Adipsin Human, Sf9

What is the molecular structure of Adipsin Human, Sf9?

Adipsin produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 241 amino acids (residues 21-253) with a molecular mass of 26.01 kDa. When visualized using SDS-PAGE, it typically appears at approximately 28-40 kDa due to glycosylation effects. The recombinant protein is expressed with an 8 amino acid histidine tag at the C-terminus to facilitate purification and experimental applications .

The protein maintains the structural integrity necessary for its serine protease function while in the recombinant form. Researchers should note that the glycosylation pattern in insect-derived proteins may differ from that in human-derived adipsin, which could impact certain functional studies. This difference should be considered when designing experiments that focus on protein-protein interactions or enzymatic activity assessments.

What are the optimal storage conditions for Adipsin Human, Sf9?

For short-term applications (2-4 weeks), Adipsin Human, Sf9 can be stored at 4°C. For longer periods, storing at -20°C is recommended. To maintain protein stability during long-term storage, it is advisable to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) . Multiple freeze-thaw cycles should be strictly avoided as they can lead to protein degradation and loss of activity.

Evidence suggests that when stored properly, the protein maintains its structural integrity and functionality for up to 12 months. Researchers should conduct activity assays after extended storage periods to verify protein functionality before proceeding with critical experiments.

How does Adipsin Human, Sf9 differ from natural human Adipsin?

Adipsin Human, Sf9 is a recombinant protein expressed in an insect cell system, whereas natural human Adipsin is produced primarily by adipocytes in vivo. The main differences include:

Despite these differences, Adipsin Human, Sf9 maintains the functional properties necessary for most research applications, particularly for in vitro studies of complement activation, protein-protein interaction analyses, and as a standard in quantitative assays.

How can Adipsin Human, Sf9 be used in cardiovascular disease research?

Adipsin has been implicated in various cardiovascular disease (CVD) processes based on epidemiological and experimental evidence. Researchers can employ Adipsin Human, Sf9 in their cardiovascular studies through several approaches:

• As a biomarker: Multiple studies have identified adipsin as a prognostic marker for coronary artery disease (CAD). Adipsin levels are significantly associated with all-cause death and rehospitalization in CAD patients . Researchers can use purified Adipsin Human, Sf9 as a standard in immunoassays for human plasma samples.

• For mechanistic studies: Adipsin has been linked to pulmonary arterial hypertension (PAH), abdominal aortic aneurysm, and pre-eclampsia . Researchers can use the recombinant protein in cell culture studies to investigate its direct effects on endothelial cells, vascular smooth muscle cells, and immune cells.

• For molecular interaction studies: Understanding how adipsin interacts with other components of the complement system in the context of vascular inflammation could provide insights into therapeutic approaches. The histidine-tagged Adipsin Human, Sf9 can be immobilized for pull-down assays or protein-protein interaction studies.

In experimental models, researchers have demonstrated that CFD deficiency reduces right ventricular remodeling and fibrosis in pulmonary artery constriction models, suggesting adipsin's direct involvement in CVD pathophysiology .

What are the validated methods for measuring Adipsin activity in experimental systems?

Measuring adipsin activity requires specific approaches due to its role as a serine protease in the alternative complement pathway. Validated methods include:

• Complement Activation Assay: Adipsin/CFD activates factor B when bound to C3b, forming the C3bBb complex (C3 convertase). Researchers can measure the formation of this complex or subsequent complement components (C3a, C5a) using ELISA or Western blotting techniques.

• Proteolytic Activity Assay: Using synthetic substrates specific for serine proteases with similar specificity to adipsin. Activity is typically measured through colorimetric or fluorometric detection of cleaved substrate.

• Cell-Based Functional Assays: Measuring adipsin's effects on adipocytes, endothelial cells, or immune cells through readouts such as cytokine production, cell adhesion, or metabolic changes.

When designing these assays, researchers should consider using appropriate controls, including heat-inactivated adipsin (to confirm specificity) and known adipsin inhibitors if available. The recombinant nature of Adipsin Human, Sf9 makes it particularly suitable as a reliable standard in these assays.

How does adipsin interact with other components of the complement pathway?

