CD46 Human, Sf9

What is CD46 Human, Sf9 and how is it produced?

CD46 Human, Sf9 is a recombinant form of human Membrane Cofactor Protein (MCP) produced in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The production process involves cloning the human CD46 gene (typically amino acids 35-313) into a baculovirus transfer vector, generating recombinant baculoviruses containing the CD46 gene, and infecting Sf9 cells for protein expression. The expressed CD46 is typically fused with a 6-amino acid His-tag at the C-terminus to facilitate purification through proprietary chromatographic techniques .

The final product is a glycosylated polypeptide chain containing 268 amino acids with a molecular mass of 32.5kDa, though it appears at approximately 40-57kDa on SDS-PAGE due to glycosylation patterns in Sf9 cells . CD46 Human, Sf9 is normally formulated as a sterile filtered colorless solution in phosphate-buffered saline (pH 7.4) containing 10% glycerol .

What are the key functions of CD46 in complement regulation?

CD46 serves as a critical regulator of the complement system through several mechanisms:

CD46's primary function is acting as a cofactor for inactivation of complement components C3b and C4b by serum factor I. This cofactor activity resides in the complement control protein repeats (CCPs), with CCP1–4 being vital for this regulatory function . By facilitating the degradation of these complement components, CD46 prevents excessive activation of the complement cascade on host cells.

The protein's regulatory action disrupts the formation of the membrane attack complex (MAC) that would otherwise create pores in cell membranes and lead to cell lysis . This protective mechanism is essential for preventing complement-mediated damage to self tissues during immune responses.

Additionally, CD46 is involved in the fusion of spermatozoa with the oocyte during fertilization, indicating its multifunctional nature beyond complement regulation . Understanding these functions is crucial when designing experiments using CD46 Human, Sf9 for studying complement regulation or developing complement-resistant biological systems.

Why use Sf9 cells for CD46 expression in research applications?

Sf9 insect cells offer several distinct advantages for CD46 expression compared to mammalian systems:

Sf9 cells can produce high quantities of recombinant proteins, which is beneficial for applications requiring substantial amounts of purified CD46. The baculovirus-Sf9 system is easily scalable for larger production when needed.

An important experimental advantage is that Sf9 cells lack a conserved complement pathway like that found in mammalian cells . This absence of an endogenous complement system makes them ideal for studying human complement proteins without interference from host cell proteins that might confound results.

While not identical to mammalian glycosylation, Sf9 cells perform key post-translational modifications including glycosylation, which contributes to CD46 function . These cells can accommodate large gene inserts and express proteins that might be toxic to mammalian cells.

Researchers should note that while Sf9-expressed CD46 is functionally active, the glycosylation patterns differ from those in human cells. This consideration becomes important depending on the specific research question being addressed, particularly for studies where glycan structure may influence protein-protein interactions.

How can CD46 be combined with other complement regulatory proteins for enhanced protection?

Research has demonstrated that combining CD46 with other complement regulatory proteins (CRPs) provides superior protection against complement attack. Based on the search results, several methodological approaches have been developed:

One effective strategy involves creating fusion proteins combining CD46 with other CRPs like DAF (CD55) and CD59. Researchers have developed Sf9 cell lines that stably express CD46, DAF, or CD46-DAF-CD59 fusion proteins under the control of the OpIE2 promoter in pIB plasmids .

The methodology for developing these stable cell lines includes:

• Transfection of Sf9 cells with pIB plasmids containing CRP expression units

• Selection with blasticidin (10 μg/mL)

• FACS purification using fluorescently labeled antibodies to enrich high-expression populations

These stably expressing cells can then be infected with baculoviruses containing other functional proteins to produce virions displaying multiple protective components. Experimental evidence shows that CSP-CD46-DAF-CD59 type baculovirus vectors demonstrate significantly improved complement resistance compared to vectors with just one CRP .

What experimental controls are essential when studying CD46-mediated complement regulation?

When investigating CD46-mediated complement regulation using CD46 Human, Sf9, implementing appropriate experimental controls is crucial for interpretable results:

Serum-related controls should include both intact and heat-inactivated serum comparisons to confirm complement-specific effects. The research indicates that C3-depleted human serum fails to activate complement-mediated lysis, making it a valuable negative control . Testing varying serum concentrations establishes dose-dependent effects and helps determine optimal experimental conditions.

For protein-related controls, researchers should include:

• Non-functional CD46 mutants that lack cofactor activity

• Concentration gradients to establish dose-response relationships

• Alternative CRPs (e.g., DAF/CD55 alone) for comparing inhibitory mechanisms

• Control proteins expressed in the same Sf9 system to account for expression system artifacts

When testing CD46-displaying baculovirus vectors, control vectors without CD46 or with other CRPs provide essential comparisons. The research specifically describes using CSP type BV (without CRPs) and control type BV (no CSP, no CRPs) as comparative standards .

Time-course experiments are valuable for distinguishing between delayed and prevented complement activation, as some inhibitors may only delay rather than block MAC formation. When using fluorescent labeling techniques, appropriate isotype controls should be included to account for non-specific binding .

