Recombinant Human Angiopoietin-Like Protein 3

Recombinant human angiopoietin-like protein 3 (ANGPTL3) is a valuable research tool with multiple compelling applications across lipid metabolism, cardiovascular biology, and hematopoietic research.

Primary Research Applications

Lipid Metabolism Studies

ANGPTL3 is best known for its function as an inhibitor of lipoprotein and endothelial lipases, making it essential for investigating lipid homeostasis mechanisms. The protein directly inhibits lipoprotein lipase (LPL) and endothelial lipase (EL), enzymes responsible for hydrolyzing circulating triglycerides and HDL phospholipids. This inhibitory capacity allows researchers to study how lipase activity is regulated and how circulating lipid levels are controlled. Additionally, recombinant ANGPTL3 can be used to examine the binding interactions between ANGPTL3 and lipoprotein particles, as the protein preferentially resides on HDL and LDL particles, and this binding is a prerequisite for potentiating its lipase-inhibitory effects.

Hepatic Function and Metabolism

Beyond its extracellular functions, ANGPTL3 exhibits cell-autonomous functions within hepatocytes. Recombinant protein can be used in cell-based assays to investigate hepatocyte glucose and fatty acid uptake, insulin sensitivity, LDL/VLDL remnant uptake, VLDL assembly and secretion, and polyunsaturated fatty acid (PUFA) metabolism. This makes it particularly useful for studying metabolic dysfunction in disease models.

Hematopoietic Stem Cell Research

ANGPTL3 secreted by fetal liver promotes the expansion of hematopoietic stem cells (HSCs). The protein protects hematopoietic cells from exhaustion by toxic agents, making recombinant ANGPTL3 valuable for research involving stem cell maintenance, quiescence, and protection from chemotoxic stress.

Experimental Advantages

Recombinant ANGPTL3 enables controlled, dose-dependent studies in both in vitro and in vivo contexts. The protein can be used at defined concentrations (typically in the range of 40-200 ng/mL for functional assays) to investigate dose-response relationships. It is particularly useful for bioassay applications where you need to measure lipoprotein lipase inhibition or examine cellular responses to ANGPTL3 signaling in a reproducible manner.

The recombinant form also allows researchers to study ANGPTL3 function independently of genetic manipulation, providing a complementary approach to knockout or knockdown studies for understanding this protein's role in cardiometabolic disease and metabolic regulation.

Recombinant Human Angiopoietin-Like Protein 3 (ANGPTL3) can be used as a standard for quantification or calibration in ELISA assays, provided it is validated for this purpose and matches the assay’s requirements.

Key considerations and supporting details:

• Commercial ELISA kits for human ANGPTL3 typically use recombinant human ANGPTL3 as the calibrator or standard. For example, the Quantikine Human ANGPTL3 ELISA is calibrated against a recombinant human ANGPTL3 standard, and the kit documentation states that it "contains recombinant human ANGPTL3 and has been shown to accurately quantitate the recombinant factor" and that "results obtained using natural human ANGPTL3 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards" . This demonstrates that recombinant ANGPTL3 is suitable for generating standard curves in these assays.

• Parallelism between recombinant and native protein is critical. The standard curve generated with recombinant ANGPTL3 should be parallel to the response obtained with native ANGPTL3 in your samples. This ensures that the assay quantifies both forms equivalently, which is a standard validation step for ELISA assays .

• Source and form of recombinant protein matter. The recombinant ANGPTL3 used as a standard should be of high purity, correctly folded, and ideally glycosylated if the native protein is glycosylated, as post-translational modifications can affect antibody recognition and assay performance. Most commercial kits use recombinant proteins produced in mammalian expression systems to ensure proper folding and modifications .

• Assay validation is essential. If you are developing your own ELISA or substituting the standard in a commercial kit, you must validate that your recombinant ANGPTL3 standard produces a standard curve with appropriate sensitivity, linearity, and parallelism to native samples. Some kit manuals explicitly state that their kits are validated only with their supplied standards and do not recommend using other recombinant proteins unless validated .

• Check for cross-reactivity and specificity. Most well-designed ELISA kits for ANGPTL3 report no significant cross-reactivity or interference between ANGPTL3 and analogues, but this should be confirmed for your specific assay and recombinant standard .

In summary:
You can use recombinant human ANGPTL3 as a standard for ELISA quantification if it is validated for your assay, produces a parallel standard curve to native ANGPTL3, and is of appropriate quality and purity. Always confirm with a parallelism test and consult your assay’s documentation for any specific recommendations or restrictions regarding standard substitution .

Recombinant Human Angiopoietin-Like Protein 3 (ANGPTL3) has been validated in published research for several key applications, primarily in the fields of lipid metabolism, cardiovascular disease, and, more recently, cartilage regeneration.

