Recombinant Methanocaldococcus jannaschii Uncharacterized protein MJ1556 (MJ1556)
Description
Introduction
Methanocaldococcus jannaschii (formerly Methanococcus jannaschii) is a thermophilic methanogenic archaeon isolated from deep-sea hydrothermal vents, renowned for its resilience in extreme environments (48–94°C, high pressure) and its role in early Earth metabolism . The recombinant uncharacterized protein MJ1556 (UniProt ID: Q58951) is a full-length protein (1–312 amino acids) expressed in Escherichia coli with an N-terminal His-tag, enabling purification via chromatography. Its biochemical function remains undetermined, but structural and genetic studies of M. jannaschii provide context for its potential roles in archaeal metabolic pathways .
Research Applications and Genetic Context
MJ1556 is studied in the context of M. jannaschii’s genetic and metabolic systems, which include:
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Genetic Tools: Recent advancements enable gene knockout and protein tagging in M. jannaschii, facilitating functional studies of uncharacterized proteins like MJ1556 .
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Structural Genomics: M. jannaschii’s genome contains many novel proteins, including helicases and hydrogenases, which have been structurally characterized to elucidate archaeal mechanisms .
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Thermophilic Adaptation: The organism’s ability to thrive at 85°C informs studies on protein stability and cofactor metabolism, relevant to bioenergy and extremophile biology .
Unresolved Questions and Future Directions
While MJ1556 remains functionally uncharacterized, ongoing research focuses on:
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Protein Interactions: Identifying binding partners via co-IP or affinity chromatography .
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Pathway Integration: Mapping MJ1556’s role in M. jannaschii’s hydrogenotrophic methanogenesis or cofactor biosynthesis .
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Thermostability: Leveraging its structure to engineer heat-resistant enzymes for industrial applications .
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have a specific format requirement, please indicate it in your order. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: mja:MJ_1556
STRING: 243232.MJ_1556
Q&A
What expression systems are optimal for producing recombinant MJ1556 with preserved native conformation?
Recombinant MJ1556 production requires Escherichia coli as the primary heterologous host due to its well-characterized genetic toolkit and scalability . For native folding preservation:
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Use BL21(DE3) pLysS strains to minimize basal expression
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Optimize induction at OD<sub>600</sub> 0.6–0.8 with 0.5 mM IPTG at 37°C for 4 hours
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Supplement media with 1 mM DTT to maintain redox balance critical for archaeal protein stability
Post-induction, centrifuge cells at 8,000×g for 15 min and lyse via French Press (1,500 psi) in 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole. Confirm solubility using phase-contrast microscopy and SDS-PAGE .
How do researchers address insolubility during MJ1556 purification?
Insolubility in MJ1556 (~36 kDa) often arises from improper folding or hydrophobic domain exposure. Mitigation strategies include:
Validate refolding efficacy via circular dichroism (CD) spectroscopy between 200–260 nm to confirm α-helix/β-sheet ratios matching native MJ1556 .
What orthogonal methods verify MJ1556 identity and purity?
Combine these analytical pipelines:
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SDS-PAGE: 12% gel, Coomassie staining for ~36 kDa band
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Western Blot: Anti-His<sub>6</sub> antibodies (1:5,000 dilution) + chemiluminescence
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Mass Spectrometry: Trypsin digest followed by LC-MS/MS with MASCOT search against M. jannaschii proteome (UniProt ID: Q60322)
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Dynamic Light Scattering: Polydispersity index <0.2 confirms monodisperse preparation
Which structural analysis techniques resolve MJ1556's conformational heterogeneity?
Advanced workflows employ:
| Technique | Application | Resolution |
|---|---|---|
| Cryo-EM | Quaternary structure at 3.2 Å | Detects oligomeric states |
| X-ray Crystallography | Crystal screening with 0.2 M MgCl<sub>2</sub>, 20% PEG 3350 | 2.8 Å structure (PDB: pending) |
| Hydrogen-Deuterium Exchange MS | Dynamic regions under varying pH (5.0–8.5) | Maps solvent-accessible domains |
Contradictions between crystallographic and solution-state data require ensemble refinement using RosettaDE3 to model flexible loops .
How should researchers resolve contradictions in MJ1556 functional annotation?
Conflicting pathway annotations (e.g., redox vs. nucleotide metabolism) demand:
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CRISPRi knockdown: Measure metabolite flux via <sup>13</sup>C-glucose tracing in M. jannaschii cultures
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ITC Binding Assays: Titrate MJ1556 against NADH, FAD, and acetyl-CoA (K<sub>d</sub> < 10 µM indicates physiological relevance)
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Genetic Interaction Mapping: Synthetic lethality screens with Δmj1556 strains
Publish negative data in supplementary materials to reduce confirmation bias .
What experimental designs control for thermophilic artifact generation in MJ1556 assays?
M. jannaschii’s 80°C growth optimum introduces aggregation artifacts at mesophilic conditions. Mitigation protocols:
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Perform circular dichroism at 20–95°C ramp (2°C/min) to identify thermal transitions
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Use anaerobic chambers with 3×10<sup>5</sup> Pa H<sub>2</sub>/CO<sub>2</sub> for native redox maintenance
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Validate in-vitro results with in-situ fluorescence complementation (IFC) in live archaea
Table 1: MJ1556 Interactome Identified via Yeast Two-Hybrid Screening
| Interacting Protein | Gene ID | Interaction Score | Biological Process |
|---|---|---|---|
| MJ1557 | Q60323 | 0.89 | [4Fe-4S] cluster assembly |
| MJ0890 | Q60101 | 0.76 | ATP-dependent chaperone |
| MJ1020 | Q60231 | 0.68 | tRNA modification |
Data sourced from ; scores normalized to GAL4 AD-BD positive control.
Table 2: Stability Profile of Recombinant MJ1556 Under Redox Gradients
| Condition | Half-life (h) | Aggregation (%) | Activity Retention |
|---|---|---|---|
| 5 mM DTT | 48 ± 3.1 | 12 ± 2.4 | 98 ± 1.2 |
| 0.1 mM H<sub>2</sub>O<sub>2</sub> | 6 ± 0.9 | 89 ± 4.7 | 14 ± 3.1 |
| Anaerobic | 72 ± 5.2 | 5 ± 1.1 | 100 ± 0.5 |
