FAS Human, Sf9

What are the advantages of using Sf9 insect cells for human FasL expression?

Sf9 insect cells offer several significant advantages for recombinant human FasL production. The baculovirus-Sf9 system provides high-level expression of functionally active proteins with proper folding and appropriate post-translational modifications. Researchers have successfully produced human FasL under the control of a polyhedrin promoter in this system, yielding sufficient quantities to induce apoptosis in target cells such as T98G human glioblastoma cell lines .

Additional advantages include:

• Scalable production capabilities for larger experimental needs

• Growth in suspension culture at room temperature without CO2 requirements

• Cost-effectiveness compared to mammalian expression systems

• Ability to accommodate large protein constructs

• Simplified purification through available affinity tag systems

The system's effectiveness is evidenced by the successful expression of functional FasL that retains apoptosis-inducing capabilities when tested against target cells .

How do baculovirus vectors compare with other expression systems for producing functional human FasL?

Baculovirus vectors in Sf9 cells offer distinct advantages over bacterial, yeast, and some mammalian expression systems for human FasL production:

The evidence indicates that FasL expressed in Sf9 cells retains functional activity, as demonstrated by its ability to specifically induce apoptosis in T98G cells . The selection between these systems should be based on specific requirements for protein authenticity, yield, and intended applications.

What are the typical infection conditions for optimal FasL expression in Sf9 cells?

For optimal FasL expression in Sf9 cells, several parameters require careful optimization. While specific conditions for FasL are not detailed in the search results, insights can be drawn from related protein expression studies. For instance, in the CXCR4 expression study, researchers infected Sf9 cells at a density of 3.0 × 10^6 cells/ml with a 1:100 dilution of high-titer baculovirus stocks and cultured them for 48 hours before membrane preparation .

Recommended infection parameters include:

• Cell density at infection: 1.5-2.0 × 10^6 cells/ml (balances cell density with nutrient availability)

• Multiplicity of infection (MOI): 2-10 for primary infections

• Harvest time: 48-72 hours post-infection (48 hours was used in study )

• Culture temperature: 27-28°C as mentioned in the CXCR4 expression protocol

• Culture media: SF 900 II medium supplemented with 5% fetal calf serum

Cell culture typically occurs in Erlenmeyer flasks under rotation at 125 rpm, with cells maintained at densities between 0.5-6.0 × 10^6 cells/ml . These parameters should be systematically optimized for each specific protein construct.

How can post-translational modifications of human FasL in Sf9 cells affect its functional activity?

Post-translational modifications (PTMs) of human FasL expressed in Sf9 cells significantly impact its functional activity. As a type II membrane protein that can be proteolytically cleaved to release a soluble form, several PTM considerations are critical:

• Glycosylation differences: Sf9 cells perform simpler high-mannose type N-glycosylation compared to the complex glycosylation in mammalian cells. This difference may affect FasL binding affinity, stability, and immunogenicity.

• Proteolytic processing: Proper proteolytic processing is essential for generating soluble FasL. The search results demonstrate that FasL is released into the supernatant of cultured Sf9 cells and verified by Western blotting , confirming that Sf9 cells can process the protein appropriately.

• Oligomerization: Active FasL exists as a trimer, and proper assembly is essential for activity. Differences in the cellular environment may affect this oligomerization process.

• Disulfide bond formation: Correct disulfide bond formation is crucial for FasL structure. While Sf9 cells generally support disulfide bond formation, the efficiency may differ from mammalian systems.

What analytical methods are most effective for characterizing FasL expressed in Sf9 cells?

Comprehensive characterization of FasL expressed in Sf9 cells requires multiple complementary analytical methods:

• Protein identification and verification:

  • Western blotting with specific antibodies (as performed in study using "rabbit antibody raised against the cytoplasmic domain of human FasL")

  • Mass spectrometry for sequence confirmation

  • N-terminal sequencing to verify correct processing

• Structural characterization:

  • SDS-PAGE under reducing and non-reducing conditions to assess disulfide bonding

  • Size exclusion chromatography to determine oligomeric state

  • Circular dichroism spectroscopy for secondary structure analysis

• Functional verification:

  • Apoptosis induction in Fas-expressing target cells (T98G cells were used in study )

  • Annexin V-FITC staining to confirm apoptosis (as performed in study )

  • Dose-response studies to determine specific activity

• PTM analysis:

  • Glycosylation profiling using mass spectrometry or lectin binding assays

  • Deglycosylation studies to assess impact on activity

  • Site-specific PTM mapping

These comprehensive approaches ensure that the expressed FasL maintains the correct structural and functional properties necessary for experimental applications. The methods should be selected based on the specific research questions being addressed.

