Recombinant Mouse NAD-dependent protein deacylase sirtuin-5 (Sirt5) is produced using a baculovirus expression system. The construct contains the full-length mature protein sequence (37-310aa). To help with purification and detection, this protein carries both an N-terminal 10xHis tag and a C-terminal Myc tag. SDS-PAGE analysis confirms the product achieves greater than 85% purity, which should provide reliable results for research work.
Sirt5 appears to be a mitochondrial protein that may regulate several metabolic processes through its NAD-dependent deacylase activity. The protein's main function seems to involve removing acyl groups from lysine residues on target proteins—a process that likely influences cellular metabolism and energy production. Given its apparent role in mitochondrial function and cellular homeostasis, Sirt5 has become an important research focus, though some aspects of its regulatory mechanisms remain unclear.
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.
Mouse Sirt5 is a mitochondrial NAD+-dependent deacylase that requires precise folding, proper cofactor binding (NAD+), and correct subcellular targeting for its enzymatic activity. The baculovirus-insect cell expression system provides a eukaryotic environment that supports proper protein folding, post-translational modifications, and disulfide bond formation, significantly increasing the probability of correct folding. However, as a mitochondrial protein, Sirt5 may require mitochondrial-specific chaperones or processing for full functional activity, which might not be fully replicated in the insect cell system. The dual N-terminal 10xHis-tag and C-terminal Myc-tag are relatively small but may potentially interfere with the protein's mitochondrial targeting or functional domains. While the baculovirus system offers favorable folding conditions, experimental validation remains essential to confirm functional deacylase activity.
1. Protein-Protein Interaction Studies Using Pull-Down Assays
This application's reliability depends on proper folding and functional validation. Sirt5 interactions with mitochondrial proteins or metabolic enzymes require precise tertiary structure and enzymatic competence. If correctly folded and active (verified), the protein is highly suitable for identifying physiological interaction partners in mitochondrial deacylation pathways. If misfolded/inactive (unverified), there is a high risk of non-specific binding or failure to identify genuine interactions, making results biologically misleading.
2. Antibody Development and Validation
Antibody development primarily relies on antigenic sequence recognition. If correctly folded (verified), the protein excels for generating conformation-sensitive antibodies that recognize native Sirt5 epitopes. If misfolded/unverified, it remains highly suitable for producing antibodies against linear epitopes, which are still valuable for detection applications in Western blot or immunoassays.
3. Biochemical Characterization and Substrate Screening
These studies are essential for determining folding status and functional competence. If correctly folded and active (verified), characterization provides reliable data on deacylase activity, substrate specificity, kinetic parameters, and cofactor requirements. If misfolded/inactive (unverified), biochemical assays will yield negative or misleading results, though basic physical properties can still be characterized.
4. Protein Stability and Folding Studies
These studies are critical for assessing protein quality regardless of functional state. If correctly folded (verified), analysis provides insights into structural stability and functional requirements. If misfolded/unverified, characterization yields valuable physical property data for this specific preparation and informs handling protocols.
Final Recommendation & Action Plan
The baculovirus expression system provides a favorable eukaryotic environment for this mitochondrial deacylase, but experimental validation of structural integrity and enzymatic activity is essential before reliable use in functional studies. Begin with Applications 3 and 4 (Biochemical Characterization and Stability Studies) to assess folding quality through size-exclusion chromatography, circular dichroism spectroscopy, and validate deacylase activity using NAD+ and known acylated substrates (e.g., succinyllysine or malonyllysine peptides). Once correct folding and functional activity are verified, proceed cautiously with Application 1 for interaction studies, ensuring appropriate controls are included. Application 2 (antibody development) can proceed immediately regardless of folding status. If misfolding or lack of enzymatic activity is detected, limit applications to linear epitope antibody production and basic biophysical characterization, avoiding all functional interaction and substrate screening studies. For reliable Sirt5 research, always include appropriate activity controls using validated deacylation assays and consider the protein's mitochondrial context in experimental design.
