Recombinant Human Granzyme K (GZMK) is produced in E.coli, spanning the full length of the mature protein from amino acids 27 to 264. It includes an N-terminal 6xHis-tag for easier purification and detection. The product achieves a purity level exceeding 90%, as verified by SDS-PAGE, which appears to make it suitable for various research applications requiring high-quality protein. This product is intended for research use only.
Granzyme K is a serine protease that's predominantly expressed by cytotoxic T cells and natural killer cells. It likely plays a crucial role in immune response by contributing to the induction of apoptosis in target cells. Its involvement in the immune system makes it a significant focus of research in understanding immune regulation and potential therapeutic interventions.
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.
The human GZMK is a serine protease that requires precise folding, disulfide bond formation, and activation (typically involving proteolytic cleavage of a pro-domain) for enzymatic activity. E. coli, as a prokaryotic system, lacks the eukaryotic chaperones and post-translational modification machinery necessary for proper folding and activation of complex serine proteases. While the His tag is small and may not significantly interfere, the absence of proper disulfide bond formation in the reducing cytoplasm of E. coli likely results in a misfolded, inactive protein. Without experimental validation (e.g., enzymatic activity assays), the protein cannot be assumed to be correctly folded or bioactive.
1. Antibody Development and Validation
The recombinant GZMK can serve as an immunogen for generating antibodies against linear epitopes, even if misfolded. The high purity supports consistent immunization. However, antibodies generated may not recognize conformational epitopes of native, properly folded and activated GZMK. Validate antibody specificity against native GZMK from human cytotoxic lymphocytes.
2. Protein-Protein Interaction Studies
Use with caution. The His-tag enables pull-down assays, but if GZMK is misfolded, interactions may be non-physiological. The enzyme's substrate specificity depends on correct active site formation. Validate any identified interactions using active GZMK from eukaryotic expression systems.
3. Biochemical Characterization and Enzyme Kinetics
Basic biophysical characterization (e.g., thermal stability, aggregation state) is feasible. However, enzyme kinetics studies require confirmed proteolytic activity. Without validation of enzymatic function, kinetic parameters are meaningless. First, test activity using synthetic substrates (e.g., Boc-Ala-Ala-Asp-S-Bzl) before kinetic analysis.
Final Recommendation & Action Plan
Before using this recombinant GZMK for functional applications, validate its folding and enzymatic activity. Start with a protease activity assay using Granzyme K-specific substrates (e.g., Boc-Ala-Ala-Asp-S-Bzl) to confirm enzymatic function. If active, proceed with interaction or kinetic studies; if inactive (as expected from E. coli expression), limit use to non-functional applications like antibody production (with validation against native GZMK). For reliable functional studies, express GZMK in mammalian or insect cell systems that support proper folding, disulfide bond formation, and activation. Always include appropriate controls with known active GZMK when possible.