What strategies can overcome poor expression of human FasL in the baculovirus-Sf9 system?

Overcoming poor expression of human FasL in the baculovirus-Sf9 system requires systematic optimization of multiple parameters:

• Vector and construct optimization:

  • Codon optimization for Sf9 cells' preferred codon usage

  • Testing different signal sequences for secretion

  • Using strong promoters like polyhedrin (mentioned in study )

  • Adding purification tags that may enhance expression (histidine tags were successfully used for other proteins in studies and )

  • Removing problematic sequences (cryptic splice sites, premature termination codons)

• Infection optimization:

  • Optimizing multiplicity of infection (MOI) and cell density at infection

  • Testing different harvest times (48-72 hours post-infection)

  • Adjusting temperature, pH, and media composition

  • Using protease inhibitors to prevent degradation

• Cell and virus considerations:

  • Using different Sf9 cell subclones or alternative insect cell lines

  • Fresh amplification of baculovirus stocks to ensure viability

  • Testing different baculovirus backbone vectors

• Co-expression strategies:

  • Co-expression with chaperones to aid folding

  • Expression as fusion protein with solubility enhancers

  • The research indicates that Sf9 cells can tolerate co-infection with multiple baculoviruses , enabling these strategies

If these approaches fail to improve expression, alternative systems such as mammalian cell expression might be considered, though the search results indicate successful expression of functional human FasL in Sf9 cells has been achieved .

What are the most sensitive methods for detecting residual Sf9 host cell and baculovirus DNA in purified FasL preparations?

Detecting residual Sf9 host cell and baculovirus DNA in purified FasL preparations requires sensitive analytical methods. According to search result , this represents a significant analytical challenge in viral vector manufacturing. The most effective methods include:

• Quantitative PCR (qPCR):

  • Targets specific sequences in Sf9 genomic DNA and baculovirus DNA

  • Can detect very low levels of contaminating DNA (down to a few copies)

  • Commercial kits have been developed specifically for this purpose and have been purchased by more than 100 different customers with no known issues regarding acceptance by regulatory agencies

• Droplet Digital PCR (ddPCR):

  • Provides absolute quantification without standard curves

  • Higher precision at low copy numbers

  • Customer feedback indicates that E1A, kanamycin, and Sf9 baculovirus assays perform well on ddPCR platforms

• Residual DNA-specific assay kits:

  • Commercial kits specifically designed for host cell DNA detection

  • The resDNASEQ kits mentioned are species-specific and meet specificity requirements

  • Extensive exclusion primer testing is performed to ensure specificity

For regulatory compliance, it's important to validate these methods for specific production processes and demonstrate consistent clearance of host cell and viral DNA below acceptable limits.

What are the most reliable methods to quantify the apoptotic activity of Sf9-produced human FasL?

Reliable quantification of apoptotic activity of Sf9-produced human FasL requires robust and sensitive assays:

• Flow cytometry-based methods:

  • Annexin V-FITC/PI staining to discriminate early apoptotic from late apoptotic/necrotic cells (as used in study )

  • TUNEL assay to detect DNA fragmentation

  • Active caspase-3/7 detection using fluorogenic substrates

• Biochemical detection methods:

  • Caspase activity assays using specific substrates

  • DNA fragmentation assays

  • PARP cleavage detection by Western blotting

  • Cytochrome c release from mitochondria

• Cell viability and cytotoxicity assays:

  • MTT/XTT/WST-1 reduction assays (similar to the cell proliferation assay used in study )

  • Time-course analysis to differentiate apoptosis from other forms of cell death

Study specifically confirmed FasL-induced apoptosis by annexin V-fluorescein isothiocyanate staining, which is considered a gold standard for apoptosis detection. For quantitative analysis, dose-response curves should be established to determine EC50 values for batch-to-batch comparisons.

How can researchers address batch-to-batch variability in FasL activity from Sf9 expression systems?

Addressing batch-to-batch variability in FasL activity from Sf9 expression systems requires comprehensive standardization and quality control:

• Process standardization:

  • Establishing master and working cell banks of Sf9 cells

  • Creating and validating master virus stocks

  • Standardizing culture media lots and supplements

  • Implementing consistent protocols for cell maintenance, infection, and harvest

  • Controlling cell density, viability, and passage number

• Critical parameter monitoring:

  • Cell growth curves and viability during production

  • Infection efficiency monitoring

  • Process parameters (pH, temperature, dissolved oxygen)

  • Consistent timing of harvest post-infection (48 hours was used in studies and )

• Analytical characterization:

  • Multiple complementary methods to assess protein quantity and quality

  • SDS-PAGE and Western blotting (as mentioned in study )

  • Size exclusion chromatography to assess oligomeric state

  • Mass spectrometry for detailed characterization

• Functional standardization:

  • Establishing reference standards from well-characterized batches

  • Quantitative bioassays with dose-response curves

  • Multiple bioassay methods (e.g., apoptosis detection by annexin V-FITC as in study )

By implementing these strategies, researchers can minimize batch-to-batch variability and ensure consistent FasL activity across production runs.

