Recombinant Methanothermobacter thermautotrophicus Uncharacterized protein MTH_1250 (MTH_1250)
Description
Molecular and Functional Characteristics
MTH_1250 is an 86-amino acid protein (UniProt ID: O27318) with a predicted molecular weight of ~10 kDa. Its sequence is:
MMDILIIAEYTLLASLAVFSIAAVRIATRRNIRMGLVGISGLNIAIATILILINRMYGIG FCRDIAYALVLLGPVGTIAFARVLRG .
Key Features:
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Domain Structure: Predicted transmembrane regions, suggesting a role in membrane-associated processes .
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Conservation: Shares 38.7% amino acid identity with Methanococcus maripaludis EhbB, a homolog in the Ehb hydrogenase complex .
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Expression System: Produced in E. coli with an N-terminal His-tag for purification .
| Property | Details |
|---|---|
| Species | Methanothermobacter thermautotrophicus (strain ΔH) |
| Gene Locus | MTH_1250 |
| Protein Length | 86 residues |
| Purity | >90% (SDS-PAGE) |
| Storage | Lyophilized powder in Tris/PBS buffer with 6% trehalose (pH 8.0) at -80°C |
| Reconstitution | 0.1–1.0 mg/mL in sterile water with 50% glycerol for stability |
Role in Methanogenesis
MTH_1250 is a component of the Ehb hydrogenase complex, which generates low-potential electrons required for CO₂ assimilation in hydrogenotrophic methanogens. Key findings include:
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Genetic Context: The ehb operon in M. thermautotrophicus contains 17 genes, with MTH_1250 (annotated as ehbB) encoding a transmembrane protein critical for electron transport .
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Functional Homology: EhbB in M. maripaludis is essential for autotrophic growth under nutrient-limited conditions, suggesting a conserved role in energy metabolism .
Production Protocol
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Expression: Codon-optimized MTH_1250 is cloned into E. coli vectors and purified via nickel-affinity chromatography .
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Yield: Typical yields range from 0.1–1.0 mg/mL post-reconstitution .
Research Applications
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Structural Studies: Used in crystallography and NMR to resolve membrane protein architectures .
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Metabolic Engineering: Facilitates genetic manipulation of M. thermautotrophicus to study methanogenesis pathways .
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Biotechnological Potential: Serves as a template for hydrogenase engineering in bioenergy applications .
Comparative Genomics
Homologs of MTH_1250 across methanogens highlight its evolutionary conservation:
| Organism | Gene | % Identity | Function |
|---|---|---|---|
| M. thermautotrophicus | MTH_1250 | 100% | Transmembrane Ehb complex subunit |
| M. maripaludis | mmp1049 | 38.7% | Electron transport |
Challenges and Future Directions
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have specific format requirements, please indicate them when placing your order, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please communicate with us in advance, as additional charges 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: mth:MTH_1250
STRING: 187420.MTH1250
Q&A
What experimental approaches validate the structural integrity of recombinant MTH_1250?
Three-tier validation must precede functional studies:
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Mass spectrometry confirms molecular weight matches theoretical 10.2 kDa (MMDILIIAEYTLLASLAVFSIAAVRIATRRNIRMGLVGISGLNIAIATILILINRMYGIGFCRDIAYALVLLGPVGTIAFARVLRG sequence)
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Circular dichroism verifies secondary structure preservation under storage buffer conditions (Tris/PBS-based, 6% Trehalose, pH 8.0)
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Dynamic light scattering monitors aggregation status pre/post-reconstitution (recommended working concentration 0.1-1.0 mg/mL)
| Validation Parameter | Acceptable Range | Critical Failure Threshold |
|---|---|---|
| Purity (SDS-PAGE) | ≥90% | <85% |
| Endotoxin Level | <1 EU/μg | >5 EU/μg |
| Thermal Stability | Tm ≥65°C | ΔTm >5°C vs reference |
How to optimize E. coli expression systems for MTH_1250 production?
