Recombinant Human PAI-1 (E. coli Expressed)

Recombinant Human PAI-1 (E. coli Expressed) is widely used in research because it provides a reliable, scalable, and cost-effective source of functionally active PAI-1 for biochemical, cellular, and structural studies. The E. coli expression system enables production of large quantities of PAI-1 that is suitable for a broad range of experimental applications.

Key reasons to use this reagent in your research include:

• Functional Activity Without Glycosylation: PAI-1 remains active even in the absence of glycosylation, which is not possible in E. coli, making bacterial expression a practical choice for producing active protein.
• High Yield and Scalability: E. coli systems allow for large-scale production, facilitating studies that require significant amounts of protein, such as high-throughput screening, mutagenesis, or structural biology.
• Structural and Biochemical Studies: Recombinant PAI-1 from E. coli has been used for crystallization and structural analysis, including studies of the latent form and mutational scanning to dissect structure-function relationships.
• Standardization in Assays: E. coli-expressed PAI-1 is commonly used as a standard in quantitative assays (e.g., ELISA, activity assays), ensuring reproducibility and comparability across experiments.
• Versatility in Functional Studies: Recombinant PAI-1 is used to study its role in processes such as fibrinolysis, immune modulation, and cell signaling, and to test the effects of inhibitors or mutations on its activity.
• Therapeutic and Pathophysiological Research: It is essential for investigating PAI-1’s involvement in diseases like cancer, cardiovascular disease, fibrosis, and inflammation, and for evaluating potential therapeutic interventions.

Technical advantages of E. coli-expressed PAI-1:

• No native cysteine residues: This simplifies protein folding and reduces aggregation, enhancing yield and purity.
• Facilitates mutagenesis: The system is amenable to site-directed mutagenesis and library generation for functional screening.

Limitations to consider:

• Lack of post-translational modifications: While PAI-1 is functional without glycosylation, certain subtle regulatory features present in mammalian-expressed protein may be absent.
• Potential for latent form predominance: E. coli-expressed PAI-1 can adopt the latent conformation, which may require refolding or activation protocols depending on your application.

In summary, Recombinant Human PAI-1 (E. coli Expressed) is a robust tool for mechanistic, structural, and translational research, offering high yield, functional activity, and flexibility for diverse experimental needs.

You can use recombinant human PAI-1 expressed in E. coli as a standard for quantification or calibration in ELISA assays, provided that the recombinant protein is properly characterized, its concentration is accurately determined, and it is recognized by the antibodies used in your ELISA system. This is a common practice in quantitative ELISA protocols.

Key considerations and supporting details:

• Recombinant PAI-1 proteins (including those expressed in E. coli) are widely used as standards in commercial ELISA kits for human PAI-1 quantification. The standard curve is typically generated using serial dilutions of the recombinant protein, and sample concentrations are interpolated from this curve.
• The antibodies in your ELISA kit must recognize the recombinant PAI-1 in the same way as the native protein. Most sandwich ELISA kits are designed to detect both natural and recombinant forms, but you should confirm this in your kit documentation or by running a parallelism test.
• Protein conformation and post-translational modifications: E. coli-expressed PAI-1 may lack certain post-translational modifications (e.g., glycosylation) present in native human PAI-1, which could affect antibody recognition or activity in some assays. However, many ELISA kits are validated with recombinant proteins from various sources, including E. coli, and are suitable for quantification as long as the epitopes are preserved.
• Standard preparation: Ensure the recombinant PAI-1 is highly purified, accurately quantified, and reconstituted according to best practices for ELISA standards. Follow the same dilution and handling procedures as you would for a kit-provided standard.
• Validation: For critical applications, validate your standard by comparing the standard curve generated with your recombinant PAI-1 to that of a commercial ELISA kit standard or a reference material, if available.

Summary Table: Use of Recombinant PAI-1 as ELISA Standard

Best practice:
Always verify that your ELISA kit or custom assay detects the recombinant PAI-1 standard equivalently to native PAI-1, and ensure your standard is well-characterized and handled under appropriate conditions. If your assay is for regulatory or clinical purposes, consider referencing an international standard if available.