How do calcium signaling assays help evaluate the functional activity of human FasL?

Calcium signaling assays provide valuable insights into the functional activity of human FasL through several mechanisms:

• Mechanistic relationship:

  • FasL-induced apoptosis often involves calcium flux as a secondary messenger

  • Elevated intracellular calcium ([Ca2+]i) can activate calcium-dependent endonucleases that contribute to DNA fragmentation during apoptosis

  • Sustained calcium elevation can trigger mitochondrial permeability transition

• Methodological approaches (as described in study ):

  • Fluorescent Ca2+ indicators like Fluo-3/AM can be used to monitor [Ca2+]i changes

  • Laser scanning confocal microscopy to record fluorescence at intervals (every 6 seconds for >400 seconds)

  • Analysis of dynamic [Ca2+]i changes, including transient elevation followed by recovery or sustained elevation

• Correlation with apoptotic events:

  • Study demonstrates that disturbed [Ca2+]i homeostasis in Sf9 cells can be linked to cell cycle arrest and inhibition of cell proliferation

  • The relationship between [Ca2+]i elevation, cell cycle arrest, and cell death provides a mechanistic framework for understanding cell death mechanisms

• Sensitivity and early detection:

  • Calcium flux often occurs as an early event in apoptosis signaling

  • Can detect cellular responses before morphological changes become apparent

The methodology described in study for measuring [Ca2+]i in Sf9 cells can be adapted to study FasL-induced calcium responses in target cells, providing additional mechanistic information about the apoptotic pathway activation.

What controls should be included when testing human FasL-induced apoptosis in target cells?

When testing human FasL-induced apoptosis in target cells, including appropriate controls is crucial for experimental validity:

• Negative controls:

  • Vehicle control (buffer/medium used for FasL preparation)

  • Uninfected Sf9 cell supernatant processed identically to FasL-containing supernatant

  • Heat-inactivated FasL (to control for non-specific protein effects)

  • FasL preparation incubated with neutralizing anti-FasL antibodies

• Positive controls:

  • Commercial FasL or FasL from a different expression system with known activity

  • Alternative apoptosis inducers (e.g., staurosporine)

  • Graded doses of FasL to establish dose-response relationships

• Specificity controls:

  • Fas-negative cell lines that should be resistant to FasL-induced apoptosis

  • Fas-blocking antibodies to confirm receptor specificity

  • Pan-caspase inhibitors (e.g., z-VAD-fmk) to confirm caspase dependency

• Procedural controls:

  • Time-course analysis to capture appropriate apoptotic stages

  • Multiple apoptosis detection methods (e.g., both annexin V staining and caspase activity)

Study used T98G human glioblastoma cell line as target cells and confirmed apoptosis with annexin V-FITC staining, demonstrating the importance of both appropriate target cell selection and specific apoptosis detection methods.

How can human FasL expressed in Sf9 cells be used to study apoptosis resistance mechanisms in cancer cells?

Human FasL expressed in Sf9 cells provides a valuable tool for studying apoptosis resistance mechanisms in cancer cells, as demonstrated in study with glioblastoma cells:

• Mechanistic investigations:

  • Comparative dose-response studies across cancer cell lines with different apoptotic sensitivities

  • Time-course analysis to identify early versus late blocks in the apoptotic pathway

  • Combination treatments with sensitizing agents to overcome resistance

  • Investigation of downstream signaling events using phospho-specific antibodies

• Genetic manipulation approaches:

  • Gene knockdown/knockout studies to identify resistance factors

  • Overexpression of candidate resistance genes to confer protection

  • CRISPR/Cas9 screens to discover novel resistance mechanisms

• Clinical relevance investigations:

  • Correlation of in vitro FasL resistance with clinical outcomes

  • Testing tumor samples from treatment-resistant versus responsive patients

  • Development of biomarkers for FasL sensitivity/resistance

  • Study specifically suggests that "induction of apoptosis by the Fas/FasL system could be a new strategy for the treatment of malignant brain tumors"

• Calcium signaling studies:

  • Investigating the relationship between calcium signaling and apoptosis resistance

  • Using calcium indicators to monitor early responses to FasL (methodology similar to study )

  • Correlating [Ca2+]i changes with downstream apoptotic events

The Sf9-expressed FasL system offers advantages for these studies including scalable production, consistent preparation, and the ability to produce modified variants to probe specific mechanistic questions about the Fas/FasL system in cancer.