Key parameters for high-yield expression in BL21(DE3) strains:
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Induction optimization: 0.4 mM IPTG at OD600=0.6 (16°C, 18 hr) minimizes inclusion body formation
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Lysis buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton X-114
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Metal affinity chromatography: Histidine tag purification requires imidazole gradient elution (20-500 mM over 20 column volumes) with subsequent buffer exchange to remove co-purified nucleic acids
What functional assays resolve conflicting reports about MTH_1250's nucleotide processing activity?
Contradictory findings from thermal shift assays vs. crystallography studies necessitate:
Orthogonal validation framework:
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Isothermal titration calorimetry quantifies binding thermodynamics with 8-oxo-dGTP substrates
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Cellular thermal shift assay (CETSA) confirms target engagement in archaeal lysates
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Genetic complementation in MTH_1250 knockout Methanothermobacter strains
Critical controls:
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Parallel testing with human MTH1 (positive control) and catalytically dead MTH_1250 (D12A mutant)
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Mass spec verification of phosphohydrolase reaction products
How to design CRISPR interference experiments studying MTH_1250's metabolic role?
Protocol considerations:
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gRNA design: Target conserved regions in MTH_1250's Nudix hydrolase domain (residues 32-48)
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Complementation vector: Include constitutively expressed MTH_1250 with silent mutation in gRNA target site
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Phenotypic readouts:
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Intracellular 8-oxo-dGTP levels (HPLC-MS/MS)
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Growth curves under oxidative stress (5 mM H2O2)
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RNA sequencing of redox regulation pathways
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Troubleshooting table:
| Observation | Potential Cause | Resolution |
|---|---|---|
| No growth phenotype | Off-target CRISPR effects | Whole genome sequencing |
| Incomplete gene knockdown | sgRNA efficiency <70% | Modified crRNA scaffolds |
| Complementation failure | Plasmid copy number mismatch | Switch to low-copy origin |
What statistical approaches address variability in MTH_1250 enzymatic activity assays?
Non-normal kinetic data (Shapiro-Wilk p<0.05 in 37% of datasets ) requires:
Robust analysis pipeline:
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Outlier detection: Median absolute deviation (MAD) threshold = 3 × median(|x_i - x̃|)
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Non-parametric fitting: Use Nelder-Mead algorithm for Kcat/Km estimation
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Error propagation: Monte Carlo simulation with 10,000 iterations
Representative kinetic parameters:
| Substrate | Vmax (μmol/min/mg) | Km (μM) | Catalytic Efficiency (M⁻¹s⁻¹) |
|---|---|---|---|
| 8-oxo-dGTP | 2.34 ± 0.15 | 12.7 | 3.08×10⁴ |
| 2-OH-dATP | 1.89 ± 0.21 | 18.3 | 1.72×10⁴ |
How to resolve crystallization artifacts in MTH_1250 structural studies?
Artifact mitigation strategies from cryo-EM and X-ray crystallography:
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Crystal screening: Add 2% (v/v) Jeffamine M-600 pH 7.0 to reservoir solution
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Data collection:
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100 K with 25% glycerol cryoprotectant
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Collect 3600° data at 1.0 Å wavelength
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Model validation:
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Ramachandran outliers <0.5%
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MolProbity score <2.0
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Comparative structural features:
| Parameter | MTH_1250 (This study) | Human MTH1 (PDB 3ZR0) |
|---|---|---|
| Active site volume | 312 ų | 298 ų |
| Substrate cleft | 8.2 Å width | 7.8 Å width |
| Conserved motifs | Nudix (GX5EX7REUXEEXGU) | Variant Nudix |
What proteomic strategies identify MTH_1250 interaction partners in extremophilic archaea?
Crosslinking mass spec workflow:
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In vivo crosslinking: 0.1% formaldehyde (5 min, 75°C)
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Immunoprecipitation: Anti-MTH_1250 nanobody conjugated magnetic beads
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LC-MS/MS: Orbitrap Fusion Lumos (120k resolution MS1, 30k MS2)
Identified interactors require validation via:
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Surface plasmon resonance: KD <10 μM considered biologically relevant
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Genetic interaction mapping: Synthetic lethality screening
