SELE Human, Sf9

What is SELE Human Recombinant protein and what are its basic properties?

SELE Human Recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 541 amino acids (22-556 a.a) with a molecular mass of 59.4kDa. The protein is typically fused to a 6 amino acid His-tag at the C-terminus to facilitate purification by chromatographic techniques . SELE is also known by several other names including E-selectin, Endothelial leukocyte adhesion molecule 1 (ELAM-1), Leukocyte-endothelial cell adhesion molecule 2 (LECAM2), and CD62E antigen . This recombinant protein maintains biological activity, as measured by its ability to support the adhesion of U937 human histiocytic lymphoma cells when immobilized on plates at 2μg/ml, with an effect greater than 40% . The complete amino acid sequence is available and includes various structural domains necessary for its adhesion functions.

How should SELE Human, Sf9 protein be stored to maintain optimal activity?

For optimal stability and retention of biological activity, SELE Human, Sf9 should be stored according to specific protocols depending on the intended usage timeframe. For short-term use (within 2-4 weeks), the protein can be stored at 4°C in its original formulation . For longer-term storage, it is recommended to freeze the protein at -20°C, ideally with the addition of a carrier protein such as 0.1% HSA (Human Serum Albumin) or BSA (Bovine Serum Albumin) to enhance stability and prevent loss of activity . Multiple freeze-thaw cycles should be carefully avoided as they can lead to protein denaturation, aggregation, and loss of biological function. The protein's biological activity can be verified through functional assays measuring its ability to support adhesion of U937 cells, with properly maintained samples showing greater than 40% effect in this assay . Researchers should consider aliquoting the purified protein before freezing to minimize the need for repeated freeze-thaw cycles.

What expression and purification methods yield high-quality SELE Human in Sf9 cells?

High-quality SELE Human production in Sf9 cells relies on optimized baculovirus expression systems and multi-step purification techniques. The expression process typically involves infecting Sf9 cells cultured in serum-supplemented or serum-free media with a recombinant baculovirus carrying the SELE gene, similar to other baculovirus expression systems described in the literature . For optimal expression, cells are generally maintained at 28°C in either adherent culture using Grace's Insect Medium supplemented with 10% FBS or in suspension using Sf-900II SFM in shake flasks at 130 rpm . Transfection of Sf9 cells is commonly performed using specialized reagents like Cellfectin II . The purification process leverages the C-terminal His-tag, employing immobilized metal affinity chromatography followed by additional chromatographic techniques to achieve greater than 95% purity as determined by SDS-PAGE . The purification strategy must account for potential contaminants specific to the Sf9/baculovirus system, including residual Sf9 host cell proteins, DNA, and baculovirus components .

How do post-translational modifications of Sf9-expressed SELE differ from mammalian-expressed versions?

The post-translational modification profile of SELE expressed in Sf9 cells differs substantially from mammalian-expressed versions, particularly regarding glycosylation patterns. Sf9 cells, being of insect origin, produce predominantly high-mannose type N-glycans rather than the complex, sialylated glycans characteristic of mammalian expression systems. This fundamental difference stems from the limited glycosylation machinery in insect cells, which lack several glycosyltransferases responsible for complex glycan formation. The altered glycosylation pattern may affect protein-protein interactions, particularly those involving the lectin domain of SELE which recognizes carbohydrate structures on leukocytes. Despite these differences, Sf9-expressed SELE maintains core biological functionality, as evidenced by its ability to support U937 cell adhesion . For research requiring mammalian-like glycosylation, engineered Sf9 cell lines expressing mammalian glycosyltransferases can be employed, similar to specialized Sf9 lines developed for other applications . Researchers should consider these glycosylation differences when interpreting binding studies or when investigating subtle regulatory mechanisms dependent on specific glycan structures.

What analytical techniques are most appropriate for characterizing SELE Human, Sf9 protein quality?

Comprehensive characterization of SELE Human, Sf9 requires multiple complementary analytical techniques addressing different quality attributes. For purity assessment, SDS-PAGE and size exclusion chromatography are fundamental techniques, with the product specification indicating greater than 95% purity by SDS-PAGE . Glycosylation profiling can be performed using mass spectrometry-based glycoproteomics or lectin binding assays to characterize the high-mannose glycan structures typical of Sf9 expression. Identity confirmation involves peptide mapping by liquid chromatography-mass spectrometry (LC-MS) to verify the 541 amino acid sequence (22-556 a.a) as specified in the product data . Functional characterization requires cell-based adhesion assays, specifically measuring the protein's ability to support U937 cell adhesion when immobilized at 2μg/ml with an expected effect greater than 40% . For researchers concerned about host cell impurities, specialized assays similar to the residual Sf9 host cell and baculovirus DNA detection methods mentioned for viral vector production could be adapted . Advanced structural characterization might include circular dichroism spectroscopy to assess secondary structure integrity or differential scanning calorimetry to evaluate thermal stability and proper folding.

How can researchers optimize baculovirus vectors specifically for high-yield SELE expression?

