Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ0007 (MJ0007)

What is Methanocaldococcus jannaschii and why is it significant for research?

Methanocaldococcus jannaschii is an extremophilic archaeon originally isolated from submarine hydrothermal vents in the East Pacific Rise . It has significant research importance due to several distinctive characteristics:

• It is a hyperthermophile with optimal growth at 80°C under strictly anaerobic conditions

• It demonstrates remarkable metabolic efficiency with a minimum generation time of only 26 minutes

• It was the first archaeal organism to have its complete genome sequenced, providing crucial insights into archaeal biology and evolution

• Its proteins exhibit exceptional thermostability, making them valuable models for studying protein structure-function relationships under extreme conditions

Researchers cultivating M. jannaschii should note that cultures grow rapidly to stationary phase, thus requiring regular monitoring rather than overnight incubation . The organism is classified as Risk Group 1, indicating minimal hazard under standard laboratory conditions .

What is currently known about the uncharacterized protein MJ0007?

MJ0007 is an uncharacterized protein from M. jannaschii (strain ATCC 43067/DSM 2661/JAL-1/JCM 10045/NBRC 100440) with the following characteristics:

• It is annotated as a "putative dehydratase subunit"

• The full-length protein consists of 373 amino acids (expression region 1-373)

• Its complete amino acid sequence is available (as shown in the search results)

• Despite its annotation, the precise biochemical function and physiological role remain experimentally unconfirmed

• The protein may be involved in metabolic pathways related to M. jannaschii's adaptation to extreme environments

For researchers seeking to investigate this protein, recombinant forms are available with an N-terminal 10xHis-tag for purification purposes . The uncharacterized status of MJ0007 presents opportunities for novel discoveries regarding archaeal metabolism and adaptation strategies.

What experimental design considerations are important when working with recombinant MJ0007?

When designing experiments with recombinant MJ0007, consider implementing a factorial or randomized complete block design to account for multiple variables that may affect protein behavior:

• Temperature variables: Test protein stability and activity across a range of temperatures (ambient to 80°C) to reflect M. jannaschii's natural environment

• Buffer composition: Evaluate performance in different buffer systems, considering:

  • pH ranges (typically 6.5-8.5)

  • Salt concentrations (reflecting marine conditions)

  • Reducing agents (to maintain anaerobic conditions)

• Experimental controls:

  • Positive controls: Well-characterized dehydratases from related organisms

  • Negative controls: Heat-denatured protein or site-directed mutants

A factorial design allows assessment of both main effects and interactions between variables . For example:

Power analysis should be conducted prior to experimentation to determine appropriate sample sizes, particularly when working with potentially variable recombinant protein preparations .

What expression systems are most effective for producing recombinant M. jannaschii proteins?

While E. coli remains the most commonly used expression system for M. jannaschii proteins like MJ0007 , researchers should consider several methodological approaches:

• E. coli expression optimization:

  • Use codon-optimized sequences to overcome codon usage bias between archaea and bacteria

  • Employ specialized strains like Rosetta or BL21(DE3) pLysS to provide rare tRNAs and reduce toxicity

  • Test induction at lower temperatures (15-25°C) to enhance proper folding

  • Co-express archaeal chaperones to assist folding of hyperthermophilic proteins

• Alternative expression systems:

  • Yeast systems like Pichia pastoris for proteins requiring eukaryotic post-translational modifications

  • Cell-free protein synthesis systems supplemented with archaeal components

  • Archaeal host systems for proteins requiring specific archaeal machinery

• Expression parameters table:

When evaluating expression systems, researchers should implement split-plot experimental designs to efficiently assess multiple variables while minimizing resource use .

What purification challenges are specific to MJ0007 and how can they be addressed?

Purifying recombinant MJ0007 presents several considerations unique to archaeal hyperthermophilic proteins:

• Heat treatment advantage: Perform initial purification step at 60-70°C to denature most E. coli proteins while preserving the thermostable MJ0007, significantly reducing contaminants

• His-tag purification strategy:

  • The N-terminal 10xHis-tag on recombinant MJ0007 enables efficient immobilized metal affinity chromatography (IMAC)

  • Consider using TALON® resin (cobalt-based) rather than Ni-NTA for higher specificity

  • Test various imidazole concentrations in wash buffers to minimize non-specific binding

• Recommended purification workflow:

  • Heat treatment (65°C, 20 minutes)

  • IMAC chromatography

  • Size exclusion chromatography for final polishing

  • Quality assessment via SDS-PAGE and mass spectrometry

• Storage considerations:

  • Short-term: Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0

  • Long-term: Lyophilize from the same buffer formulation

  • Avoid repeated freeze-thaw cycles to maintain protein integrity

For experimental design, implement a randomized complete block design when testing multiple purification conditions to control for batch-to-batch variation .

What bioinformatic approaches can predict potential functions of MJ0007?

