Acrp30 Porcine, HEK

What is Acrp30 Porcine and how does it differ from human Adiponectin?

Acrp30 Porcine (porcine adiponectin) is a 238-amino acid protein with a molecular mass of 26 kDa that belongs to the soluble defense collagen superfamily. When produced in HEK293 cells, it is typically fused to a 13-amino acid N-terminal FLAG tag for purification and detection purposes . Structurally, it has a collagen-like domain homologous with collagen VIII and X and a complement factor C1q-like globular domain .

The porcine variant shares approximately 80-85% sequence homology with human adiponectin (244 amino acids), though both contain characteristic collagen and globular domains essential for their function. These species-specific differences may affect epitope recognition and receptor binding, which researchers should consider when designing cross-reactivity experiments or using porcine models for translational research.

Why is the HEK293 cell line preferred for Acrp30 expression?

The HEK293 cell line is preferred for Acrp30 expression for several reasons:

• Post-translational modifications: HEK293 cells provide mammalian-type glycosylation patterns important for adiponectin function

• Proper protein folding: Unlike bacterial systems, HEK293 cells correctly form disulfide bonds essential for native conformation

• Secretion efficiency: HEK293 cells efficiently secrete the protein into culture medium, facilitating purification

• Biological activity: HEK-derived adiponectin demonstrates confirmed functionality in in vitro assays, such as inhibiting glucose production in hepatocytes with an ED50 of approximately 6 μg/ml

• Oligomerization: The mammalian expression system enables formation of proper homotrimers, which are building blocks for higher-order complexes found in serum

Research has shown that HEK293 cells effectively produce biologically active Acrp30 that maintains its physiological properties, making this expression system valuable for generating research-grade protein.

What are the structural characteristics of Acrp30?

Acrp30 has several distinctive structural features that are crucial to its function:

• Domain organization: Contains a collagen-like domain and a C1q-like globular domain

• Oligomerization: Forms homotrimers that serve as building blocks for higher-order complexes

• Post-translational modifications: Undergoes glycosylation that affects oligomerization and function

• Molecular weight: The monomeric form of porcine Acrp30 has a molecular mass of 26 kDa

• Tag fusion: Recombinant versions often include N-terminal tags (like FLAG) that add approximately 1-2 kDa to the molecular weight

When analyzed by SDS-PAGE under reducing conditions, the protein appears as a single band at approximately 26-30 kDa, but under native conditions, it forms higher molecular weight complexes. The collagen domain facilitates triple helix formation in oligomeric structures, while the globular domain is responsible for receptor binding.

How should Acrp30 Porcine be stored and handled to maintain stability?

For optimal stability and functionality of Acrp30 Porcine, researchers should follow these guidelines:

• Long-term storage: Store lyophilized protein at -20°C, where it remains stable for up to 24 months

• Reconstitution: Add deionized water to the lyophilized pellet and allow it to dissolve completely

• Aliquoting: After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles

• Short-term storage: Reconstituted protein can be stored at 4°C for up to two weeks without significant change

• Buffer composition: Typically formulated in buffers such as 20 mM Tris with 50 mM NaCl at pH 7.5

Proper handling is critical as adiponectin is sensitive to denaturation. Avoid vigorous shaking that may disrupt protein structure, and use low-protein binding tubes to prevent adsorption to container surfaces when working with dilute solutions.

What experimental applications are suitable for Acrp30 Porcine expressed in HEK cells?

Acrp30 Porcine expressed in HEK cells can be used in various research applications:

• ELISA development: For quantitative measurement of adiponectin levels, using systems like the DuoSet ELISA

• Western blotting: For detection and analysis of adiponectin expression and post-translational modifications

• Cell culture studies: To investigate adiponectin signaling pathways, particularly in relation to insulin sensitivity

• Gluconeogenesis assays: To study inhibitory effects on glucose production in hepatocytes, with demonstrated ED50 of ~6 μg/ml

• Gene therapy models: As evidenced by studies using adeno-associated virus vectors encoding adiponectin to treat obesity and insulin resistance

• Metabolic pathway analysis: To examine effects on PEPCK and SREBP-1c expression in the liver, key genes in gluconeogenesis and lipogenesis

Recombinant Acrp30 has been successfully used in studies demonstrating significant reductions in body weight, food intake, and improved peripheral insulin sensitivity in animal models .

What mechanisms explain Acrp30's effect on hepatic glucose metabolism?

Acrp30 regulates hepatic glucose metabolism through several molecular mechanisms:

• PEPCK suppression: Acrp30 significantly reduces phosphoenolpyruvate carboxykinase (PEPCK) expression in the liver, which is the rate-limiting enzyme in gluconeogenesis

• AMPK activation: Adiponectin activates AMP-activated protein kinase (AMPK), leading to phosphorylation and inactivation of acetyl-CoA carboxylase

• Lipogenesis regulation: Acrp30 modulates SREBP-1c (sterol regulatory element-binding protein 1c) expression, affecting hepatic lipid metabolism

• Insulin sensitization: Enhances insulin signaling pathways in hepatocytes

Research has demonstrated that in Sprague-Dawley rats, high-fat diet significantly increases PEPCK levels, while Acrp30 expression reduces PEPCK expression even below levels observed in normal diet control animals . This powerful suppression of gluconeogenesis contributes to the protein's ability to improve glucose homeostasis and insulin sensitivity.

