Recombinant Human Relaxin-2

Recombinant human relaxin-2 offers several compelling advantages for cardiovascular research applications, particularly in studying heart failure and related pathologies.

Physiological Relevance and Mechanism of Action

Recombinant relaxin-2 provides a well-characterized tool for investigating endogenous hormone signaling through the relaxin family peptide receptor 1 (RXFP1). This G protein-coupled receptor activation triggers multiple downstream signaling cascades that are directly relevant to cardiovascular physiology, making it an excellent model system for understanding hormone-receptor interactions in disease states.

Broad Spectrum of Cardioprotective Effects

The protein demonstrates multiple therapeutic mechanisms that are valuable for mechanistic research:

Hemodynamic and vascular effects: Recombinant relaxin-2 induces vasodilation, reduces systemic vascular resistance, increases arterial compliance, and enhances cardiac output. These properties make it ideal for studying vascular biology and hemodynamic regulation.

Anti-fibrotic and anti-hypertrophic activity: The protein attenuates myocardial fibrosis and cardiac hypertrophy through regulation of extracellular matrix turnover and remodeling. This is particularly valuable for investigating pathological cardiac remodeling in heart failure models.

Anti-inflammatory and cytoprotective functions: Recombinant relaxin-2 reduces inflammation, oxidative stress, and apoptosis while promoting angiogenesis. These properties enable comprehensive studies of cardioprotection mechanisms.

Metabolic modulation: The protein influences glucose uptake in cardiomyocytes and modulates lipid metabolism, providing opportunities to study metabolic aspects of cardiac dysfunction.

Established Preclinical and Clinical Track Record

The protein has demonstrated efficacy in multiple animal models, including mouse cardiac injury models and non-human primate models with cardiac dysfunction. Clinical trials have established its safety profile and shown improvements in clinical symptoms in acute heart failure patients, providing confidence in its biological activity and translational relevance.

Extended Half-Life Variants Available

While native recombinant relaxin-2 has a short serum half-life, engineered variants—such as fusion proteins combining relaxin-2 with antibody Fc fragments—now achieve circulating half-lives of 3-5 days in mice while maintaining full RXFP1 agonist activity. This enables more physiologically relevant dosing regimens in long-term studies.

These characteristics make recombinant human relaxin-2 a valuable research tool for cardiovascular disease modeling, drug development, and mechanistic studies of heart failure pathology.

Recombinant Human Relaxin-2 can be used as a standard for quantification or calibration in ELISA assays, provided the assay is validated for recombinant protein and the standard curve is established using the same recombinant form.

Most commercial ELISA kits for human Relaxin-2 are designed to detect both native and recombinant forms, and several protocols specifically mention the use of recombinant human Relaxin-2 as a standard:

• The Quantikine Human Relaxin-2 ELISA kit uses E. coli-expressed recombinant human Relaxin-2 as a standard and demonstrates that results obtained with natural human Relaxin-2 are parallel to those obtained with the recombinant standard curve, indicating suitability for quantification.
• Recombinant human Relaxin-2 is recommended for use as an ELISA standard, especially when formulated with carrier proteins like BSA to enhance stability and solubility.
• The Human Relaxin-2 DuoSet ELISA includes a recombinant standard for quantification, confirming its appropriateness for calibration.

Key considerations for using recombinant Relaxin-2 as a standard:

• Ensure the recombinant protein is of high purity and properly quantified.
• Validate that your ELISA kit or custom assay detects recombinant Relaxin-2 with comparable sensitivity and specificity as native protein.
• Prepare a standard curve using serial dilutions of the recombinant protein in the same matrix as your samples to account for potential matrix effects.
• Confirm that the assay’s antibodies recognize epitopes present on the recombinant form, especially if the recombinant protein differs in post-translational modifications or folding from the native protein.

Limitations:

• Some ELISA kits are optimized for native Relaxin-2 and may not guarantee identical reactivity with recombinant forms; always consult the kit’s technical documentation.
• If your recombinant protein is a fusion or truncated variant, verify that it retains the relevant epitopes for antibody recognition.

Best practice:
Use recombinant human Relaxin-2 as a standard only after confirming compatibility with your specific ELISA system, and always establish a standard curve with the recombinant protein under your assay conditions for accurate quantification.

Recombinant human relaxin-2 has been validated for a diverse range of applications across multiple therapeutic and research domains, as demonstrated in published literature.

Functional and Bioassay Applications

Recombinant relaxin-2 is validated for functional assays and bioassays, serving as a research tool to investigate the hormone's biological activity and receptor interactions. These applications are fundamental for characterizing the protein's ability to activate the relaxin family peptide receptor 1 (RXFP1) and downstream signaling cascades.

