Recombinant Human Isthmin-2 (ISM2) is produced using the E.coli expression system, covering the partial length of the protein from amino acids 89 to 571. This recombinant protein features an N-terminal 10xHis-tag and a C-terminal Myc-tag, which help with detection and purification. SDS-PAGE analysis confirms it achieves a purity level greater than 85%. This quality appears suitable for various research applications that need reliable protein reagents.
Isthmin-2 is a secreted protein that seems to be involved in cellular processes related to angiogenesis and tissue development. Research suggests it may play a role in modulating cell adhesion, migration, and proliferation, which makes it an interesting target for studies focused on vascular biology and developmental pathways. Understanding how Isthmin-2 functions could potentially provide insights into its impact on tissue regeneration and repair mechanisms.
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 Human Isthmin-2 (ISM2) is expressed in E. coli, a prokaryotic system that may not support proper folding of eukaryotic proteins like ISM2, which likely requires specific post-translational modifications (e.g., glycosylation) and chaperone-assisted folding for native conformation. The protein is expressed as a partial fragment (89-571aa), which may lack critical domains needed for full functionality, and the dual tags (N-terminal 10xHis and C-terminal Myc) could potentially interfere with folding. Since activity is unverified, the protein cannot be assumed to be correctly folded or bioactive. While E. coli can sometimes produce functional proteins, the mismatch between the eukaryotic protein and the prokaryotic system increases the risk of misfolding. Experimental validation is essential to confirm folding and bioactivity.
1. Antibody Development and Validation Studies
This recombinant ISM2 protein can work as an immunogen for generating antibodies, as antibodies may recognize linear epitopes even if the protein is misfolded. The dual tags provide epitopes for characterization, and >85% purity is sufficient for immunization. However, if misfolded, antibodies may not recognize the native, full-length ISM2 in physiological contexts, so validation against native protein is recommended.
2. Protein-Protein Interaction Studies
The dual-tag system allows for pull-down assays, but if ISM2 is misfolded, it may not interact physiologically with binding partners, leading to non-specific or false interactions. The tags themselves could influence binding specificity. This application should only be pursued after confirming protein folding through biochemical assays. If folded correctly, it is feasible; otherwise, results may be unreliable.
3. Biochemical Characterization and Stability Studies
This application is appropriate and should be prioritized to assess folding. Techniques like size exclusion chromatography and dynamic light scattering can directly evaluate oligomerization and stability. The His-tag facilitates purification, and these studies are valuable even if the protein is misfolded, as they characterize the recombinant product.
4. ELISA-Based Quantitative Assays
The recombinant protein can be used as a standard in ELISA, but if misfolded, it may not behave like native ISM2, leading to inaccurate quantitation. The Myc-tag enables detection, but assays must be validated against native ISM2 to ensure similarity. This application is feasible for development but requires cross-validation for reliability.
Final Recommendation & Action Plan
Given the uncertainty in folding and bioactivity, it is crucial to first perform biochemical characterization (e.g., circular dichroism for secondary structure, size-exclusion chromatography for oligomeric state) and functional assays (e.g., binding studies with known partners) to validate the protein's conformation. If the protein is correctly folded, it can be used for interaction studies and quantitative assays with confidence; if misfolded, focus on applications like antibody development for linear epitopes or as a control for biochemical studies. Always include controls such as native ISM2 when possible, and consider using mammalian expression systems for activity-dependent applications. Prioritize folding validation before investing in complex functional experiments.