Optimizing baculovirus vectors for high-yield SELE expression requires strategic engineering of multiple vector elements. Researchers can implement a dual-function baculovirus expression vector design similar to the BEV/Cap-(ITR-GOI) system described for rAAV production , which provides flexibility and high expression levels. The vector should incorporate strong promoters such as the polyhedrin or p10 promoters, which drive high-level expression in the late phase of baculovirus infection. Codon optimization of the SELE sequence for Sf9 cells can significantly enhance translation efficiency and protein yields. Including an efficient signal sequence at the N-terminus facilitates proper translocation into the secretory pathway, critical for a glycoprotein like SELE. The C-terminal His-tag, as used in the commercially available SELE , enables efficient purification while minimally affecting protein function. For challenging expression cases, incorporation of chaperone co-expression cassettes can improve folding efficiency. Development of stable Sf9 cell lines expressing components of the expression system, similar to the Sf9-GFP/Rep packaging cell line described for rAAV production , can provide consistent protein quality and eliminate batch-to-batch variability associated with transient baculovirus infection.

What considerations are important when designing functional assays using SELE Human, Sf9?

When designing functional assays with SELE Human, Sf9, researchers must account for several critical factors to ensure reliable and physiologically relevant results. First, the immobilization strategy significantly impacts protein activity - the standard functional assay described uses 2μg/ml coating concentration for plate-based cell adhesion studies , but alternative strategies like oriented immobilization through the His-tag might preserve activity better for certain applications. Researchers should consider the effects of different buffer compositions, particularly divalent cation concentrations (Ca²⁺, Mg²⁺), which are essential for SELE's lectin domain function. Appropriate positive controls (such as mammalian-expressed SELE) and negative controls (non-functional SELE mutants or irrelevant proteins) should be included to validate assay specificity. Since SELE-mediated adhesion is often studied under flow conditions in vivo, researchers developing more physiologically relevant assays might consider flow-based systems rather than static adhesion assays alone. Temperature control during assays is critical - while SELE is produced in Sf9 cells at 28°C , functional assays should be conducted at physiological temperatures (37°C) to reflect human biology. For studies evaluating potential inhibitors of SELE-mediated adhesion, researchers should establish clear dose-response relationships and consider testing across multiple cell types expressing different SELE ligands.

What are common challenges in expressing full-length functional SELE in Sf9 cells and how can they be addressed?

Expression of full-length functional SELE in Sf9 cells presents several challenges due to its complex structure and glycoprotein nature. Protein misfolding often occurs with multi-domain proteins like SELE; this can be mitigated by lowering the expression temperature to 25-27°C (instead of the standard 28°C) to slow protein synthesis and allow proper folding. Proteolytic degradation can be problematic for large proteins expressed at high levels; researchers should consider adding protease inhibitors to culture media and optimizing harvest timing (typically 48-72 hours post-infection) to maximize intact protein yield. Low expression levels may result from suboptimal codon usage; implementing Sf9-optimized codon sequences can enhance translation efficiency. Aggregation during expression or purification is common with membrane-associated proteins like SELE; this can be addressed by adding low concentrations of non-ionic detergents or stabilizing agents to extraction and purification buffers. Insufficient glycosylation or improper disulfide bond formation affects protein function and stability; employing specialized Sf9 cell lines engineered to enhance these post-translational modifications, similar to the engineered lines described for other applications , can produce more authentic protein. For purification challenges, implementing on-column refolding techniques during affinity chromatography can rescue misfolded protein and increase the yield of functional SELE.

What analytical methods are effective for detecting and quantifying host cell contaminants in purified SELE preparations?

Effective detection and quantification of host cell contaminants in purified SELE preparations require sensitive analytical methods targeted at specific impurity classes. For Sf9 host cell proteins (HCPs), enzyme-linked immunosorbent assays (ELISAs) using antibodies raised against Sf9 cell lysates can detect residual host proteins with high sensitivity. More comprehensive HCP profiling can be achieved using liquid chromatography-mass spectrometry (LC-MS/MS) with data-dependent acquisition. For residual Sf9 host cell DNA contamination, quantitative PCR (qPCR) assays can be employed, similar to those mentioned for viral vector production from Sf9 cells . The search results specifically mention that "customer feedback indicates that the E1A, kanamycin, and Sf9 baculovirus strips also perform well on dPCR platforms" , suggesting that droplet digital PCR (dPCR) provides a sensitive alternative for detecting specific DNA sequences. Residual baculovirus detection is critical for research applications; this can be accomplished through qPCR targeting baculovirus-specific sequences or through in vitro transduction assays with heat-treated samples as described in the search results, where "heat-treated and untreated control samples were then added to 96-well plates to infect either HEK293 cells or Sf9 cells" . For endotoxin contamination, the standard Limulus Amebocyte Lysate (LAL) assay provides sensitive detection, though this is generally less critical for insect cell-derived proteins compared to bacterial expression systems.

What experimental controls should be included when using SELE Human, Sf9 for cell adhesion studies?

