Acrp30 Human, Sf9

What is Acrp30 Human, Sf9 and how does it differ from native adiponectin?

Acrp30 Human, Sf9 refers to human adiponectin recombinantly produced in Sf9 Baculovirus cells. This recombinant form is a single, glycosylated polypeptide chain containing 146 amino acids (residues 106-242) with a molecular mass of 16.9kDa, expressed with a 6-amino acid His tag at the C-terminus for purification purposes .

In contrast, native human adiponectin is a 226-amino acid protein with a molecular mass of 24.54 kDa that is secreted by adipose cells . The key structural difference is that the recombinant form typically represents a truncated version focusing on the globular domain, while native adiponectin contains both a collagen-like domain and a C1q-like globular domain .

Native adiponectin circulates in serum as higher-order complexes (high, medium, and low molecular weight fractions) formed from homotrimeric building blocks , whereas the recombinant form may exhibit different oligomerization properties. These differences should be carefully considered when designing experiments, as they may affect binding properties and physiological responses.

What is the structural composition of Acrp30 Human produced in Sf9 cells?

Acrp30 Human produced in Sf9 cells is a single, glycosylated polypeptide containing 146 amino acids (residues 106-242) with a molecular mass of 16.9kDa . On SDS-PAGE, it typically appears at approximately 13.5-18kDa due to its glycosylation pattern . The protein includes a 6-amino acid His-tag at the C-terminus to facilitate purification .

Structurally, this recombinant form primarily represents the TNF-like or globular domain of adiponectin. The amino acid sequence includes the characteristic globular domain involved in receptor binding and activation. The protein's glycosylation profile in Sf9 cells differs from mammalian expression systems, with insect cells producing simpler glycan structures.

The purity of commercially available Acrp30 Human, Sf9 is typically greater than 95.0% as determined by SDS-PAGE . For structural studies, it's important to note that Sf9 cells contain high levels of oleic acid (C18:1, 48.0%) and stearic acid (C18:0, 17.9%), which may associate with the protein .

How should Acrp30 Human, Sf9 be stored and handled in laboratory settings?

For optimal stability and activity of Acrp30 Human, Sf9, implement the following storage and handling protocols:

• Store lyophilized protein at -20°C for long-term storage .

• After reconstitution, store at 4°C for short-term usage (within a few days) .

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity and integrity .

• For reconstitution, use sterile buffer conditions - typically 20mM TRIS, 50mM NaCl, and 1mM CaCl2, pH 7.5 or similar buffering systems .

• For extended storage, consider adding carrier proteins (0.1% HSA or BSA) to prevent adhesion to tubes and minimize degradation .

• Prepare working aliquots immediately after reconstitution to avoid repeated freezing and thawing.

• Prior to experiments, centrifuge reconstituted protein briefly to remove any potential aggregates.

• When using in cell culture applications, ensure sterile handling techniques and consider filter-sterilization (0.22μm) for sterile culture environments.

• Document lot numbers, reconstitution dates, and freeze-thaw cycles to maintain experimental reproducibility.

What are the recommended applications for Acrp30 Human, Sf9 in cell culture experiments?

Acrp30 Human, Sf9 is suitable for several specialized cell culture applications:

• Receptor binding assays: Effective for studying AdipoR1 and AdipoR2 interactions, particularly the binding properties of the globular domain .

• Metabolic signaling studies: Valuable for investigating AMPK activation, fatty acid oxidation, and glucose uptake in muscle, liver, or adipocyte cell lines .

• Inflammatory response investigations: Can be used to examine inhibitory effects on TNF-alpha-induced expression of endothelial adhesion molecules and NF-kappa-B signaling through cAMP-dependent pathways .

• Ceramidase activity assays: Based on structural insights suggesting ADIPOR ceramidase activity .

Note that the truncated, His-tagged nature of the recombinant protein may influence certain signaling outcomes compared to full-length native adiponectin.

