tPA Human, Sf9

What is tPA Human recombinant protein expressed in Sf9 cells?

tPA Human recombinant produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 545 amino acids (positions 24-562) with a theoretical molecular mass of 61.3 kDa, though it typically appears between 50-70 kDa on SDS-PAGE due to glycosylation patterns. This protein is fused to a 6 amino acid His-tag at the C-terminus and is purified using proprietary chromatographic techniques. Functionally, tPA (also known as PLAT) is a secreted serine protease that converts the proenzyme plasminogen to plasmin, a fibrinolytic enzyme involved in the breakdown of blood clots .

How does tPA Human function physiologically?

tPA plays a critical role in cell migration and tissue remodeling through its proteolytic activity. The conversion of plasminogen to plasmin by tPA creates a two-chain disulfide-linked protein that functions in the fibrinolytic pathway. Physiologically, tPA activity exists in a delicate balance: increased enzymatic activity can cause hyperfibrinolysis, which manifests as excessive bleeding, while decreased activity leads to hypofibrinolysis, potentially resulting in thrombosis or embolism. This balance makes tPA a critical therapeutic target for conditions involving blood clotting disorders .

What are the optimal storage conditions for tPA Human from Sf9 cells?

For short-term use (2-4 weeks), tPA Human from Sf9 cells can be stored at 4°C. For longer periods, storage at -20°C is recommended. The protein is typically provided in a formulation containing 50mM MES buffer (pH 5.5), 40% glycerol, 5mM CaCl₂, 1mM DTT, and 0.5M NaCl, which helps maintain stability. For extended storage, researchers should add a carrier protein (0.1% HSA or BSA) to prevent protein degradation. Multiple freeze-thaw cycles should be avoided as they can significantly reduce protein activity and integrity .

What formulation is tPA Human typically provided in?

tPA protein solution is typically supplied at a concentration of 0.25mg/ml in a buffer containing 50mM MES (pH 5.5), 40% glycerol, 5mM CaCl₂, 1mM DTT, and 0.5M NaCl. This formulation is designed to maintain protein stability and enzymatic activity. The solution appears as a sterile filtered colorless liquid. The high glycerol content (40%) serves as a cryoprotectant, while the reducing agent DTT helps maintain the protein's disulfide bonds in their proper configuration .

What are the key characteristics of Sf9 cells used for tPA expression?

Sf9 cells are derived from Spodoptera frugiperda (fall armyworm) ovarian tissue and are widely used in the baculovirus expression vector system. These cells exhibit distinct growth phases and proliferation kinetics that change with passage number. Low passage (Lp) Sf9 cultures undergo a switch in proliferation kinetics after 30-40 passages, characterized by a shorter lag phase and an increased maximum specific proliferation rate (μN,max) from 0.03/h to 0.04/h. Sf9 cell cycle dynamics typically include an initial G2/M arrest that synchronizes the cells, a feature more pronounced in high passage (Hp) cells than in low passage cells .

What strategies can optimize protein yields in Sf9 expression systems?

Several approaches can enhance tPA expression in Sf9 systems:

• Cell Synchronization: Artificial synchronization through yeastolate limitation can maintain high specific product formation rates over extended periods, potentially increasing volumetric yield by up to 69%.

• Timing of Infection: Infecting cells during their maximum specific growth rate (μN,max) phase yields the highest specific product formation rate.

• Medium Management: While medium renewal at infection prolongs the productivity phase, the benefit may be limited to a modest 10% increase in yield.

• Conditioned Medium Effects: For low passage cells, addition of 20% conditioned medium (CM) or 10 kDa CM filtrate decreased specific product formation by 30-50%, whereas high passage cells were unaffected, suggesting different regulatory mechanisms based on culture age .

How does chemical induction affect Sf9 cells and potentially impact protein production?

Chemical induction using agents like 5-iodo-2′-deoxyuridine (IUdR), 5-azacytidine (AzaC), and sodium butyrate (NaB) can significantly alter Sf9 cellular physiology. IUdR treatment results in higher reverse transcriptase activity compared to AzaC and NaB treatments. The optimal treatment protocol involves exposing cells at the beginning of log phase (1.6 × 10^6 cells) to chemicals for 48 hours (approximately 1.25 times the population doubling time), followed by washing and continuation in fresh medium until confluence. For IUdR specifically, concentrations ranging from 25-400 μg/mL have been tested, with higher concentrations inducing more significant cellular responses. These chemical treatments may affect the expression of recombinant proteins by altering cellular transcription patterns and should be considered when designing expression protocols for tPA production .

What analytical methods are recommended for characterizing tPA from Sf9 cells?

A comprehensive analytical approach for tPA characterization should include:

• SDS-PAGE Analysis: To confirm molecular weight (approximately 50-70 kDa) and purity (>90%).

• Activity Assays: Functional assays measuring the conversion of plasminogen to plasmin to confirm biological activity.

