaeal organism first classified as Methanococcus jannaschii and referenced in multiple strain collections including ATCC 43067, DSM 2661, JAL-1, JCM 10045, and NBRC 100440 . This archaeon has been a subject of scientific interest since its genome was sequenced, revealing numerous proteins with unknown functions, including the MJ1249.1 protein examined in this review. The organism serves as an important model for studying archaeal biology and extremophilic adaptations.
The protein MJ1249.1 remains uncharacterized in terms of its precise biological function, despite being identified and cataloged in protein databases. This lack of functional characterization is common among many archaeal proteins, particularly those from extremophiles like M. jannaschii. The recombinant form of this protein has been produced to enable structural and functional studies that may eventually elucidate its role in archaeal biology.
Recombinant protein technology has proven essential for studying proteins from difficult-to-culture organisms such as M. jannaschii. Through heterologous expression systems, typically E. coli, researchers can produce sufficient quantities of these proteins for biochemical, structural, and functional analyses. This approach circumvents the challenges of directly isolating proteins from archaeal sources that require specialized growth conditions .
Context Within Archaeal Genomics
The genome of M. jannaschii contains numerous genes encoding proteins with unknown functions. These uncharacterized proteins represent potential insights into unique archaeal biochemical pathways, particularly those related to methanogenesis and adaptation to extreme environments. MJ1249.1 is one such protein that may contribute to the distinct biology of this archaeon.
Genomic analysis of M. jannaschii has revealed multiple hypothetical proteins that await functional characterization. For example, MJ1247, another hypothetical protein from the same organism, has been structurally determined and its function inferred as 3-hexulose-6-phosphate isomerase based on structural homology and biochemical assays . This suggests that similar approaches might eventually reveal the function of MJ1249.1.
Sequence Analysis and Domain Predictions
While specific structural studies on MJ1249.1 are not detailed in the provided search results, sequence analysis can provide insights into potential structural features. The high proportion of hydrophobic amino acids in certain regions suggests multiple membrane-spanning domains. The sequence also contains charged residues likely positioned at membrane interfaces or in soluble domains.
Sequence analysis tools often used in protein structure prediction would likely identify this protein as having multiple transmembrane helices based on the distribution of hydrophobic amino acids. These predictions would need experimental validation through techniques such as crystallography or NMR spectroscopy, similar to approaches used for other M. jannaschii proteins like MJ1247 .
Expression and Production Systems
The recombinant MJ1249.1 protein is produced using in vitro E. coli expression systems, as indicated in the product specifications . This approach is common for archaeal proteins, especially those from extremophiles like M. jannaschii, whose native growth conditions are difficult to replicate in laboratory settings.
Expression Vector Considerations
While the specific expression vector used for commercial production of MJ1249.1 is not detailed in the search results, general principles of recombinant protein expression in E. coli would apply. The choice of expression vector significantly impacts protein yield and quality. Research on recombinant protein expression indicates that the balance between vector copy number and promoter strength is crucial for optimal protein production .
High copy number plasmids derived from pMB1 origins (500-700 copies/cell) combined with strong promoters can lead to high protein expression but may also increase metabolic burden on the host cells. In contrast, lower copy number vectors like those with p15A origins (approximately 10 copies/cell) might result in lower but more stable expression . This balance would be particularly important for membrane proteins like MJ1249.1, which can be challenging to express in functional forms.
Tags and Purification Strategy
The commercially available recombinant MJ1249.1 protein includes an N-terminal 10xHis tag . This affinity tag facilitates purification through immobilized metal affinity chromatography (IMAC) and may enhance protein solubility. The choice of an N-terminal tag might be strategic based on the protein's topology, suggesting that the N-terminus is likely to be accessible in the folded protein.
Table 1: Specifications of Recombinant MJ1249.1 Protein
Production Challenges
The expression of archaeal membrane proteins in bacterial hosts presents several challenges. Differences in membrane composition, protein folding machinery, and codon usage between archaea and bacteria can affect the yield and functionality of recombinant proteins. Studies on recombinant protein expression highlight that the metabolic burden associated with high-level expression of foreign genes can significantly impact host cell growth and protein production .