Investigating the function of uncharacterized proteins like MJ0007 requires a comprehensive bioinformatic strategy:

• Sequence-based analysis:

  • BLAST against characterized proteins across all domains of life

  • Multiple sequence alignment with putative dehydratases to identify conserved catalytic residues

  • Domain architecture analysis using PFAM, SMART, and CDD

  • Genomic context analysis to identify operons or gene clusters providing functional clues

• Structure-based prediction:

  • Generate 3D structure predictions using AlphaFold2 or RoseTTAFold

  • Perform structural alignment against known dehydratases

  • Identify potential active site pockets and substrate-binding regions

  • Conduct molecular docking with potential substrates

• Integrative approaches:

  • Metabolic pathway reconstruction for M. jannaschii

  • Gene co-expression analysis using available transcriptomic data

  • Phylogenetic profiling to identify co-evolving genes

When implementing these approaches, use both positive controls (known dehydratases) and negative controls (functionally unrelated proteins) to validate predictions. Document all software versions, parameters, and statistical thresholds to ensure reproducibility.

How can researchers design activity assays for the putative dehydratase function of MJ0007?

Designing robust activity assays for an uncharacterized putative dehydratase requires a systematic approach:

• Substrate screening strategy:

  • Test a panel of potential dehydratase substrates based on M. jannaschii metabolic pathways

  • Employ both direct and coupled enzyme assays to detect product formation

  • Utilize isotope-labeled substrates to confirm reaction specificity

• Assay optimization factors table:

• Detection methods:

  • Spectrophotometric assays for chromogenic products

  • HPLC or LC-MS for direct product identification

  • NMR for structural confirmation of products

  • Polarimetric methods for stereospecific reactions

• Control experiments:

  • Heat-denatured MJ0007 (negative control)

  • Site-directed mutants of predicted catalytic residues

  • Characterized dehydratases from related organisms (positive control)

Implement a factorial experimental design to efficiently test multiple assay conditions while identifying potential interactions between factors . Begin with a broad screening approach, then refine conditions based on initial results.

What strategies can address the challenges of studying protein-protein interactions involving MJ0007?

Investigating protein-protein interactions for MJ0007 requires specialized approaches due to its archaeal origin and thermophilic nature:

• In vitro interaction methods:

  • Pull-down assays using the His-tagged MJ0007 as bait

  • Surface plasmon resonance (SPR) with temperature-controlled flow cells

  • Isothermal titration calorimetry (ITC) capable of high-temperature measurements

  • Microscale thermophoresis (MST) for label-free interaction analysis

• Experimental design considerations:

  • Test interactions at both standard (25°C) and physiological (80°C) temperatures

  • Include appropriate buffer controls with matching ionic strength

  • Implement a randomized block design to control for protein batch variation

• Candidate interactor identification:

  • Genomic context analysis for genes adjacent to MJ0007

  • Structural modeling to predict potential interaction interfaces

  • Cross-linking mass spectrometry (XL-MS) with M. jannaschii lysates

  • Bacterial two-hybrid systems modified for archaeal proteins

• Validation approach:

  • Employ multiple complementary techniques

  • Confirm specificity with competitive binding assays

  • Verify biological relevance through mutagenesis of predicted interface residues

When analyzing interaction data, implement robust statistical analysis accounting for technical and biological replicates, with clear distinction between specific and non-specific interactions through appropriate negative controls .

How can structural biology approaches be optimized for thermostable proteins like MJ0007?

Thermostable proteins from M. jannaschii present both challenges and opportunities for structural biology:

• X-ray crystallography optimization:

  • Screen crystallization conditions at both ambient and elevated temperatures

  • Include stabilizing agents like trehalose in crystallization buffers

  • Consider in situ crystallization approaches to capture native conformations

  • Test both vapor diffusion and lipidic cubic phase methods

• Cryo-EM considerations:

  • Optimize grid preparation to minimize preferred orientation issues

  • Test protein stability in various buffer conditions during freezing

  • Consider collecting data at multiple temperatures to capture conformational diversity

• NMR spectroscopy approaches:

  • Develop thermally stable buffer systems compatible with long acquisition times

  • Consider selective isotopic labeling strategies to reduce spectral complexity

  • Test high-temperature NMR experiments to capture native-like dynamics

• Integrated structural biology workflow:

Design experiments using split-plot approaches to efficiently test multiple crystallization or sample preparation conditions while controlling for protein batch effects . Implement rigorous statistical validation of structural data using established metrics like R-factors, FSC curves, or RMSD values where appropriate.

What methodological approaches can determine MJ0007's role in extremophile adaptation?

Investigating MJ0007's potential role in M. jannaschii's adaptation to extreme environments requires integrating multiple experimental approaches:

• Comparative genomic analysis:

  • Analyze presence/absence of MJ0007 homologs across archaea with varying growth conditions

  • Examine sequence conservation patterns in relation to environmental factors

  • Identify potential coevolution with other proteins involved in stress response

• Expression analysis under stress conditions:

  • Design experiments to test MJ0007 expression under various stressors:

    • Temperature shifts (both above and below optimal 80°C)

    • Pressure variations (simulating hydrothermal vent conditions)

    • Oxidative stress

    • Nutrient limitation

• Functional characterization under extreme conditions:

  • Compare enzymatic activity across temperature range (25-95°C)

  • Assess stability under combined stressors using factorial design

  • Examine potential moonlighting functions under different conditions

• Structural analysis of adaptation mechanisms:

  • Identify structural features contributing to thermostability

  • Compare with mesophilic homologs to identify adaptation-specific modifications

  • Conduct molecular dynamics simulations at various temperatures

For such complex investigations, utilize nested experimental designs to account for hierarchical factors while maintaining statistical power . When comparing MJ0007 with homologs from different environments, implement appropriate phylogenetic corrections to account for evolutionary relationships rather than direct adaptation.