• Glycosylation Analysis: As tPA is a glycosylated protein, techniques like lectin blotting or mass spectrometry can characterize post-translational modifications.

• Inhibition Studies: Using specific inhibitors to confirm specificity of enzymatic activity.

• Density Gradient Analysis: For monitoring protein aggregation and quality.

• RT Activity Testing: PCR-enhanced reverse transcriptase (PERT) assay can be used to monitor potential RT activity from endogenous retroviral-like particles in the preparation, particularly important for therapeutic applications .

How can researchers differentiate between native tPA and His-tagged recombinant tPA from Sf9 cells?

The recombinant tPA from Sf9 cells includes a C-terminal 6-amino acid His-tag that distinguishes it from native tPA. This modification enables purification using nickel or cobalt affinity chromatography but may potentially affect protein behavior in certain applications. To differentiate between native and His-tagged tPA, researchers can employ anti-His antibodies in Western blot analysis or perform mass spectrometry to detect the additional mass contributed by the His-tag. For applications where the His-tag might interfere, enzymatic removal using specific proteases can be considered, though this requires subsequent purification steps to separate the cleaved tag from the protein of interest .

What potential contaminants should researchers monitor in tPA preparations from Sf9 cells?

When working with tPA expressed in Sf9 cells, researchers should monitor several potential contaminants:

• Baculovirus Particles: Residual baculovirus used for infection may remain in preparations.

• Endogenous Retroviral-like Particles: Sf9 cells produce particles with reverse transcriptase activity that may co-purify with the target protein.

• Cathepsin L: Sf9 cells secrete both 49 kDa proform and 39 kDa active form of cathepsin L that may affect the stability and activity of the recombinant protein.

• Extracellular Vesicles: Diverse particle sizes associated with RT activity, including viral-like and extracellular vesicles, have been observed in Sf9 cultures.

• Antimicrobial Factors: Sf9 cells produce ~10 kDa bactericidal factors that could affect downstream applications if not removed during purification .

How do the proliferation characteristics of Sf9 cells impact experimental planning for tPA production?

Understanding Sf9 proliferation dynamics is crucial for optimizing tPA production schedules. Conditioned medium (CM) promotes proliferation in low passage Sf9 cells but has no effect after cells undergo their kinetic switch (typically after 30-40 passages). The initial G2/M arrest that synchronizes cells is more pronounced in high passage cultures. For optimal protein yields, researchers should consider:

• Culture Age: High passage cells exhibit higher specific productivity but different responses to culture conditions.

• Cell Cycle Synchronization: Either natural (through G2/M arrest) or artificial (through yeastolate limitation) synchronization can significantly improve productivity.

• Timing Windows: The volumetric product yield increases linearly only up to 68-75 hours of culture post-infection, creating a critical harvest window.

• Medium Composition: The presence of <10 kDa peptides and procathepsin L in conditioned medium affects proliferation through potential autocrine signaling systems .

What are common challenges in maintaining Sf9 cell viability for consistent tPA expression?

Maintaining optimal Sf9 cell viability for consistent tPA expression requires addressing several challenges. Cell passage number significantly impacts culture behavior, with a switch in proliferation kinetics occurring after 30-40 passages. Initial G2/M arrest synchronizes cells but varies in intensity between low and high passage cultures. Conditioned medium components affect low passage cells but not high passage cells, suggesting fundamental changes in cellular requirements over time. Additionally, researchers should be aware that an octaploid population can emerge during G2/M arrests, potentially affecting production consistency. Monitoring cell cycle dynamics throughout the culture period is essential for predicting and optimizing expression windows. Regular assessment of cathepsin L levels may also be valuable, as this enzyme appears to play a role in Sf9 proliferation .

How can researchers mitigate the risk of endogenous retroviral-like particles in therapeutic applications of tPA?

While infectivity studies show no evidence of replicating retrovirus transmission to human cells, researchers developing therapeutic applications of tPA from Sf9 cells should implement several risk mitigation strategies:

• Purification Strategy: Implement orthogonal purification steps specifically designed to remove viral particles based on their physical properties (density of ~1.08 g/mL).

• RT Activity Testing: Incorporate PCR-enhanced reverse transcriptase (PERT) assay as a quality control measure for final products.

• Inhibition Testing: Validate the effectiveness of inhibitors like AzTTP against any detected RT activity.

• Ultracentrifugation: Consider ultracentrifugation steps since RT activity can be pelleted at 124,406× g.

• Filtration Strategy: Implement size-based filtration since RT activity is associated with particles of various sizes.

• Chemical Treatment Avoidance: Minimize use of agents like IUdR that can significantly increase RT activity (up to 33-fold) .

These approaches, particularly when combined, can substantially reduce potential risks associated with endogenous retroviral-like particles in Sf9-derived products intended for therapeutic use.