For transmembrane proteins like MJ1249.1, ensuring proper folding and membrane insertion in the heterologous host is particularly challenging. Optimizing expression conditions, including temperature, induction timing, and host strain selection, becomes crucial for obtaining functional protein. The commercial availability of recombinant MJ1249.1 suggests that these challenges have been at least partially addressed for this protein.
Shelf Life Considerations
The shelf life of recombinant proteins depends on various factors including storage conditions, buffer composition, and the intrinsic stability of the protein. Generally, liquid formulations have a shelf life of approximately 6 months when stored at -20°C or -80°C, while lyophilized forms can remain stable for up to 12 months under the same conditions .
For MJ1249.1 specifically, the high glycerol content in the storage buffer likely contributes to extended stability by preventing ice crystal formation during freezing and providing a stabilizing environment for the hydrophobic regions of this transmembrane protein.
Table 2: Storage and Stability Information for Recombinant MJ1249.1
| Parameter | Recommendation | Notes |
|---|---|---|
| Short-term Storage | -20°C | Standard condition for regular use |
| Long-term Storage | -20°C or -80°C | For extended preservation |
| Working Storage | 4°C | Up to one week |
| Storage Buffer | Tris-based with 50% glycerol | Optimized for this protein |
| Freeze-Thaw Cycles | Minimize | Repeated cycles not recommended |
| Liquid Form Shelf Life | ~6 months at -20°C/-80°C | Dependent on multiple factors |
| Lyophilized Form Shelf Life | ~12 months at -20°C/-80°C | If available in this format |
Predicted Functional Roles
Based on its transmembrane nature, MJ1249.1 might function in processes such as:
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Membrane transport of ions or small molecules
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Signal transduction across archaeal cell membranes
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Maintenance of membrane structure or integrity in extreme environments
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Energy conservation in methanogenic pathways
The approach used to infer the function of other M. jannaschii proteins could serve as a model for MJ1249.1. For example, the crystal structure of MJ1247 combined with sequence analysis and biochemical assays led to its identification as a 3-hexulose-6-phosphate isomerase involved in the dissimilatory ribulose monophosphate cycle . Similar multidisciplinary approaches might reveal the function of MJ1249.1.
Research Applications
The availability of recombinant MJ1249.1 enables various research applications:
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Structural studies using X-ray crystallography or NMR spectroscopy
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Functional characterization through biochemical assays
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Investigation of membrane protein adaptation to extreme conditions
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Comparative studies with homologous proteins from other organisms
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Development of antibodies for localization studies
These applications could contribute to our understanding of archaeal biology and potentially reveal novel biochemical pathways or mechanisms of adaptation to extreme environments.
Comparative Analysis
While direct comparisons between MJ1249.1 and other proteins are not provided in the search results, approaches for such comparisons can be inferred from methodologies used for protein structure classification and analysis.
Product Specs
Note: While we preferentially ship the format currently in stock, we are happy to accommodate your specific format needs. Please indicate your preference when placing the order, and we will do our best to fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
KEGG: mja:MJ_1249.1
STRING: 243232.MJ_1249.1
Q&A
What is Methanocaldococcus jannaschii and why is it significant for research?
Methanocaldococcus jannaschii is a hyperthermophilic methanogenic archaeon that has become an important model organism for studying archaeal biology and metabolism. This organism has been the subject of comprehensive metabolic reconstruction efforts, which have predicted the existence of 609 metabolic reactions assembled into 113 metabolic pathways and 17 super-pathways . As one of the first archaeal genomes to be completely sequenced, M. jannaschii provides crucial insights into archaeal metabolism and evolution across different domains of life . Its proteins are particularly interesting due to their adaptation to extreme environments, making them valuable for both fundamental research and potential biotechnological applications.
What are the basic characteristics of the MJ1249.1 protein?