What are the implications of using His-tagged Acrp30 Human, Sf9 in receptor binding studies?

When using His-tagged Acrp30 Human, Sf9 in receptor binding studies, researchers should consider several important methodological implications:

• The C-terminal His-tag may potentially influence receptor binding kinetics or affinity, as it introduces six additional positively charged histidine residues near the globular domain that interacts with adiponectin receptors .

• Control experiments comparing tagged and untagged versions or using tag cleavage approaches should be conducted to assess any tag-related artifacts.

• The tag provides advantages for orientation-specific immobilization in surface plasmon resonance (SPR) or pull-down assays, allowing more controlled binding site presentation than random coupling methods.

• Tag-based purification selects for properly folded proteins that expose the tag, potentially enriching for specific conformational states that may not represent the full spectrum of conformations found in native adiponectin.

• The His-tag can be exploited for quantitative receptor binding assays using anti-His antibodies or Ni-NTA conjugates as detection systems.

• When designing competition assays between tagged recombinant protein and native adiponectin, consider that differential receptor binding may reflect tag effects rather than true biological differences.

How does Acrp30 Human, Sf9 interact with free fatty acids in structural and functional studies?

The interaction between Acrp30 Human, Sf9 and free fatty acids (FFAs) represents an important consideration in both structural and functional studies:

• Structural analyses of adiponectin receptors have revealed the presence of FFAs, particularly oleic acid (C18:1), within a large internal cavity of the receptor .

• This is significant because oleic acid is the predominant unsaturated fatty acid in Sf9 insect cells (48.0%), followed by stearic acid (C18:0, 17.9%) .

• The aliphatic chain binding pocket for FFAs is formed by hydrophobic residues from the transmembrane helices TM5, TM6, and TM7 of the adiponectin receptor .

• Different crystal structures have shown distinct binding poses for the carboxylic acid moiety of FFAs:

  • In one configuration (S1), it directly coordinates with the zinc ion

  • In others (S2 and S3), it positions away from zinc and forms alternative polar contacts

• When designing functional assays with Acrp30 Human, Sf9, researchers should consider that co-purified FFAs might influence binding kinetics, receptor activation, or downstream signaling events.

• For accurate interpretation of experimental outcomes, control experiments using defatted protein preparations or competitive binding with defined fatty acid species may help differentiate intrinsic adiponectin effects from those influenced by associated lipids.

What are the considerations for using Acrp30 Human, Sf9 in studies examining adiponectin receptor activity and ceramidase function?

When investigating adiponectin receptor activity and ceramidase function using Acrp30 Human, Sf9, researchers should implement the following methodological approaches:

• Recent structural insights revealed that adiponectin receptors (ADIPORs) possess ceramidase activity, suggesting that Acrp30 Human, Sf9 can be used to study this enzymatic function .

• The truncated nature of the recombinant protein (containing only the globular domain) may affect its ability to activate the full spectrum of ADIPOR-mediated ceramidase activity compared to full-length adiponectin .

• When designing ceramidase activity assays, account for the presence of free fatty acids (FFAs) that may co-purify with the recombinant protein from Sf9 cells, as structural studies have identified oleic acid bound within the receptor's internal cavity .

• Experimental design recommendations:

  • Control experiments using defatted protein preparations

  • Site-directed mutagenesis of key residues in the binding pocket

  • Zinc-dependent versus zinc-independent activity assessments

  • Comparative analyses with other adiponectin forms (full-length, mammalian-expressed)

• For ADIPOR2 studies, pay attention to the three distinct binding poses observed for the carboxylic acid moiety of FFAs, which may influence zinc coordination and enzymatic activity .

• Consider combining recombinant protein stimulation with structural biology techniques (X-ray crystallography) and site-directed mutagenesis for comprehensive insights into the relationship between adiponectin binding and ceramidase activity regulation.

How does the Sf9-produced Acrp30 compare to other expression systems for structural studies?