What methods verify proper folding and function of CD46 expressed in Sf9 cells?

Confirming the proper folding and function of CD46 expressed in Sf9 cells requires multiple complementary approaches:

SDS-PAGE and Western Blot analysis serve as fundamental techniques to verify the molecular weight and purity of expressed CD46. The research indicates that CD46 Human from Sf9 cells has a molecular mass of 32.5kDa but appears at approximately 40-57kDa on SDS-PAGE due to glycosylation . Purity assessments typically target >90.0% as determined by SDS-PAGE .

Flow cytometry analysis provides critical information about proper membrane localization. Researchers use fluorescently labeled anti-CD46 monoclonal antibodies (e.g., MEM258-FITC) to detect surface expression on transfected cells. This approach verifies that the protein is correctly transported to the cell membrane, which is essential for its native function .

Functional cofactor assays directly test CD46's ability to act as a cofactor for factor I-mediated cleavage of C3b and C4b. This involves measuring the degradation of purified complement components in the presence of CD46 and factor I, with degradation products visualized by SDS-PAGE and Western blotting.

Complement resistance assays evaluate the ability of CD46-expressing vectors or cells to resist complement-mediated lysis by comparing lysis rates between samples exposed to intact serum versus heat-inactivated serum. The search results describe such assays for testing baculovirus vectors incorporating CD46 .

How does CD46 Human, Sf9 contribute to baculovirus vector development for gene therapy?

The incorporation of CD46 into baculovirus vectors (BVs) represents a significant advancement for gene therapy applications, with the search results providing detailed methodological approaches:

Researchers have developed a system using stably expressing Sf9 cells that display CD46 (alone or as fusion proteins with other CRPs). These cells are transfected with pIB plasmids containing CD46 under the OpIE2 promoter, and high-expressing populations are selected using blasticidin and enriched through FACS sorting .

When these stably expressing cells are infected with baculoviruses containing therapeutic genes, the resulting viral particles incorporate CD46 into their envelope during budding. The modified viruses can then be purified from the culture medium, with titers remaining comparable to those from parental Sf9 cells (approximately 10^8–10^9 pfu/mL) .

The functional impact of this modification is substantial - CSP-CD46-DAF-CD59 type BVs exhibited a 20-fold higher transduction efficacy in HepG2 cells compared to control BVs when exposed to intact serum . This dramatic improvement overcomes one of the major limitations of baculovirus vectors: their rapid inactivation by the complement system in vivo.

This approach offers significant versatility, as it allows for the combination of multiple functional proteins in BVs without creating entirely new viral constructs. The method is particularly valuable for gene therapy targeting the liver, where complement activation can severely limit vector efficacy .

What challenges arise when using CD46 Human, Sf9 for studying reproductive biology?

CD46 plays an important role in reproductive biology, particularly in the fusion of spermatozoa with the oocyte during fertilization . When using CD46 Human, Sf9 to investigate these reproductive functions, researchers face several significant challenges:

The differing glycosylation patterns between Sf9-expressed CD46 and native human CD46 present a major consideration. Sf9 cells produce proteins with high-mannose type glycans and glycans with trimannosyl cores , whereas human cells generate complex and hybrid type N-glycans. These glycosylation differences may critically impact CD46's binding properties and interactions with gamete-specific proteins.

Another challenge relates to membrane association. Native CD46 in reproductive tissues is membrane-bound, whereas CD46 Human, Sf9 is typically produced as a soluble protein with a His-tag . For reproductive biology studies, the protein would need to be appropriately reconstituted or immobilized to mimic this membrane context.

Multiple isoforms of CD46 exist due to alternative splicing, with the search results noting that recombinant CD46 typically corresponds to a specific isoform (isoform 14) . Since reproductive functions may be isoform-specific, careful selection of the appropriate variant is essential for meaningful results.

Methodologically, researchers must design physiologically relevant assays that isolate CD46's specific role in fertilization while controlling for species-specific interactions when using human CD46 with animal gametes. Complementary approaches, such as CD46 knockdown/knockout models, can help validate findings obtained using the recombinant protein.

How can researchers troubleshoot decreased activity of CD46 Human, Sf9?

When researchers encounter decreased activity of CD46 Human, Sf9 in experimental systems, a systematic troubleshooting approach is necessary:

The first consideration should be protein quality and storage conditions. The search results specify that CD46 should be stored at 4°C if used within 2-4 weeks or at -20°C for longer periods . Excessive freeze-thaw cycles should be avoided, and the addition of carrier proteins (0.1% HSA or BSA) is recommended for long-term storage .

Structural integrity assessment is critical. Researchers should perform SDS-PAGE to check for degradation or aggregation and consider additional techniques like size exclusion chromatography to evaluate monomer/oligomer status. Circular dichroism can provide information about secondary structure integrity.

For complement regulation assays specifically, serum quality is paramount. Fresh serum sources should be used as complement activity degrades over time. Proper storage of serum samples (aliquoted, stored at -80°C) and verification of complement activity using standard hemolytic assays help ensure reliable results.