How do different expression systems affect Acrp30 functionality?

The expression system significantly impacts Acrp30 functionality through several mechanisms:

HEK293-derived Acrp30 demonstrates superior functionality in research applications due to proper post-translational modifications that preserve the native structure and oligomerization capacity . Studies have shown that HEK293 cells secreting Acrp30 exhibit improved protein production characteristics , making this system particularly valuable for generating functionally relevant adiponectin for research purposes.

What factors influence the oligomerization state of recombinant Acrp30?

The oligomerization state of recombinant Acrp30 is influenced by several factors:

• Post-translational modifications: Glycosylation and hydroxylation of lysine residues in the collagen domain are critical for higher-order structure formation

• Buffer conditions: pH, ionic strength, and presence of divalent cations affect assembly of oligomers

• Protein concentration: Higher concentrations favor formation of larger oligomeric complexes

• Reducing agents: Presence of reducing agents disrupts disulfide bonds essential for maintaining multimeric structures

• Temperature: Both storage and experimental temperatures influence oligomer stability

• Tags: N-terminal tags may slightly modify the oligomerization properties

The distribution of oligomeric forms (trimers, hexamers, and high-molecular-weight multimers) is functionally significant as research indicates that high-molecular-weight forms correlate more strongly with insulin sensitivity. Therefore, characterizing the oligomeric profile is crucial for interpreting functional outcomes in experimental systems using recombinant Acrp30.

How can researchers assess Acrp30 activity in metabolic assays?

For reliable assessment of Acrp30 activity in metabolic assays, particularly gluconeogenesis inhibition:

• Cell model selection:

  • Primary hepatocytes preferred over hepatoma cell lines

  • Freshly isolated cells maintain higher sensitivity to adiponectin

• Experimental conditions:

  • Culture cells in glucose-free medium with gluconeogenic substrates (10 mM lactate, 1 mM pyruvate)

  • Use concentration range of 1-10 μg/ml (ED50 ~6 μg/ml for HEK-derived adiponectin)

  • Optimal incubation time: 4-6 hours for glucose production effects

• Controls:

  • Positive control: Insulin (10-100 nM)

  • Negative control: Inactive protein or buffer

  • Vehicle control: Buffer used for protein reconstitution

• Readout methods:

  • Direct measurement: Glucose in culture medium using enzymatic assays

  • Molecular endpoints: PEPCK and G6Pase expression by RT-qPCR

  • Signaling verification: AMPK and ACC phosphorylation via Western blotting

Research has demonstrated that adiponectin significantly reduces PEPCK expression levels in liver, a key enzyme in the gluconeogenesis pathway, confirming its metabolic effects .

What technical considerations are important when using FLAG-tagged Acrp30?

When working with FLAG-tagged Acrp30, researchers should consider several technical aspects:

• Detection considerations:

  • FLAG tag enables consistent detection using standardized anti-FLAG antibodies

  • May alter antibody recognition of native epitopes in adiponectin-specific assays

• Functional implications:

  • N-terminal FLAG tags (DYKDDDDK) introduce negative charges that may subtly influence receptor binding

  • Most studies show FLAG-tagged adiponectin retains AMPK phosphorylation capability

  • The tag appears to preserve biological activity in gluconeogenesis inhibition assays

• Experimental design:

  • For critical functional studies, confirm key findings with untagged protein when possible

  • In immunoprecipitation experiments, consider potential steric hindrance from the tag

  • For crystallography studies, the flexible tag may impede crystal formation

• Quantitative analysis:

  • Standard curves must use the same form (tagged or untagged) as experimental samples

  • The tag adds approximately 1 kDa to the molecular weight, affecting migration in gels

The Acrp30 Porcine recombinant typically contains the 238-amino acid native protein fused to a 13-amino acid N-terminal FLAG tag, resulting in specific detection properties that researchers should account for in experimental design .

How do in vivo and in vitro Acrp30 effects correlate in research models?

The correlation between in vivo and in vitro effects of Acrp30 shows both similarities and important differences:

• Metabolic effects:

  • In vitro: Direct inhibition of gluconeogenesis in hepatocytes (ED50 ~6 μg/ml)

  • In vivo: Sustained peripheral expression reduces body weight by 8-18% and improves glucose tolerance

• Dose-response relationships:

  • In vitro: Linear dose-response curves typically observed

  • In vivo: Complex relationships due to tissue distribution, receptor regulation, and compensatory mechanisms

• Temporal dynamics:

  • In vitro: Rapid effects observed within hours

  • In vivo: Both acute effects and prolonged adaptations (over weeks to months) with sustained expression

• Long-term adaptations:

  • In vivo studies show sustained peripheral expression of Acrp30 (using viral vectors) results in significant weight reduction over 41 weeks, with reduced food intake and improved feed efficiency

  • This duration of effect isn't reproducible in vitro

• Mechanistic insights:

  • Both systems show Acrp30 reduces PEPCK expression, confirming a common mechanism of action

  • In vivo models additionally demonstrate effects on SREBP-1c expression, linking adiponectin to lipid metabolism regulation

Long-term studies using recombinant adeno-associated virus vectors encoding Acrp30 have demonstrated sustained (up to 280 days) significant reduction in body weight and improved insulin sensitivity, validating the physiological relevance of in vitro findings .