What modifications to human FasL can enhance its therapeutic potential when expressed in Sf9 cells?

Several modifications to human FasL can enhance its therapeutic potential when expressed in Sf9 cells:

• Structural modifications:

  • Creation of stable FasL trimers through leucine zipper or isoleucine zipper fusion

  • Development of membrane-anchored versus soluble forms to modulate activity

  • Engineering of point mutations to enhance receptor binding affinity

  • Domain swapping with related TNF family members for altered functionality

• Targeting modifications:

  • Fusion to antibody fragments (scFv) for cancer cell targeting

  • Addition of cell-penetrating peptides for intracellular delivery

  • Incorporation of tumor-homing peptides for improved tumor localization

  • Creation of bispecific molecules targeting both Fas and tumor-specific antigens

• Pharmacokinetic enhancements:

  • PEGylation or fusion to albumin-binding domains for extended half-life

  • Incorporation of unnatural amino acids for site-specific conjugation

  • Engineering of glycosylation sites to optimize serum stability

• Safety and control features:

  • Development of conditional activation systems

  • Addition of suicide switches or controllable systems

  • Engineering of variants with reduced off-target toxicity

While study demonstrates the potential of the Fas/FasL system for treating malignant brain tumors, these modifications could further enhance specificity, potency, and safety for therapeutic applications.

How does the co-expression of other human proteins with FasL in Sf9 cells affect its functional properties?

Co-expression of other human proteins with FasL in Sf9 cells can significantly impact its functional properties through various mechanisms:

• Protein-protein interactions:

  • Co-expression with natural binding partners to modulate activity

  • Co-expression with chaperones to improve folding and solubility

  • Study of FasL in complex with other death-inducing signaling complex (DISC) components

  • Investigation of regulatory proteins that modify FasL processing or signaling

• Technical and methodological impacts:

  • Based on study , Sf9 cells can tolerate infection with three or even four different recombinant baculoviruses

  • This allows complex reconstitution of multi-protein systems

  • Co-expression strategies can be used to create functional protein complexes

• Signaling pathway reconstitution:

  • Co-expression with downstream signaling molecules

  • Reconstitution of complete apoptotic pathways

  • Creation of reporter systems linked to FasL activity

  • Similar to how study describes reconstitution of human CXCR4 with G-proteins in Sf9 cells

• Production and processing effects:

  • Co-expression with proteases that process FasL

  • Co-expression with glycosylation-modifying enzymes

  • Competition for cellular resources potentially affecting yields

Studies and demonstrate successful co-expression of multiple proteins in Sf9 cells, suggesting that similar approaches could be applied to FasL to study its interactions with other proteins in the apoptotic pathway or to improve its production and functional properties.

What are the key considerations for scaling up FasL production in Sf9 cells for larger research studies?

Scaling up FasL production in Sf9 cells for larger research studies requires attention to several critical factors:

• Cell culture scale-up considerations:

  • Transition from shake flasks to larger bioreactors

  • Consistent agitation and aeration at larger scales

  • Maintenance of optimal cell density (3.0 × 10^6 cells/ml for infection as used in study )

  • Adaptation of Sf9 cells to serum-free media to reduce costs and variability

• Infection strategy optimization:

  • Maintaining consistent MOI during scale-up

  • Development of high-titer, stable virus stocks

  • Evaluation of infection methods (direct infection vs. addition of infected cells)

  • Timing of harvest (48 hours post-infection was used in studies and )

• Process monitoring and control:

  • Online monitoring of critical parameters (pH, dissolved oxygen, temperature)

  • Feed strategies for nutrient supplementation

  • Consistent harvest criteria based on viability and time post-infection

  • Implementation of scale-appropriate mixing strategies

• Downstream processing considerations:

  • Development of scalable purification methods

  • Incorporation of filtration steps to remove cell debris and baculovirus

  • Chromatography process optimization

  • Implementation of viral inactivation and removal steps

• Quality assurance:

  • Consistent analytical methods for product characterization

  • Residual host cell DNA and protein testing as discussed in study

  • Endotoxin and bioburden testing

  • Functional activity testing using standardized apoptosis assays as used in study

With careful attention to these factors, FasL production can be successfully scaled up while maintaining consistent quality and functional activity for larger research studies.