Validated Applications in Published Research:

• Lipid Metabolism and Lipoprotein Regulation:

  • ANGPTL3 is widely used in in vitro assays to study its role as an inhibitor of lipoprotein lipase (LPL) and endothelial lipase, which are crucial for the regulation of plasma triglyceride and cholesterol levels.
  • Recombinant ANGPTL3 has been applied to cell culture systems (e.g., T37i adipocytes) to assess its effects on LPL activity, lipid uptake, and the interaction with lipoproteins such as HDL and LDL.
  • Studies have used recombinant ANGPTL3 to investigate its binding to lipoprotein particles and its requirement for potentiating LPL inhibition.

• Cardiometabolic Disease Models:

  • ANGPTL3 is a target in preclinical and clinical studies for the development of lipid-lowering therapies, including monoclonal antibodies and antisense oligonucleotides.
  • Recombinant ANGPTL3 has been used to model the effects of genetic or pharmacologic suppression on lipid profiles and cardiovascular risk.

• Cartilage Regeneration and Chondrogenesis:

  • A derivative of ANGPTL3 (LNA043), which retains the chondrogenic domain, has been validated in in vitro assays with human mesenchymal stem cells to induce chondrogenesis and cartilage matrix synthesis.
  • LNA043 and, by extension, recombinant ANGPTL3, have been used in preclinical in vivo models of osteoarthritis and cartilage injury to demonstrate cartilage regeneration and disease-modifying effects.
  • These studies include transcriptomic profiling and safety assessments in human clinical trials.

• Hematopoietic Stem Cell (HSC) Niche Studies:

  • Recombinant ANGPTL3 has been used to support the activity of hematopoietic stem cells in bone marrow niche models, confirming its role in HSC maintenance and localization.

Experimental Techniques and Protocols:

• Cell-based assays: Incubation of recombinant ANGPTL3 with cultured cells to assess effects on lipid metabolism, LPL activity, and gene expression.
• Protein-lipoprotein binding assays: Evaluating the binding of ANGPTL3 to HDL and LDL particles using ultracentrifugation and fractionation.
• Animal models: Administration of ANGPTL3 or its derivatives in mice or other preclinical models to study systemic metabolic effects or tissue regeneration.
• Clinical studies: Use of ANGPTL3 derivatives in human trials for safety, pharmacokinetics, and efficacy in disease contexts such as osteoarthritis.

Summary Table:

These applications are supported by multiple peer-reviewed studies, demonstrating the versatility of recombinant ANGPTL3 in both basic and translational research.

To reconstitute and prepare Recombinant Human Angiopoietin-Like Protein 3 (ANGPTL3) for cell culture experiments, first centrifuge the vial briefly to collect the lyophilized powder at the bottom, then reconstitute in sterile distilled water or PBS to a concentration of 0.1–1.0 mg/mL, depending on your experimental requirements. Avoid vortexing or vigorous pipetting to prevent protein denaturation.

Step-by-step protocol:

• Centrifuge the vial before opening to ensure all protein is at the bottom.
• Add sterile distilled water or PBS to achieve a final concentration of 0.1–1.0 mg/mL (commonly 0.1–0.5 mg/mL for most applications).
  • For example, to reconstitute 100 μg of protein to 0.5 mg/mL, add 200 μL of buffer.
• Gently mix by swirling or inverting the vial; do not vortex or pipette vigorously.
• Allow the protein to dissolve at room temperature for 15–30 minutes. If necessary, gently pipette up and down with a wide-bore tip.
• Aliquot the reconstituted protein into working volumes to avoid repeated freeze-thaw cycles.
• Storage: Store aliquots at –20 °C or –70 °C for long-term storage. The protein is stable for at least one week at 4 °C after reconstitution. Avoid repeated freeze-thaw cycles.

Buffer selection:

• If the protein was lyophilized from a buffer containing PBS, NaCl, or other stabilizers, reconstitute in sterile PBS.
• If the protein was lyophilized without buffer, use sterile distilled water or a buffer compatible with your downstream application.

Preparation for cell culture:

• Before adding to cell culture, dilute the reconstituted protein to the desired working concentration using sterile cell culture medium or buffer.
• Filter-sterilize the final solution if sterility is required and the protein is not already sterile-filtered.

Additional notes:

• Always consult the specific product datasheet for any unique instructions regarding your ANGPTL3 preparation, as tags, truncations, or buffer components may vary by construct.
• For functional assays, confirm the protein is fully dissolved and free of particulates before use.

Summary of key points:

• Centrifuge, reconstitute in sterile water or PBS (0.1–1.0 mg/mL), avoid vortexing, aliquot, and store at –20 °C or below.
• Dilute to working concentration in cell culture medium before use.

If you need a specific working concentration or protocol for a particular cell type or assay, please specify for more tailored guidance.