Recombinant Human Tissue-type plasminogen activator (PLAT) (R497Q) comes from a baculovirus expression system. This version represents the complete mature protein spanning amino acids 36 to 562, though it carries a specific R497Q mutation. Scientists have added both an N-terminal 10xHis-tag and a C-terminal Myc-tag to make detection and purification more straightforward. SDS-PAGE analysis shows the product maintains purity levels above 85%, which appears suitable for most research applications.
Tissue-type plasminogen activator (tPA) functions as a serine protease that breaks down blood clots. It does this by converting plasminogen into plasmin. This protein plays a crucial role in fibrinolysis—a process that seems essential for keeping our vascular system healthy. Given its significance in thrombolytic therapy, researchers frequently study tPA in cardiovascular and blood-related research. These studies may offer valuable insights into therapeutic approaches for clot-related disorders.
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 baculovirus expression system supports complex post-translational modifications, including disulfide bond formation and partial glycosylation, both essential for proper folding and enzymatic activity of tPA. The R497Q substitution (arginine → glutamine) lies in a region associated with tPA’s proteolytic or fibrin-binding domains, and such a substitution may influence its enzymatic activity or receptor interactions, but it does not inherently prevent proper folding. Therefore, this recombinant protein is likely to fold correctly and may exhibit partial or full biological activity, depending on the extent of glycosylation fidelity compared to human native tPA.
1. Protein-Protein Interaction Studies
This dual-tagged recombinant PLAT (R497Q) can be used in pull-down and binding assays to study how the R497Q mutation influences interactions with plasminogen, fibrin, or inhibitor proteins (e.g., PAI-1). If the protein is properly folded and glycosylated, it can provide reliable insights into mutation-induced changes in binding kinetics or affinity. If bioactivity has not been verified, experiments should be treated as qualitative interaction screens, since altered folding or incomplete glycosylation may affect native binding behavior. The His-tag enables immobilization on nickel-based matrices, while the Myc-tag facilitates detection and confirmation of binding events.
2. Structural and Biochemical Characterization
This recombinant PLAT variant is suitable for structural and biochemical analyses such as circular dichroism (CD), differential scanning calorimetry (DSC), and possibly cryo-EM or crystallography, depending on protein quality. If correctly folded, it can reveal mutation-specific conformational changes and stability differences compared with wild-type PLAT. If folding or glycosylation is incomplete, it still serves as a comparative model to analyze how expression systems or point mutations affect structural integrity. The dual tags support efficient purification, concentration, and detection during analytical workflows.
3. Antibody Development and Validation
This recombinant full-length PLAT (R497Q) protein is well-suited for antibody production and screening. If the protein maintains its native-like conformation, it can elicit antibodies recognizing conformational epitopes and potentially distinguish between wild-type and R497Q mutant forms. If folding or glycosylation is atypical, it remains appropriate for generating antibodies targeting linear or tag-associated epitopes, which can be used in ELISA and Western blotting. The presence of both His and Myc tags aids in quantitative assay monitoring and specificity validation.
4. ELISA and Immunoassay Development
The dual-tagged PLAT (R497Q) variant can serve as a reference standard or control reagent in immunoassays detecting human tPA or its variants. If bioactive and properly folded, it can be used to calibrate ELISA assays for quantitative detection and to test antibody recognition across PLAT isoforms. If activity has not been validated, it should be used as a structural or immunological control rather than a quantitative standard for enzymatic assays. The His-tag enables immobilization on nickel-coated plates, while the Myc-tag supports alternative detection methods for assay optimization.
Final Recommendation & Action Plan
The recombinant human tPA (PLAT, R497Q) expressed via baculovirus is likely well-folded and partially or fully functional, given the system’s ability to perform essential post-translational modifications. However, its exact enzymatic activity and fibrin-binding capacity must be experimentally confirmed, especially since the R497Q mutation may alter catalytic behavior or interaction dynamics. For biochemical, structural, or antibody-based studies, the protein can be confidently used as a research-grade reagent. For quantitative enzymatic assays or kinetic studies, verify activity before application. Researchers should leverage its dual-tag system for efficient purification, immobilization, and detection, and use it in mutation effect analysis, immunoassay development, or comparative biophysical studies.
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