Adipsin/CFD serves as the rate-limiting factor in the alternative complement pathway. Its primary function is to cleave factor B when bound to C3b, forming the C3bBb complex. The molecular interactions involve:

• Recognition and binding to the C3b-Factor B complex

• Proteolytic cleavage of Factor B into Ba and Bb fragments

• Formation of the active C3 convertase (C3bBb)

• Amplification of complement activation through a positive feedback loop

Researchers studying these interactions can use purified components and surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to determine binding affinities and kinetics. Cell-free reconstitution assays with purified components are also valuable for dissecting the sequence of events in this pathway.

Recent investigations have suggested that adipsin may have additional roles beyond the complement pathway, potentially interacting with adipocyte-specific factors and influencing metabolic processes . This represents an emerging area of research where Adipsin Human, Sf9 could be utilized as a tool to explore novel protein-protein interactions.

How does adipsin contribute to adipocyte-specific gene expression?

Adipsin plays a role in adipocyte function and may influence gene expression patterns specific to adipose tissue. Research has identified a potent adipocyte-specific enhancer in the aP2 gene (an adipocyte-specific gene) that is activated during adipocyte differentiation . While the direct role of adipsin in regulating this enhancer hasn't been fully established, there are several research approaches to investigate this relationship:

• Chromatin Immunoprecipitation (ChIP) assays using antibodies against adipsin or its downstream effectors to identify potential binding to regulatory regions of adipocyte-specific genes.

• Gene expression analyses in adipocytes treated with Adipsin Human, Sf9 or in adipsin-deficient models to identify differentially expressed genes.

• Reporter gene assays with adipocyte-specific promoters/enhancers to determine if adipsin signaling influences their activity.

Research has shown that a 500-bp enhancer fragment upstream of the aP2 gene is activated upon differentiation of preadipocytes into adipocytes, showing at least 15-fold stimulation . This enhancer contains binding sites for transcription factors, including members of the nuclear factor 1 (NF-1) family. Investigating whether adipsin signaling influences the activity of these transcription factors represents an important research direction.

What are the challenges in studying adipsin's role in metabolic diseases?

Researchers face several significant challenges when investigating adipsin's role in metabolic diseases:

• Dual functionality: Adipsin functions both as an adipokine and as complement factor D, making it difficult to dissect which role predominates in specific pathological contexts.

• Tissue-specific effects: While primarily produced by adipocytes, adipsin may have different effects on various tissues, including pancreatic β-cells, vascular endothelium, and immune cells. Designing experiments to address this complexity requires careful planning.

• Species differences: Animal models may not fully recapitulate human adipsin biology. For instance, while adipsin deficiency has been reported in diabetic animals and humans , the functional consequences may differ between species.

• Confounding variables: When studying adipsin in human populations, factors such as age, gender, ethnicity, and comorbidities may influence adipsin levels and activity, complicating data interpretation .

To address these challenges, researchers should consider using multiple complementary approaches, including in vitro studies with Adipsin Human, Sf9, animal models with tissue-specific manipulation of adipsin expression, and carefully designed human studies with appropriate controls for confounding variables.

How can researchers differentiate between the adipokine and complement functions of adipsin?

Differentiating between adipsin's dual roles presents a significant challenge. Methodological approaches include:

• Domain-specific mutants: Creating recombinant adipsin variants with mutations in domains specific to either complement activation or potential adipokine functions, then testing their functional outcomes in relevant assays.

• Selective inhibition: Using inhibitors that specifically block the proteolytic activity of adipsin without affecting potential binding interactions with adipokine receptors.

• Comparative studies: Examining the effects of adipsin versus other complement components in adipocyte and metabolic models to identify unique effects attributable to adipsin beyond complement activation.

• Receptor identification studies: Investigating whether adipsin acts through specific receptors on target cells, distinct from its role in complement activation.

Advanced experimental techniques such as proximity labeling, interactome analysis, and single-cell transcriptomics could help identify cell- and context-specific effects of adipsin that distinguish between its complement and potential adipokine functions.

What controls should be included when working with Adipsin Human, Sf9 in experimental systems?

