Recombinant Human TIMP-2

Using Recombinant Human TIMP-2 in research applications is valuable due to its well-characterized roles as a multifunctional regulator of extracellular matrix (ECM) remodeling, cell proliferation, angiogenesis, and tumor progression, as well as its emerging therapeutic potential in cancer and other disease models.

Key scientific reasons to use recombinant human TIMP-2 include:

• Matrix Metalloproteinase (MMP) Inhibition: TIMP-2 is a potent endogenous inhibitor of MMPs, particularly MMP-2, which are enzymes involved in ECM degradation, tissue remodeling, and metastasis. Recombinant TIMP-2 allows precise modulation of MMP activity in vitro and in vivo, facilitating studies on ECM dynamics, cancer invasion, and tissue repair.

• Anti-Tumor and Anti-Metastatic Effects: Recombinant TIMP-2 has been shown to significantly inhibit tumor growth and metastasis in preclinical models, including triple-negative breast cancer and lung cancer. Its effects are mediated both by MMP inhibition and by MMP-independent mechanisms, such as suppression of growth factor-mediated cell proliferation and angiogenesis.

• Angiogenesis Regulation: TIMP-2 can suppress neovascularization (formation of new blood vessels), which is critical in tumor progression and chronic diseases. This anti-angiogenic property is partly independent of MMP inhibition and involves direct antagonism of growth factor signaling (e.g., IGF-1R).

• Immunomodulatory Functions: TIMP-2 modulates immune cell behavior, including reducing the formation of immunosuppressive cell types in the tumor microenvironment, which can be relevant for studies on tumor immunity and inflammation.

• Broader Disease Relevance: Beyond oncology, TIMP-2 is implicated in cardiovascular disease, neurodegeneration (e.g., Alzheimer’s, Parkinson’s), and degenerative joint disease, making it a versatile tool for studying diverse pathophysiological processes.

• Standardization and Reproducibility: Recombinant production ensures high purity, batch-to-batch consistency, and defined activity, which are critical for reproducible results in cell-based assays, animal models, and biochemical studies.

• Versatile Applications: Recombinant TIMP-2 is suitable for:

  • Cell culture experiments to study proliferation, migration, and invasion.
  • In vivo models to assess anti-tumor or anti-angiogenic effects.
  • Biochemical assays for MMP inhibition and protein-protein interaction studies.
  • Use as a standard in ELISA or Western blot applications.

In summary, recombinant human TIMP-2 is a powerful research tool for dissecting ECM biology, cancer progression, angiogenesis, and immune regulation, and for preclinical evaluation of novel therapeutic strategies.

Yes, recombinant human TIMP-2 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated for this purpose. Recombinant TIMP-2 is commonly used as a standard in commercial human TIMP-2 ELISA kits, and its dose-response curves are shown to be parallel to those of natural TIMP-2, supporting its suitability for quantification.

Key considerations and best practices:

• Validation: Ensure the recombinant TIMP-2 standard is validated for your specific ELISA format. Commercial ELISA kits typically use recombinant TIMP-2 as the calibrator, and their protocols include validation data such as recovery, linearity, and parallelism with natural TIMP-2.
• Standard Curve Preparation: Prepare a standard curve by serially diluting the recombinant TIMP-2 in the recommended assay diluent. The standard curve should cover the expected concentration range of your samples.
• Parallelism: Confirm that the standard curve generated with recombinant TIMP-2 is parallel to the response obtained from natural TIMP-2 in your sample matrix. This ensures accurate quantification.
• Assay Range and Sensitivity: Follow the validated concentration range and sensitivity limits provided by your ELISA protocol. For human TIMP-2, typical assay ranges are from low picogram to nanogram per milliliter, with sensitivities as low as 2 pg/mL.
• Controls: Include appropriate controls and run standards in duplicate to ensure precision and reproducibility. Calculate intra- and inter-assay coefficients of variation (CV%) to assess assay performance.

Scientific context:

• Recombinant TIMP-2 is produced in systems such as CHO cells and is highly purified for use as an ELISA standard.
• ELISA kits using recombinant TIMP-2 as a standard have demonstrated accurate detection of both recombinant and natural TIMP-2 in biological samples, with validated recovery and linearity.
• The use of recombinant standards is standard practice in quantitative ELISA, provided the standard is well-characterized and the assay demonstrates parallelism and specificity.