Robust cell adhesion studies using SELE Human, Sf9 require carefully designed experimental controls to ensure valid and interpretable results. Positive controls should include a known functional form of E-selectin, ideally mammalian-expressed SELE or purified native protein, to establish maximum binding capacity and proper assay performance. Negative controls must include both uncoated surfaces and surfaces coated with an irrelevant protein of similar size and properties to control for non-specific adhesion. Blocking controls using function-blocking antibodies against SELE provide critical evidence for binding specificity, while competitive inhibition using soluble known ligands for SELE can further validate specific interactions. Since bivalent cations are critical for SELE function, including EDTA-treated samples serves as a functional negative control. For mechanistic studies, specific glycosidase treatments of the cell surface can establish the dependency on particular carbohydrate structures. When testing cell lines for SELE binding, researchers should include both known positive cell lines (such as U937 cells, which are used in the standard activity assay showing >40% binding effect ) and negative cell lines lacking the appropriate ligands. Temperature controls are important since selectin-mediated binding has temperature dependencies; conducting parallel experiments at 4°C versus 37°C can distinguish between energy-dependent and energy-independent adhesion processes.

How can researchers optimize transfection conditions for SELE expression in Sf9 cells?

Optimizing transfection conditions for SELE expression in Sf9 cells requires systematic evaluation of multiple parameters to achieve maximal protein yield and quality. Based on the search results discussing Sf9 cell transfection, Cellfectin II reagent is commonly used for effective DNA delivery to these insect cells . For optimal results, researchers should evaluate the DNA:transfection reagent ratio, typically starting with manufacturer recommendations and then testing ratios ranging from 1:2 to 1:6 to identify the optimal proportion for SELE plasmid delivery. Cell density at transfection significantly impacts success - Sf9 cells should ideally be at 80-90% confluence for adherent cultures or at 1-2 × 10^6 cells/mL for suspension cultures to balance transfection efficiency with subsequent protein expression capacity . The medium composition during transfection affects DNA-lipid complex formation and cellular uptake; transfection should be performed in serum-free medium initially, with serum-containing medium added several hours post-transfection. The timing between transfection and baculovirus amplification is critical; typically, viral supernatants should be harvested 72-96 hours post-transfection when signs of late-stage infection are visible. For researchers developing stable Sf9 cell lines expressing SELE, selection agent concentration and timing must be optimized, similar to the approach used for the stable Sf9-GFP/Rep packaging cell line where "pools of cells were generated that stably expressed" the proteins of interest .

What statistical approaches are recommended for analyzing SELE binding and adhesion assay data?

Robust statistical analysis of SELE binding and adhesion assay data requires appropriate experimental design and analytical methods to account for the biological variability inherent in these systems. For adhesion assays measuring U937 cell binding to immobilized SELE (as described in the biological activity data showing >40% effect ), researchers should employ multivariate ANOVA to assess the effects of different experimental variables (protein concentration, buffer composition, temperature) while accounting for potential interactions between factors. Dose-response curves should be analyzed using non-linear regression to determine EC50 values, Hill coefficients, and maximum binding capacity, which provide quantitative parameters for comparing different experimental conditions. To account for the non-normal distribution often observed in cell adhesion data, non-parametric statistical tests such as Mann-Whitney U or Kruskal-Wallis should be considered when comparing experimental groups. For more complex experiments examining SELE-mediated rolling adhesion under flow conditions, time-series analysis and survival analysis methods can quantify adhesion dynamics and detachment rates. Internal standards and normalization procedures are essential for controlling plate-to-plate variability; researchers should normalize raw data to the positive control (maximum binding) on each plate. Power analysis should be conducted during experimental design to determine the appropriate number of technical and biological replicates needed to detect statistically significant differences of the expected magnitude between experimental conditions.

How can researchers integrate SELE Human, Sf9 into advanced in vitro models of inflammation?

Integration of SELE Human, Sf9 into advanced in vitro models of inflammation requires thoughtful experimental design that recapitulates key aspects of the inflammatory microenvironment. Researchers can develop parallel plate flow chambers coated with purified SELE Human, Sf9 at the biologically active concentration (2μg/ml as indicated in the product specifications ) to study leukocyte rolling and adhesion under physiologically relevant shear stress conditions. Microfluidic devices offer more sophisticated platforms where SELE can be immobilized in specific patterns or gradients to investigate spatial aspects of leukocyte recruitment. Co-immobilization of SELE with other adhesion molecules (ICAM-1, VCAM-1) and chemokines creates more complex models that better represent the multi-step leukocyte adhesion cascade. For dynamic models, researchers can use cytokine-stimulated endothelial cells transfected to overexpress SELE alongside the purified protein as comparative systems. Three-dimensional tissue models incorporating SELE-coated surfaces or particles within extracellular matrix components can examine leukocyte migration in more complex environments. Advanced imaging techniques such as real-time confocal microscopy paired with fluorescently labeled cells allow visualization of dynamic interactions between leukocytes and immobilized SELE. For studies examining SELE-mediated signal transduction, the recombinant protein can be clustered using secondary antibodies or presented on nano/microparticles to mimic the clustering that occurs during cellular interactions, potentially triggering signaling events that wouldn't occur with monomeric protein.

Table 1: Key Properties of SELE Human, Sf9 Recombinant Protein

Table 2: Sf9 Cell Culture Conditions for Optimal Protein Expression