Recombinant Mouse Enoyl-CoA hydratase, mitochondrial (Echs1) is produced in E. coli and covers the full length of the mature protein from amino acids 28 to 290 with a C225S mutation. The protein features an N-terminal 10xHis tag and a C-terminal HA tag, which should make purification and detection more straightforward. SDS-PAGE analysis indicates it reaches a purity level greater than 85%, suggesting it's well-suited for various research applications.
Enoyl-CoA hydratase, mitochondrial (Echs1) appears to play a critical role in the fatty acid oxidation pathway. It catalyzes the hydration of enoyl-CoA to hydroxyacyl-CoA—a step that seems essential for breaking down fatty acids and extracting energy from them. This makes Echs1 particularly relevant for studies examining metabolic processes and energy homeostasis, potentially offering valuable insights into how cells manage their metabolism.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
Based on the provided information, the recombinant mouse Echs1 (C225S) protein is unlikely to be correctly folded or fully bioactive without experimental validation. Echs1 is a mitochondrial enzyme involved in fatty acid β-oxidation, requiring precise folding for its enoyl-CoA hydratase activity. The E. coli expression system lacks the eukaryotic machinery for mitochondrial-specific folding, potentially leading to misfolding. The dual tags (N-terminal 10xHis and C-terminal HA) may sterically hinder proper folding, particularly as the C-terminal HA tag could disrupt the active site or oligomerization. The C225S mutation might affect structural stability or catalytic function, as cysteine residues can be involved in disulfide bonds or metal coordination. The purity >85% indicates impurities that could include misfolded species. Without validation (e.g., circular dichroism for secondary structure, enzyme activity assays), the protein's folding and bioactivity cannot be assured.
1. Biochemical Characterization of Mutant Enzyme Activity
This recombinant Echs1 (C225S) protein can be used for kinetic studies only if correct folding and enzymatic activity are experimentally verified. The dual tags may alter the active site conformation, leading to inaccurate Km or Vmax measurements. If misfolded, comparative studies with wild-type Echs1 may yield misleading results. It is recommended to first validate hydratase activity using substrates like crotonyl-CoA and remove tags if possible for reliable kinetics. The purity of>85% may require further purification for precise assays.
2. Protein-Protein Interaction Studies
The dual-tagged Echs1 mutant can be employed in pull-down assays, but interactions may be tag-mediated or non-specific if the protein is misfolded. The tags allow immobilization and detection, but biologically relevant binding partners in mitochondrial pathways may not be identified without native conformation. Validate folding using biophysical methods and include controls (e.g., tag-only proteins) to minimize artifacts. Results should be confirmed with full-length, tag-free Echs1.
3. Antibody Development and Validation
This recombinant protein can generate antibodies, but the dual tags may dominate the immune response, resulting in antibodies that primarily recognize tags rather than Echs1-specific epitopes. If misfolded, antibodies may not recognize native mitochondrial Echs1. For reliable outcomes, validate antibodies against tag-free Echs1 or endogenous protein from mouse tissues. The HA tag serves as a control but does not ensure specificity for the native enzyme.
4. Structural and Folding Studies
This protein is unsuitable for high-resolution structural studies (e.g., crystallography) without tag removal, as the tags introduce flexibility and heterogeneity. Biophysical techniques (e.g., circular dichroism) can assess general folding but may be confounded by tag contributions. The C225S mutation allows investigation of folding effects, but for meaningful insights, use tag-free protein and compare with wild-type. The purity of>85% is suboptimal for structural work.
5. In Vitro Metabolic Pathway Reconstitution
The recombinant Echs1 mutant can be incorporated into metabolic assays only if enzymatic activity is confirmed. Misfolding or tag interference may lead to inaccurate pathway efficiency measurements. Validate activity in a reconstituted system with other β-oxidation enzymes and use tag-free protein if possible. The tags aid monitoring but do not guarantee functional integrity.
Final Recommendation & Action Plan
To ensure reliable results, first validate the folding and bioactivity of the recombinant Echs1 (C225S) protein through techniques such as circular dichroism to confirm secondary structure, size-exclusion chromatography to assess oligomeric state, and enzyme activity assays using standard substrates like crotonyl-CoA. Given the potential interference from dual tags, remove them via proteolytic cleavage (if feasible) and re-purify the tag-free protein for functional studies. For applications like antibody development, use the current protein but thoroughly validate antibodies against native Echs1. In all cases, include appropriate controls, such as wild-type Echs1, tag-only proteins, and activity-negative mutants, to account for folding-related artifacts. Prioritize validation before quantitative biochemical or metabolic studies.
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