Buffer and reaction conditions can significantly impact activity. Researchers should verify buffer composition (pH, salt concentration, presence of divalent cations), temperature control during assays, and optimize incubation times for cofactor activity.

When conducting factor I cofactor assays, confirming the activity of factor I itself and the quality of C3b/C4b substrates is essential. Concentration optimization through dose-response experiments with CD46 and adjustment of component ratios may help restore activity.

What methods quantify CD46 incorporation into baculovirus vectors?

Several complementary methodologies enable researchers to quantify CD46 incorporation into baculovirus vectors:

Western blot analysis represents a fundamental approach specifically mentioned in the research for detecting CRPs in viral particles . Purified virion samples are processed through SDS-PAGE, transferred to membranes, and probed with anti-CD46 antibodies. Densitometric analysis of bands compared against standards of known CD46 quantities provides quantitative information about total CD46 incorporation per viral preparation.

Flow cytometry offers a valuable approach for surface display quantification. Though the search results specifically describe this technique for detecting cell surface expression of CRPs , it can be adapted for viral particles using bead-based systems. Viral particles captured on beads and stained with fluorescently labeled anti-CD46 antibodies can be analyzed to measure surface-displayed CD46.

Functional quantification through complement resistance assays provides information about the biological relevance of CD46 incorporation. By comparing survival rates of different viral preparations after exposure to complement, researchers can determine the functional consequence of CD46 display. The research describes comparing intact serum versus heat-inactivated serum exposure as a method for this assessment .

Advanced techniques like immunogold electron microscopy enable direct visualization of CD46 on viral surfaces using gold-labeled antibodies. This approach provides information about both the distribution and density of CD46 on individual virions, which can be quantified by counting gold particles per virion.

How do glycosylation differences between Sf9 and human cells affect CD46 function?

The glycosylation differences between Sf9-expressed and human CD46 have several important functional implications:

Sf9 cells produce high-mannose type glycans and glycans with trimannosyl cores, while human cells generate complex and hybrid type N-glycans with terminal sialic acid residues . These structural differences explain why CD46 Human, Sf9 appears at 40-57kDa on SDS-PAGE despite having a calculated size of 32.5kDa .

These glycosylation differences may affect CD46's complement regulatory efficiency by altering binding affinities for C3b and C4b. The specific glycan structures can influence protein-protein interactions that are crucial for cofactor activity, potentially modifying the kinetics of these interactions.

Protein stability is another consideration, as altered glycosylation can impact the half-life and structural integrity of CD46 in experimental systems. Different glycan patterns may provide varying degrees of protection against proteolytic degradation.

For applications involving immune cells or in vivo studies, different glycosylation patterns could potentially influence immunogenicity, as glycan structures are recognized by various pattern recognition receptors of the immune system.

When using CD46 Human, Sf9 for research, these glycosylation differences should be considered in experimental design and interpretation. Validation of functional activity in the specific experimental system being used is recommended, particularly for studies where glycosylation may significantly impact protein-protein interactions or cellular recognition.

How can stably expressing Sf9 cells be utilized beyond CD46 research?

The development of stably expressing Sf9 cells represents a versatile platform with applications extending beyond CD46 research:

The search results highlight that "the utilization of stably expressing Sf9 cells to introduce the protein products of the gene of interest would be a useful strategy to generate BVs with novel functions" . This approach parallels packaging cell methodologies used for retrovirus vectors, where cells stably express viral components.

This technology enables researchers to incorporate various proteins of interest into baculovirus particles without creating entirely new viral constructs. The methodology streamlines the development process, as researchers can infect existing stable cell lines with different baculoviruses to create customized vectors.

Applications could extend to:

• Expression of targeting ligands to direct vectors to specific cell types

• Incorporation of immune-modulatory proteins to reduce vector immunogenicity

• Display of enzymes or binding proteins that facilitate extracellular matrix penetration

• Production of vectors displaying therapeutic proteins for direct delivery

The technical advantage of this approach is that it doesn't require time-consuming development of new BV constructs for each application. The search results note that "production of this combination of BVs does not require the generation of new BV constructs, which would be time-consuming" .

What insights do CD46-transgenic models provide for complement regulation research?

While the search results don't provide direct information about CD46-transgenic models, the functional interchangeability of human and mouse complement pathway proteins has important research implications:

The search results note that "human CD46 shows some functional interchangeability between species" with mouse and human CD46 sharing approximately 48-51% identity and 64% similarity . This suggests that human CD46 can functionally regulate mouse complement activation, though with potential differences in efficiency.

In mouse models, CD46 expression is more restricted than in humans, being predominantly found in the testes and retina. Instead, another protein called Crry (complement receptor 1-related protein/gene y) is more widely expressed and performs similar functions to both CD46 and CD55 .

These cross-species interactions provide important context for interpreting data from:

• Humanized mouse models expressing human CD46

• Studies using human CD46 in mouse experimental systems

• Comparative analyses of complement regulation mechanisms across species

Understanding these interspecies differences and similarities helps researchers design appropriate controls and interpret results when studying complement regulation in various model systems, particularly when translating findings between mouse models and human applications.