Recombinant Clostridium kluyveri NAD-dependent 4-hydroxybutyrate dehydrogenase (4hbD) is expressed in E. coli and contains the complete 371 amino acid sequence. The protein includes an N-terminal 10xHis-tag and a C-terminal Myc-tag, which help with purification and detection processes. SDS-PAGE analysis confirms the product maintains greater than 85% purity, suggesting it should be reliable for research applications. This recombinant protein is intended for research use only and appears to meet high-quality standards.
NAD-dependent 4-hydroxybutyrate dehydrogenase (4hbD) from Clostridium kluyveri seems to play an important role in metabolic pathways that involve reducing 4-hydroxybutyrate. It represents a valuable enzyme for studies in metabolic engineering and biochemical research. The enzyme may provide insights into enzymatic function and potential biotechnological applications, though its activity in converting specific substrates could be key to advancing our understanding of microbial 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 Clostridium kluyveri 4-hydroxybutyrate dehydrogenase is expressed in E. coli, a prokaryotic system that is generally suitable for producing bacterial enzymes like this dehydrogenase. As a bacterial protein expressed in its native prokaryotic environment, the likelihood of proper folding is relatively high. The protein is full-length (1-371aa) with dual tags (N-terminal 10xHis and C-terminal Myc) and >85% purity. However, dehydrogenase enzymes require precise folding for their active site formation and cofactor (NAD) binding capability. The presence of dual tags may potentially interfere with the native protein structure. Since activity is unverified, the protein cannot be assumed to be correctly folded or bioactive without experimental validation of its enzymatic activity.
1. Biochemical Characterization and Enzyme Kinetics Studies
This application is appropriate but requires activity validation first. Basic biochemical characterization is feasible, but enzyme kinetics studies (Km, Vmax determinations) are only valid if the 4-hydroxybutyrate dehydrogenase is properly folded and active. The dual tags facilitate purification but may affect enzymatic properties. These studies should only proceed after confirming dehydrogenase activity with proper substrates.
2. Antibody Development and Validation
This application is well-suited as a primary use case. The recombinant 4-hydroxybutyrate dehydrogenase can serve as an effective immunogen for generating antibodies that recognize linear epitopes. The high purity and full-length sequence support antibody production. The Myc-tag enables easy detection during screening. However, antibodies may not recognize conformational epitopes if the protein is misfolded.
3. Protein-Protein Interaction Studies
This application requires caution. While the tags enable technical feasibility for pull-down assays, if the 4-hydroxybutyrate dehydrogenase is misfolded, it may not interact physiologically with true binding partners. Bacterial metabolic enzymes often form complexes that require precise conformation. This application should only be pursued after confirming proper folding and activity.
4. Comparative Enzyme Studies and Metabolic Pathway Analysis
This application is valuable but highly dependent on correct folding. Comparative studies with other bacterial dehydrogenases require proper folding to yield valid evolutionary and functional insights. Metabolic pathway reconstruction experiments will only be meaningful if the enzyme is active. This requires prior validation of enzymatic function.
Final Recommendation & Action Plan
Given that this is a bacterial enzyme expressed in its native prokaryotic system, the probability of proper folding is relatively high. However, recommend first validating enzymatic activity using standard dehydrogenase assays with 4-hydroxybutyrate and NAD+ as substrates. Perform basic biophysical characterization (size-exclusion chromatography, circular dichroism) to confirm proper folding. Antibody development can proceed as the safest application. For interaction studies and comparative analyses, await activity validation. Always include appropriate controls such as known substrates and specific inhibitors in experiments. If activity is confirmed, the protein becomes suitable for all described applications; if not, focus on antibody development and basic biochemical characterization.
