Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1155.2 (MJ1155.2)

What expression systems are optimal for producing recombinant MJ1155.2?

Escherichia coli is the predominant expression system used for producing recombinant MJ1155.2, as evidenced by commercially available preparations of this protein . For effective expression, a His-tag is commonly incorporated to facilitate purification through affinity chromatography. The E. coli system offers several advantages for expressing archaeal proteins, including:

• Rapid growth and high cell density cultivation

• Well-established genetic manipulation protocols

• Compatibility with various induction systems for controlled expression

• Scalability for different research requirements

When expressing MJ1155.2, researchers should consider optimizing the following parameters:

For thermostable proteins like MJ1155.2, expression conditions might benefit from applying full factorial design experiments to systematically identify optimal conditions across multiple variables simultaneously .

How should recombinant MJ1155.2 be stored for optimal stability?

Recombinant MJ1155.2 should be stored in a Tris-based buffer with 50% glycerol at -20°C for routine laboratory use, or at -80°C for extended storage periods . The high glycerol content prevents freeze-thaw damage to the protein structure. Researchers should note the following storage recommendations:

• Avoid repeated freeze-thaw cycles as they can significantly degrade protein integrity

• Prepare working aliquots that can be stored at 4°C for up to one week

• For long-term storage beyond one month, maintain the protein at -80°C

• Consider the addition of reducing agents if the protein contains cysteine residues that may form disulfide bridges

What experimental approaches are recommended for characterizing the function of MJ1155.2?

Characterizing the function of an uncharacterized protein like MJ1155.2 requires a multi-faceted approach combining computational predictions with experimental validation. Given the limited information available about this protein, researchers should consider implementing the following experimental strategy:

• Computational Analysis:

  • Perform sequence homology searches against characterized proteins

  • Conduct structural prediction using tools like AlphaFold

  • Analyze conserved domains and motifs

  • Assess evolutionary conservation across archaeal species

• Structural Studies:

  • X-ray crystallography or cryo-electron microscopy to determine 3D structure

  • Circular dichroism spectroscopy for secondary structure analysis

  • Nuclear magnetic resonance for solution structure determination

• Functional Assays:

  • Gene knockout or silencing in M. jannaschii (if genetic tools available)

  • Heterologous expression followed by phenotypic analysis

  • Protein-protein interaction studies using pull-down assays, co-immunoprecipitation, or yeast two-hybrid systems

  • Metabolomics analysis comparing wild-type and knockout strains

• Physiological Context Analysis:

  • Expression profiling under different growth conditions

  • Subcellular localization studies

  • Metabolic pathway analysis

A full factorial design approach would be particularly valuable when testing multiple experimental conditions simultaneously, as it would allow researchers to identify potential interaction effects between variables that might influence MJ1155.2 function .

How can comparative genomics approaches help elucidate the function of MJ1155.2?

Comparative genomics represents a powerful approach for inferring the function of uncharacterized proteins like MJ1155.2. Since direct experimental characterization data is limited, researchers can leverage evolutionary relationships and genomic context to gain functional insights.

The following methodological framework is recommended:

• Ortholog Identification:

  • Identify MJ1155.2 orthologs across archaeal and bacterial genomes

  • Construct phylogenetic trees to analyze evolutionary relationships

  • Determine if orthologs exist in organisms with different ecological niches

• Synteny Analysis:

  • Examine the genomic neighborhood of MJ1155.2 in M. jannaschii

  • Compare gene arrangements with related organisms

  • Identify consistently co-located genes that may participate in the same biological process

• Co-expression Analysis:

  • Analyze transcriptomic data to identify genes with similar expression patterns

  • Look for coordinated regulation that suggests functional relationships

• Domain Architecture Analysis:

  • Compare the domain organization of MJ1155.2 with characterized proteins

  • Identify conserved sequence motifs that might indicate function

By integrating these comparative genomics approaches, researchers can develop testable hypotheses about the biological role of MJ1155.2, even in the absence of direct experimental evidence.

What are the challenges in studying the interaction partners of MJ1155.2?