MJ1249.1 is an uncharacterized protein from M. jannaschii consisting of 166 amino acids. Its complete amino acid sequence is: MRGINPVLFKISFLLIILVSLILSLFYYNFLFAFLLSFILFGITWAYCYIKIESTDKGKLLYEIKRPGIETLRFLFILMIISVFIKSLLHSNSFFPYISFLLSNLILGLVLFDDYILGNPTIKFYEKGVVFDRVAFYNWEELDIKEDEGYLKIKIKYYPKEIMYKK . The protein has been recombinantly expressed with an N-terminal His-tag in E. coli expression systems . The recombinant product is typically supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE . Sequence analysis suggests it may contain multiple hydrophobic regions, potentially indicating a membrane-associated protein.
How does MJ1249.1 compare to other uncharacterized proteins in M. jannaschii?
M. jannaschii contains several uncharacterized proteins, including MJ0379 and MJ1249.1 . While these proteins share the common feature of being uncharacterized, they differ in their molecular characteristics. Unlike other characterized M. jannaschii proteins such as MJ1225, which contains CBS domains and has been identified as an archaeal homologue of γ-AMPK , the functional domains and potential roles of MJ1249.1 remain to be elucidated. The identification and characterization of such proteins contribute to a more comprehensive understanding of archaeal proteomes and metabolic networks.
What expression systems are optimal for producing recombinant M. jannaschii proteins?
Recombinant M. jannaschii proteins, including MJ1249.1, are typically expressed in E. coli expression systems due to the challenges of culturing extremophilic archaea in laboratory settings . Based on protocols established for other M. jannaschii proteins, such as MJ1225, successful expression has been achieved using E. coli strain BL21Star (DE3) with cloning into vectors such as pET101/D-TOPO . For MJ1225, expression has been reported without IPTG induction, with cells grown in Luria-Bertani medium containing appropriate antibiotics (e.g., 100 μg/ml ampicillin) for 12 hours at 310 K . Similar approaches could be adapted for MJ1249.1, with optimization of expression conditions based on protein-specific characteristics.
What purification strategies are effective for recombinant archaeal proteins?
Effective purification of recombinant archaeal proteins often leverages their inherent thermostability. For example, purification protocols for MJ1225 have employed the following strategy:
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Heat-shock treatment (348 K for 30 min) to eliminate heat-labile E. coli proteins
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Ion-exchange chromatography (HiTrap Q column)
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Affinity chromatography (HiTrap Blue column)
For His-tagged proteins like recombinant MJ1249.1, immobilized metal affinity chromatography (IMAC) would be an appropriate initial purification step. The purification protocol should be optimized based on the specific properties of MJ1249.1, potentially including appropriate buffer conditions (e.g., 50 mM HEPES pH 7.0, 1 mM EDTA, 1 mM β-mercaptoethanol) .
What are the optimal storage and handling conditions for recombinant MJ1249.1?
The recombinant MJ1249.1 protein requires specific storage and handling conditions to maintain stability and functionality. According to product specifications, the lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, the addition of glycerol to a final concentration of 5-50% (with 50% being the default recommendation) followed by aliquoting and storage at -20°C/-80°C is advised . It is important to avoid repeated freeze-thaw cycles, and working aliquots can be maintained at 4°C for up to one week . The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .
What crystallization approaches are suitable for archaeal proteins like MJ1249.1?
Based on successful crystallization of other M. jannaschii proteins, such as MJ1225, the following approaches may be suitable for MJ1249.1:
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Concentration of purified protein to a high level (e.g., 160 mg/ml for MJ1225)
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Crystallization using the hanging-drop vapor-diffusion method
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Optimization of crystallization conditions specific to the protein's properties
For MJ1225, crystals were obtained that diffracted to 2.3 Å resolution using synchrotron radiation, belonging to space group H32 with unit-cell parameters a = b = 108.95, c = 148.08 Å, α = β = 90.00, γ = 120.00° . Similar approaches could be adapted for MJ1249.1, although its potential membrane protein characteristics might require specialized crystallization methods such as lipidic cubic phase techniques or detergent-based crystallization approaches.
How can mass spectrometry be applied to verify the identity and modifications of MJ1249.1?