Robust experimental design requires appropriate controls when working with Adipsin Human, Sf9:

• Negative controls:

  • Heat-inactivated Adipsin Human, Sf9 (typically 95°C for 10 minutes)

  • Empty vector-transfected Sf9 cell-derived protein fraction

  • Vehicle controls for buffer components

• Positive controls:

  • Commercial complement factor D (from human sources when available)

  • Known activators of the complement pathway

  • Standards with validated activity measurements

• Specificity controls:

  • Specific inhibitors of adipsin proteolytic activity

  • Antibodies that neutralize adipsin function

  • Competitive substrates or binding partners

• System validation controls:

  • Cell lines or primary cells known to respond to adipsin

  • Readouts with established sensitivity to complement activation

  • Internal standards for quantitative measurements

Researchers should also address potential endotoxin contamination in recombinant protein preparations, as this could confound results, especially in immune cell-based assays.

How can researchers address the challenge of adipsin degradation during experimental procedures?

Adipsin, like many proteases, can be sensitive to experimental conditions that may affect its stability and activity. To minimize degradation and maintain functional integrity:

• Storage optimization:

  • Use carrier proteins (0.1% HSA or BSA) for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store at recommended temperatures (-20°C for long-term)

• Handling during experiments:

  • Maintain samples on ice when possible

  • Use protease inhibitor cocktails (without serine protease inhibitors if measuring enzymatic activity)

  • Process samples quickly and minimize exposure to room temperature

• Analytical considerations:

  • Include positive controls in each experimental run to confirm activity

  • Consider time-course studies to account for potential degradation

  • Verify protein integrity by SDS-PAGE before critical experiments

• Formulation optimization:

  • Buffer composition can significantly impact stability (consider pH, salt concentration, and additives)

  • Addition of stabilizers like glycerol (10-20%) may enhance stability

Researchers should validate adipsin activity in their specific experimental system at the beginning and end of critical experiments to account for potential activity loss.

What emerging technologies might advance our understanding of adipsin biology?

Several cutting-edge technologies hold promise for deepening our understanding of adipsin biology:

• CRISPR-Cas9 gene editing: Creating precise modifications in adipsin or related genes in relevant cell types to study functional consequences. This approach allows for endogenous tagging of adipsin for real-time visualization or the introduction of specific mutations identified in human populations.

• Cryo-electron microscopy: Determining high-resolution structures of adipsin in complex with its binding partners could provide insights into its molecular mechanisms and guide the development of specific inhibitors or activators.

• Spatial transcriptomics and proteomics: Mapping adipsin expression and activity in tissue contexts with spatial resolution could reveal microenvironmental factors that regulate its function in different physiological and pathological states.

• Single-cell multi-omics: Integrating transcriptomic, proteomic, and metabolomic data at the single-cell level could identify cell populations particularly responsive to adipsin and characterize the downstream molecular events.

• Organoids and microphysiological systems: Studying adipsin in more physiologically relevant 3D culture systems could bridge the gap between traditional cell culture and in vivo models, particularly for investigating adipose tissue-specific effects.

These technologies, combined with computational approaches like machine learning for pattern recognition in large datasets, could help reconcile the sometimes contradictory findings in adipsin research and clarify its diverse roles in health and disease.

How might adipsin research contribute to novel therapeutic approaches for cardiovascular diseases?

Based on the emerging understanding of adipsin's role in cardiovascular pathophysiology, several therapeutic approaches could be developed:

• Diagnostic applications: Serum adipsin has been recognized as a prognostic marker for cardiovascular diseases . Further research could lead to the development of adipsin-based diagnostic panels, potentially combined with other biomarkers like brain natriuretic peptide (BNP) or high-sensitive C-reactive protein (hs-CRP) for improved predictive value.

• Targeted inhibition strategies: For conditions associated with complement overactivation, selective inhibition of adipsin's catalytic activity could reduce alternative pathway activation while preserving classical and lectin pathway function for immune defense.

• Cell-specific modulation: Technologies that allow for tissue-specific regulation of adipsin expression or activity could help address the pathological effects of adipsin in specific cardiovascular contexts while minimizing systemic effects.

• Metabolic intervention: As adipsin levels have been reported to be low in diabetic animals and humans , investigating whether adipsin supplementation could ameliorate metabolic dysfunction in diabetes and consequently reduce cardiovascular risk represents an important research direction.

Given the complex and sometimes contradictory roles of adipsin in different contexts, future therapeutic approaches will likely need to be carefully tailored to specific disease states and patient populations. Continued research with tools like Adipsin Human, Sf9 is essential for developing these precision medicine approaches.