Studying the interaction partners of an uncharacterized protein from a hyperthermophilic archaeon presents several unique challenges that researchers must address through specialized methodological approaches.

The primary challenges include:

• Thermostability Requirements: Standard protein interaction assays are typically optimized for mesophilic conditions (25-37°C), whereas interactions involving proteins from hyperthermophiles like M. jannaschii may require significantly higher temperatures to maintain native conformations and interactions.

• Expression System Limitations: Heterologous expression in E. coli may not provide the appropriate cellular environment or post-translational modifications necessary for authentic interactions.

• Limited Knowledge Base: The scarcity of characterized proteins from M. jannaschii creates a significant barrier to identifying potential interaction partners through bioinformatic approaches.

• Technical Challenges:

  • Traditional pull-down assays may require modification for thermostable proteins

  • Yeast two-hybrid systems may not accurately represent archaeal protein interactions

  • Mass spectrometry-based approaches may be complicated by sample preparation requirements

To overcome these challenges, researchers should consider the following methodological strategies:

• Modified Pull-down Assays:

  • Use thermostable affinity tags

  • Perform interaction studies at elevated temperatures

  • Include archaeal-specific lipids or cofactors that might be required for interaction

• Crosslinking Mass Spectrometry:

  • Apply in vivo crosslinking in native M. jannaschii cultures

  • Use MS-compatible crosslinkers to capture transient interactions

  • Analyze data with specialized algorithms designed for crosslinking studies

• Reconstituted Systems:

  • Develop liposome-based systems incorporating archaeal lipids

  • Reconstitute potential interaction complexes in vitro

  • Use microscale thermophoresis or surface plasmon resonance at elevated temperatures

• Computational Predictions:

  • Employ structure-based docking simulations

  • Use co-evolution analysis to predict interaction interfaces

  • Apply machine learning approaches trained on archaeal protein-protein interactions

These methodological approaches should be applied systematically to overcome the inherent challenges in studying the interaction network of MJ1155.2.

How can full factorial design be applied to optimize expression and purification of MJ1155.2?

Full factorial design represents an ideal experimental approach for optimizing the expression and purification of recombinant MJ1155.2, as it enables systematic evaluation of multiple factors simultaneously, including their interactions, which simple one-factor-at-a-time experiments cannot detect .

For MJ1155.2 expression optimization, the following full factorial design can be implemented:

• Factor Selection:

  • Temperature (20°C, 30°C, 37°C)

  • Inducer concentration (0.1 mM, 0.5 mM, 1.0 mM IPTG)

  • Media composition (LB, TB, autoinduction media)

  • Host strain (BL21(DE3), Rosetta, Arctic Express)

  • Induction time (4 hours, 8 hours, overnight)

• Experimental Design Matrix:
  A complete full factorial design with these factors would require 3^5 = 243 experiments. Researchers may consider a fractional factorial design to reduce the number of experiments while still capturing main effects and critical interactions.

  Experiment	Temperature	IPTG	Media	Strain	Time	Yield (mg/L)	Solubility (%)
  1	20°C	0.1	LB	BL21	4h	Measured	Measured
  2	20°C	0.1	LB	BL21	8h	Measured	Measured
  ...	...	...	...	...	...	Measured	Measured
  243	37°C	1.0	Auto	Arctic	O/N	Measured	Measured

• Response Variables:

  • Protein yield (mg per liter of culture)

  • Protein solubility (percentage of total expressed protein)

  • Protein purity after initial purification

  • Biological activity (if an assay is available)

• Statistical Analysis:

  • Analysis of variance (ANOVA) to determine significant factors and interactions

  • Response surface methodology to model the relationship between factors and responses

  • Optimization algorithms to identify the optimal combination of conditions

Full factorial design allows researchers to understand not only which factors individually affect protein expression but also how these factors interact with each other . For example, the optimal temperature might differ depending on the host strain used, or the effect of IPTG concentration might depend on the induction time - relationships that would not be apparent in simpler experimental designs.

The insights gained from this systematic approach can dramatically improve both the quantity and quality of the recombinant MJ1155.2 protein obtained, creating a solid foundation for subsequent structural and functional studies.