Mass spectrometric analysis of MJ1249.1 could follow protocols similar to those employed for MJ1225:
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In-gel tryptic digestion of SDS-PAGE bands containing the protein
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Sample preparation with appropriate matrix solution (e.g., α-cyano-4-hydroxycinnamic acid)
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Analysis by MALDI-TOF mass spectrometry
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Database searching using tools like Mascot to confirm protein identity
The mass spectrometry parameters would typically include considerations for potential modifications such as carbamidomethylation of cysteine (complete) and oxidation of methionine (partial) . This approach can verify both the identity of the recombinant protein and confirm that no mutations or unexpected modifications have occurred during expression.
What computational tools can predict the structure and potential function of MJ1249.1?
Several computational approaches can be employed to predict the structure and potential function of uncharacterized proteins like MJ1249.1:
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Homology modeling using proteins with similar sequences as templates
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Ab initio structure prediction tools such as AlphaFold
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Transmembrane topology prediction algorithms (particularly relevant given the likely membrane-associated nature of MJ1249.1)
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Functional prediction through metabolic pathway analysis similar to the PathoLogic approach used for M. jannaschii metabolic reconstruction
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Protein family classification and conserved domain analysis
These computational predictions can guide experimental approaches and provide initial hypotheses about the protein's role in archaeal biology.
How can functional characterization of uncharacterized proteins like MJ1249.1 be approached?
Functional characterization of uncharacterized archaeal proteins requires a multi-faceted approach:
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Bioinformatic analysis: Sequence comparison, structural prediction, and genomic context analysis
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Expression pattern analysis: Determining conditions under which the protein is expressed in M. jannaschii
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Protein-protein interaction studies: Identifying binding partners that might suggest function
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Structural determination: X-ray crystallography or NMR analysis following methods similar to those used for MJ1225
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Biochemical assays: Testing for specific activities based on structural predictions or genomic context
Integrating these approaches can provide complementary lines of evidence about the protein's function and biological role.
What is known about the potential membrane association of MJ1249.1?
The amino acid sequence of MJ1249.1 suggests it may be a membrane-associated protein, based on the presence of multiple hydrophobic regions that could form transmembrane domains . While the search results don't provide direct experimental evidence of membrane association, sequence analysis tools can predict the number and orientation of potential transmembrane helices. For membrane proteins from extremophiles like M. jannaschii, special attention should be given to features that might contribute to stability in extreme conditions, such as increased hydrophobicity and specific amino acid compositions that favor stability at high temperatures.
What evolutionary insights can be gained from studying uncharacterized archaeal proteins?
Uncharacterized proteins like MJ1249.1 can provide valuable insights into archaeal evolution and the adaptation of organisms to extreme environments. Comparative analysis with homologs in other archaea, bacteria, or eukaryotes can reveal evolutionary patterns and domain-specific adaptations. M. jannaschii proteins have been studied to facilitate comparative analyses of archaeal metabolism and evolution across different organisms and domains of life . The identification of conserved regions or unique features in proteins like MJ1249.1 can highlight functional constraints and evolutionary innovations in archaeal lineages.
How does the genomic context of MJ1249.1 compare to related genes in other archaeal species?
Genomic context analysis—examining the organization of genes surrounding MJ1249.1 in the M. jannaschii genome and comparing this arrangement to related species—can provide clues about functional relationships and evolutionary history. While the search results don't provide specific information about the genomic context of MJ1249.1, this type of analysis has been valuable for other archaeal proteins. Conservation of gene neighborhoods across species often indicates functional relationships, such as involvement in the same metabolic pathway or protein complex.
What protein domains or structural features might be conserved between MJ1249.1 and other archaeal proteins?
While the search results don't identify specific conserved domains in MJ1249.1, comparative analysis with other archaeal proteins could reveal shared structural features or functional motifs. For example, MJ1225 has been characterized as containing CBS domains and serving as an archaeal homologue of γ-AMPK . Similar detailed characterization of MJ1249.1 could identify previously unrecognized domains or structural motifs that are conserved across archaeal species, potentially providing clues about function and evolutionary history.