When conducting structural studies with Acrp30, the choice of expression system significantly impacts protein properties:

• The Sf9 baculovirus expression system produces recombinant Acrp30 with specific post-translational modifications and lipid associations that may affect structural analyses .

• Crystallographic studies of ADIPOR2 complexed with Sf9-expressed proteins revealed important structural features including:

  • The 7TM architecture of the receptor

  • A zinc-binding site crucial for function

  • The presence of bound fatty acids within an internal cavity

• Data processing for structural determination of Sf9-expressed adiponectin complexes typically requires specialized approaches:

  • Simultaneous processing of data from multiple crystals using programs like XDS

  • Cluster analysis procedures using BLEND to find optimal merging combinations

  • Considerations for anisotropic diffraction patterns

• Refinement of structural models with Sf9-expressed proteins often involves:

  • Iterative building in software like Coot

  • Refinement cycles using programs such as AutoBuster

  • Application of translation libration screw-motion (TLS) parameters

• When interpreting electron density maps from Acrp30-receptor complexes, researchers should consider omitting fatty acids in initial building cycles to obtain unbiased difference maps for subsequent lipid modeling .

What molecular approaches can be used to study multimerization of Acrp30 in experimental systems?

Adiponectin naturally forms multimeric structures, and researchers can explore this property using various molecular approaches:

• The Acrp30 molecule can be engineered to produce defined multimeric forms by fusion with multimerization domains:

  • Fusion with the body of Acrp30 itself can produce 2-trimer forms

  • Fusion with surfactant protein D can produce 4-trimer forms

• For constructing plasmids encoding multimeric forms, researchers can use approaches such as:

  • PCR amplification of the 5' untranslated region and coding sequence of Acrp30

  • Creating fusion constructs using overlapping PCR primers

  • Designing appropriate linker sequences at fusion junctions

• A specific example of a fusion junction sequence would be: ...KGEPGELQGDEDPQIA..., where:

  • The N-terminal portion derives from Acrp30 (amino acids 1-109)

  • LQ serves as a linker sequence

  • The C-terminal portion represents the TNF-like domain

• Expression and detection methodologies:

  • Transient transfection of 293T cells using Lipofectamine 2000

  • Purification using biotinylated antibodies bound to streptavidin-coated magnetic beads

  • Analysis by SDS-PAGE followed by Western blotting

• For detecting multimeric forms, researchers can use:

  • Primary antibodies such as goat anti-mouse CD40L or rabbit anti-mouse GITRL

  • Secondary detection with horseradish peroxidase-conjugated antibodies

  • Visualization via chemiluminescence

How can researchers verify the quality and activity of Acrp30 Human, Sf9 preparations?

Comprehensive quality control for Acrp30 Human, Sf9 preparations should include:

• Purity assessment:

  • SDS-PAGE analysis under reducing and non-reducing conditions (expected purity >95%)

  • Size exclusion chromatography to evaluate oligomeric state distribution

  • Mass spectrometry to confirm molecular weight and post-translational modifications

• Structural integrity verification:

  • Circular dichroism spectroscopy to assess secondary structure

  • Intrinsic fluorescence spectroscopy to evaluate tertiary structure

  • Limited proteolysis to confirm proper folding

• Functional activity assays:

  • Receptor binding assays using cells overexpressing AdipoR1 or AdipoR2

  • AMPK phosphorylation in responsive cell lines (e.g., C2C12, HepG2)

  • NF-κB inhibition assays in endothelial cells

• Contaminant analysis:

  • Endotoxin testing (Limulus Amebocyte Lysate assay) to ensure preparations are endotoxin-free

  • Host cell protein ELISA to quantify residual Sf9 cell proteins

  • Lipidomic analysis to characterize and quantify co-purified lipids

• Stability testing:

  • Accelerated stability studies at different temperatures

  • Monitoring activity retention over time using functional assays

  • Analyzing freeze-thaw stability to establish optimal aliquoting guidelines