Recombinant Human PAI-1 (E. coli Expressed) has been validated for several key applications in published research, including enzyme activity assays, bioassays, ELISA (as a standard), Western blotting, immunocytochemistry, and structural studies.

Validated Applications in Published Research:

• Enzyme Activity Assays:
  Used to measure its ability to inhibit urokinase-type plasminogen activator (uPA) cleavage of peptide substrates, confirming its functional activity as a serine protease inhibitor.

• Bioassays:
  Applied in cell-based assays to study cellular responses, such as senescence, migration, invasion, and niche formation in cancer and stem cell biology.

• ELISA (Standard):
  Utilized as a quantitative standard in multiplex immunoassays for profiling human adipokines and detecting circulating PAI-1 complexes in plasma.

• Western Blotting & Immunocytochemistry:
  Used for detection and quantification of PAI-1 in cell lysates and tissue samples, including analysis of protein complexes (e.g., FXIa–PAI-1) in endothelial cells.

• Structural Studies:
  Recombinant PAI-1 expressed in E. coli has been crystallized in its latent form, enabling structural characterization and conformational analysis.

• Fibrinolysis and Plasminogen Activation Studies:
  Employed in in vitro assays to investigate its role in blocking/inhibition of fibrinolysis and plasminogen activation, as well as receptor binding studies.

Additional Context:

• Sample Types:
  Validated in whole cells, cell lysates, and as purified recombinant protein.

• Disease and Mechanistic Studies:
  Used in research on cancer prognosis, cardiovascular disease, wound healing, and metabolic disorders due to its regulatory role in fibrinolysis and tissue remodeling.

• Protein Conformation:
  Studies have distinguished between active and latent forms of PAI-1, with E. coli-expressed protein enabling analysis of these states.

Summary Table of Applications

These applications are supported by peer-reviewed publications and product validation data, confirming the utility of recombinant human PAI-1 (E. coli expressed) in diverse experimental settings.

To reconstitute and prepare Recombinant Human PAI-1 (E. coli Expressed) protein for cell culture experiments, follow these steps to ensure protein stability and biological activity:

1. Allow the lyophilized protein to equilibrate to room temperature before opening the vial to prevent condensation from forming inside the vial.

2. Reconstitution:

   • Use sterile, distilled water or the buffer recommended in the product datasheet (commonly PBS, pH 7.4–7.5, or 20 mM sodium acetate, 0.5 M NaCl, pH 7.4–7.5).
   • Add the appropriate volume to achieve the desired concentration (e.g., 0.25 mg/mL is typical for PAI-1).
   • Gently mix by pipetting or swirling; avoid vigorous vortexing to prevent protein denaturation.

3. Stabilization (optional but recommended for cell culture use):

   • Add 0.1% BSA (bovine serum albumin) to the reconstituted solution to stabilize the protein and prevent adsorption to plastic surfaces.
   • Filter-sterilize the solution using a 0.2 μm filter if sterility is required for cell culture.

4. Aliquoting and Storage:

   • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can reduce activity.
   • Store aliquots at –20°C to –70°C for long-term storage or at 2–8°C for up to one month.
   • Avoid frost-free freezers, as temperature fluctuations can degrade the protein.

5. Preparation for Cell Culture:

   • Thaw aliquots on ice and use immediately.
   • Dilute the protein into cell culture medium just before use to the final working concentration required for your experiment.
   • Ensure the final buffer composition is compatible with your cells (e.g., avoid high salt concentrations or non-physiological pH).

Additional Notes:

• Always consult the specific product datasheet for any lot-specific instructions, as buffer composition and recommended concentrations may vary.
• If the protein is to be used in functional assays, confirm activity post-reconstitution, as some recombinant PAI-1 preparations may be partially latent or inactive depending on storage and handling.

Summary Table: Key Steps for Recombinant Human PAI-1 (E. coli) Reconstitution

This protocol ensures that recombinant PAI-1 remains stable and active for cell culture applications.