Summary of protocol steps for using recombinant TIMP-2 as a standard:

1. Reconstitute the lyophilized recombinant TIMP-2 in the recommended assay buffer.
2. Prepare serial dilutions to generate a standard curve covering the assay’s dynamic range.
3. Run standards and samples in duplicate or triplicate for accuracy.
4. Analyze data using appropriate curve fitting (e.g., 4-parameter logistic regression).
5. Confirm assay performance with recovery, linearity, and parallelism studies.

Conclusion:
Recombinant human TIMP-2 is suitable as a standard for ELISA quantification and calibration, provided you follow validated protocols and confirm assay performance parameters such as parallelism, recovery, and linearity.

Applications of Recombinant Human TIMP-2 in Published Research

Recombinant human TIMP-2 has been validated across multiple therapeutic and research applications, with particularly strong evidence in oncology and tissue biology.

Cancer Therapeutics

Tumor Growth and Metastasis Inhibition

Recombinant human TIMP-2 has demonstrated significant antitumor activity in preclinical models. In an orthotopic triple-negative breast cancer model, daily injection of recombinant human TIMP-2 significantly inhibited both tumor growth and metastasis. The protein functions as a potent inhibitor of invadopodia formation in in vitro models of pancreatic and breast carcinomas, which are critical structures that cancer cells use for invasion.

Lung Cancer Cell Proliferation

Recombinant human TIMP-2 has been validated for inhibiting growth factor-induced proliferation in lung adenocarcinoma cells. Pretreatment of A549 lung cancer cells with recombinant human TIMP-2 in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. The protein demonstrated dose-dependent inhibition of MMP-2 enzymatic activity with an IC₅₀ of approximately 1.4 nM.

Triple-Negative Breast Cancer

Similar to lung cancer applications, recombinant human TIMP-2 pretreatment of JygMC(A) triple-negative breast cancer cells inhibited EGF-induced proliferation.

Angiogenesis Inhibition

Recombinant human TIMP-2 has been validated for inhibiting angiogenesis in various lung cancer types through inhibition of VEGF-A and FGF-2 both in vitro and in vivo. The protein can bind the α3β1 integrin heterodimer and stimulate SHP1-mediated dephosphorylation of FGFR1 or EGFR, contributing to its anti-angiogenic effects.

Ocular Applications

Myopia Prevention

In vivo exogenous addition of recombinant human TIMP-2 significantly reduced myopia development by reducing vitreous chamber elongation. The treatment also significantly inhibited posterior scleral collagen degradation, maintaining levels similar to those in control eyes.

Bioanalytical Applications

Recombinant human TIMP-2 has been validated for use as a Western blot standard protein for reproducible quantification in western blot assays. The protein is also suitable for ELISA applications and serves as a reference standard for protein quantification.

Research Applications

The protein has been validated for bioassay applications in cell and tissue culture systems, where it can be used to study MMP inhibition, cell proliferation dynamics, and integrin-mediated signaling pathways in various cell types.

To reconstitute and prepare Recombinant Human TIMP-2 protein for cell culture experiments, centrifuge the vial before opening, then gently dissolve the lyophilized protein in sterile distilled water to a concentration of 0.1–1.0 mg/mL. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Step-by-step protocol:

• Centrifuge the vial briefly to collect the lyophilized powder at the bottom before opening.
• Add sterile distilled water to achieve a final concentration between 0.1–1.0 mg/mL (do not reconstitute below 100 μg/mL for stability).
• Mix gently by slow pipetting or gentle inversion; do not vortex.
• Let the solution stand at room temperature for 10–15 minutes to ensure complete dissolution.
• For cell culture applications, further dilute the reconstituted stock in your desired cell culture medium or buffer. It is recommended to include a carrier protein (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) in the dilution buffer to stabilize the protein and minimize adsorption losses.
• Aliquot the solution into single-use volumes to avoid repeated freeze-thaw cycles, which can reduce activity.
• Storage:
  • Short-term: Store at 4°C for up to 1 week.
  • Long-term: Store aliquots at −20°C or −80°C with carrier protein/stabilizer.

Additional notes:

• Always consult the specific Certificate of Analysis (CoA) for your batch for optimal reconstitution buffer and concentration recommendations.
• For functional assays, TIMP-2 is typically active at concentrations in the low μg/mL range, but optimal dosing should be empirically determined for your cell type and experimental setup.
• Ensure all solutions and buffers are sterile and endotoxin-free for cell culture use.

Summary of best practices:

• Use gentle handling throughout.
• Include carrier protein for stability.
• Aliquot to avoid freeze-thaw cycles.
• Store appropriately for your experimental timeline.

These steps will help maintain the integrity and biological activity of recombinant TIMP-2 for cell culture experiments.