RLN2 Human, Sf9

What are the known physiological functions of RLN2?

RLN2 is a peptide hormone with diverse physiological roles. Primarily produced by the ovary, it targets the mammalian reproductive system where it:

• Ripens the cervix during pregnancy

• Elongates the pubic symphysis

• Inhibits uterine contractions

Beyond reproductive functions, RLN2 is involved in several other physiological processes:

• Enhancement of sperm motility

• Regulation of blood pressure

• Control of heart rate

• Release of oxytocin and vasopressin

At the molecular level, RLN2 is linked to therapeutically relevant effects including regulation of collagen metabolism and involvement in multiple vascular control pathways . These diverse functions make RLN2 an important target for reproductive biology, cardiovascular research, and tissue remodeling studies.

How does RLN2 relate to other members of the insulin superfamily?

RLN2 belongs to the insulin gene superfamily, which includes insulin, insulin-like growth factors, and other relaxin peptides. In humans, there are three non-allelic relaxin genes: RLN1, RLN2, and RLN3 . RLN1 and RLN2 share high sequence homology, suggesting functional redundancy or specialization .

The structural similarity between relaxins and insulin includes:

• The characteristic A and B chain arrangement

• Disulfide bond connectivity patterns

• Similar tertiary folding patterns

Despite these similarities, RLN2 binds to distinct G protein-coupled receptors rather than receptor tyrosine kinases used by insulin, which accounts for its different physiological effects. The evolutionary conservation of RLN2 across species highlights its fundamental biological importance, though its precise role may vary between organisms .

What experimental approaches can be used to study RLN2 activity in physiological systems?

To investigate RLN2 activity in physiological systems, researchers can employ several methodological approaches:

Cell-based assays:

• cAMP accumulation assays using cells expressing RXFP1/RXFP2 receptors to measure receptor activation

• ERK1/2 phosphorylation assays to assess downstream signaling

• Collagen production/degradation assays in fibroblasts to study effects on extracellular matrix remodeling

Ex vivo tissue models:

• Myometrial contractility studies using isolated uterine strips

• Vascular reactivity assays with isolated blood vessels to measure vasodilatory effects

• Cardiac tissue preparations to assess chronotropic and inotropic effects

In vivo models:

• Targeted delivery of recombinant RLN2 to specific tissues

• Transgenic models with controlled RLN2 expression

• Receptor knockout models to evaluate specificity of observed effects

When designing these experiments, researchers should consider using appropriate positive controls and specific inhibitors to validate RLN2-specific effects. The recombinant RLN2 protein expressed in Sf9 cells provides a valuable tool for such studies due to its high purity (>85% by SDS-PAGE) and proper post-translational modifications .

How do post-translational modifications of RLN2 produced in Sf9 cells compare to native human RLN2?

Recombinant RLN2 produced in Sf9 Baculovirus cells undergoes insect cell-specific post-translational modifications, which differ in some aspects from native human RLN2:

Glycosylation patterns:
The Sf9-expressed RLN2 is glycosylated, but insect cells produce simpler, high-mannose type N-glycans compared to the complex glycans found in human cells. This results in the SDS-PAGE mobility difference, with the protein appearing between 18-28 kDa despite its calculated mass of 19.3 kDa .

Proteolytic processing:
While native RLN2 requires specific proteolytic processing to generate the functional A and B chains, recombinant RLN2 may have different cleavage patterns. The exact cleavage sites for generating mature RLN2 are not definitively described yet .

Disulfide bond formation:
The insect cell expression system generally supports proper disulfide bond formation, which is critical for RLN2 function. The recombinant protein likely maintains the correct disulfide bonding pattern connecting the A and B chains .

When performing functional studies, researchers should consider these differences and potentially validate key findings with native human RLN2 or alternative expression systems. For structural studies, the His-tagged recombinant protein offers advantages in terms of purification efficiency and yield .

What are the challenges in distinguishing between RLN2-specific effects and those of other relaxin family members?

Distinguishing RLN2-specific effects from those of other relaxin family members presents several methodological challenges:

Receptor promiscuity:
RLN2 primarily signals through RXFP1 receptors but can also activate RXFP2 with lower affinity. Similarly, other relaxin family peptides may cross-react with these receptors, complicating the interpretation of observed physiological responses.