Cardiovascular Disease Research

The most extensively studied application involves cardiovascular disease models. Recombinant relaxin-2 has been validated in preclinical studies demonstrating its capacity to prevent and reverse isoproterenol-induced cardiac hypertrophy and fibrosis in mouse models. The protein has shown efficacy in attenuating endothelial dysfunction, reducing myocardial inflammation, inhibiting fibrosis, and promoting angiogenesis. Clinical investigations have progressed through more than 15 human trials examining relaxin-2's therapeutic potential in acute and chronic heart failure, preeclampsia, and systemic sclerosis.

Hepatic Disease Models

Recombinant relaxin-2 has been validated in in vivo applications for non-alcoholic fatty liver disease, where it demonstrated the ability to attenuate hepatic steatosis and fibrosis in mouse models.

Musculoskeletal and Joint Applications

The protein has been validated for intraarticular injection therapy, with published research demonstrating its effectiveness in alleviating shoulder arthrofibrosis (frozen shoulder) in murine models. Additionally, sustained-release formulations of relaxin-2 microparticles have been validated for restoring range of motion in joint applications, with effects sustained for up to 8 weeks following a single injection.

Bone Regeneration

Recombinant relaxin-2 carried by magnetically directed liposomes has been validated in in vivo rat models for accelerating midpalatal suture expansion and subsequent new bone formation.

Structural and Biochemical Characterization

The protein is suitable for biochemical applications including SDS-PAGE analysis, enabling validation of protein purity, molecular weight confirmation, and structural integrity assessment.

Reconstitution Protocol

Recombinant Human Relaxin-2 is typically supplied as a lyophilized powder and requires proper reconstitution before use in cell culture experiments. The protein is a non-glycosylated, 6.0 kDa disulfide-linked heterodimer consisting of a 24 amino acid A-chain and a 29 amino acid B-chain.

Initial Preparation Steps

Begin by equilibrating both the protein vial and reconstitution buffer to room temperature. This step is critical to prevent osmotic stress on the protein. Gently centrifuge the vial to collect any powder from the cap and walls.

Reconstitution Buffer and Concentration

Reconstitute the protein in phosphate-buffered saline (PBS) at pH 7.4 to achieve a working concentration of 0.1–1.0 mg/mL. Alternatively, some formulations recommend reconstituting at 100 μg/mL in PBS containing at least 0.1% human or bovine serum albumin (BSA) as a carrier protein. The inclusion of BSA helps stabilize the protein and prevents non-specific adsorption to container surfaces.

Reconstitution Procedure

Add the appropriate volume of buffer to the vial and avoid vortexing or vigorous mixing, as this can cause foaming and protein denaturation. Instead, gently agitate the vial for 15–30 minutes at room temperature. If visible flakes remain after this period, continue mixing for up to 2 hours at room temperature with gentle agitation until complete dissolution occurs.

Allow the reconstituted stock solution to sit for a minimum of 15 minutes with gentle agitation before preparing working dilutions.

Storage and Stability

Short-term Storage

After reconstitution, the protein can be stored at 2–8°C for up to one month under sterile conditions. This temperature range is suitable for experiments planned within a short timeframe.

Long-term Storage

For extended storage, maintain the reconstituted protein at −20°C to −70°C in a manual defrost freezer. The lyophilized powder itself remains stable for 12 months from the date of receipt when stored at −20°C to −70°C.

Critical Handling Considerations

Avoid repeated freeze-thaw cycles, as these can compromise protein integrity and biological activity. To minimize this risk, apportion the reconstituted protein into working aliquots before freezing. This approach allows you to thaw only the volume needed for each experiment without repeatedly exposing the entire stock to temperature fluctuations.

Sample Preparation for Cell Culture

When using reconstituted Relaxin-2 in cell culture experiments, prepare biological samples appropriately. For cell culture supernatants or other biological fluids, centrifuge samples for 20 minutes at 1,000×g at 2–8°C and collect the supernatant for assay. For cell lysates, use 3–4 freeze-thaw cycles in liquid nitrogen to release cellular components, then centrifuge at 4°C for 20 minutes at 2,000–3,000 rpm to pellet debris.

Key Precautions

• Never vortex the reconstituted protein solution
• Change pipette tips between additions to avoid cross-contamination
• Use sterile, low-endotoxin buffers for dilutions if preparing for sensitive cell culture applications
• Protect from light during extended incubations, particularly if using enzyme-linked detection methods downstream

Following these protocols ensures optimal protein stability, maintains biological activity, and provides reliable results in your cell culture experiments.