Sequence homology:
The high sequence homology between RLN1 and RLN2 (approximately 80%) makes it difficult to develop truly specific antibodies or detection methods . This becomes particularly challenging when studying endogenous expression patterns.

Methodological approaches to address these challenges:

• Receptor-specific antagonists: Employ selective receptor antagonists to block specific relaxin-receptor interactions

• siRNA/shRNA knockdown: Use RNA interference approaches targeting specific relaxin family members or receptors

• CRISPR-Cas9 gene editing: Generate cell lines or animal models with specific relaxin family gene knockouts

• Recombinant protein controls: Include carefully designed experiments with recombinant RLN1, RLN2, and RLN3 to demonstrate specificity

• Receptor expression profiling: Characterize receptor expression in target tissues to interpret observed effects

By combining these approaches, researchers can more confidently attribute observed effects to RLN2 specifically, rather than to other relaxin family members or non-specific interactions .

What are the optimal storage and handling conditions for recombinant RLN2 Human, Sf9?

To maintain the structural integrity and biological activity of recombinant RLN2 Human, Sf9, researchers should follow these evidence-based storage and handling guidelines:

Storage conditions:

• Store the protein in small aliquots at -80°C for long-term storage

• Avoid repeated freeze-thaw cycles as this can lead to protein denaturation and loss of activity

• For working solutions, store at 4°C for up to 1 week

Buffer composition:
The recombinant protein is typically supplied as a sterile filtered colorless solution . Optimal buffer conditions include:

• pH 7.2-7.4 phosphate or Tris-buffered saline

• Addition of 0.1% BSA or HSA as a carrier protein to prevent adsorption to surfaces

• Consider adding protease inhibitors for extended storage

Handling recommendations:

• Use low-binding microcentrifuge tubes and pipette tips

• Centrifuge the vial briefly before opening to ensure all liquid is at the bottom

• Handle the protein on ice when preparing working solutions

• Validate protein integrity by SDS-PAGE before critical experiments

By adhering to these storage and handling protocols, researchers can maximize the stability and functionality of recombinant RLN2 Human, Sf9 in their experimental systems .

What analytical techniques are recommended for validating RLN2 Human, Sf9 structure and activity?

Several complementary analytical techniques are recommended for comprehensive validation of recombinant RLN2 Human, Sf9:

Structural validation:

Functional validation:

• Receptor binding assays: Using cells expressing RXFP1 or RXFP2 receptors with radiolabeled or fluorescently labeled RLN2

• cAMP signaling assays: Measuring cAMP accumulation in receptor-expressing cells following RLN2 stimulation

• Cell-based functional assays: Including:

  • Collagen production in fibroblasts

  • Vasodilation in vascular smooth muscle cells

  • Chronotropic effects in cardiomyocytes

The supplied recombinant RLN2 Human, Sf9 is tested by SDS-PAGE and has a purity >85% . Researchers should consider performing additional validation specific to their experimental systems to ensure consistency across studies .

How can researchers optimize the use of recombinant RLN2 in cell culture experiments?

To optimize the use of recombinant RLN2 Human, Sf9 in cell culture experiments, researchers should consider these methodological aspects:

Dose determination:

• Perform dose-response experiments (typically 0.1-100 nM range) to determine optimal concentrations for specific cell types

• Consider that primary cells may respond differently than immortalized cell lines

• Include positive controls with known RLN2-responsive cell types

Treatment duration:

• Acute responses (cAMP, calcium signaling): 5-60 minutes

• Intermediate responses (gene expression): 4-24 hours

• Long-term responses (collagen remodeling): 24-72 hours

Experimental conditions:

• Serum considerations: Reduce or eliminate serum during RLN2 treatment to minimize interference from serum factors

• Cell density: Optimize cell confluency (typically 70-80%) for consistent responses

• Medium composition: Consider using phenol red-free medium for fluorescence-based assays

• Receptor expression: Verify RXFP1/RXFP2 receptor expression in your cell model

Controls and validation:

• Include vehicle controls with the same buffer composition as the RLN2 preparation

• Use receptor antagonists or siRNA knockdown to confirm receptor specificity

• Consider including related peptides (insulin, IGF-1) as specificity controls

By methodically addressing these considerations, researchers can establish robust cell culture protocols for studying RLN2 biology and ensure reproducibility across experiments .