Insect Cell Proten Expression |
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Expression Vector System for Insect Cell
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 Insect Cell Expression |
How does use of the InsectDirect® System benefit me?
transfer plasmid plus linearized AcNPV vector DNA |
plasmid using Insect GeneJuice® Transfection Reagent |
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Researchers dealing with high-throughput (HT) expression screening face the fundamental challenge of trying to rapidly identify the appropriate expression system for many targets in parallel. Often known or unknown open reading frames (ORFs) are amplified by PCR, cloned into a variety of vectors, and the recombinants used to direct target protein expression in E. coli, mammalian cells, insect cells, or yeast. The focus has traditionally not involved insect expression systems due to their complexity and time-consuming protocols. However, insect cell systems provide an alternative means for expressing proteins that require specific post-translational modifications to retain solubility and activity.
What Does the System Include?
Insect Cell Expression Vectors
The pIEx™ series of transient expression vectors are designed for rapid, high-level protein expression in insect cells, while eliminating the need to create recombinant baculovirus for protein expression. They feature an optimal combination of AcNPV baculovirus-derived transcription elements, the hr5 (homologous region 5) enhancer and the ie1 (immediate early) promoter, to direct expression in insect cells. This promoter/enhancer combination recruits endogenous insect cell transcription machinery, thereby avoiding baculovirus infection and its associated cytopathic effects.
| Vector | Promoter(s) | Signal sequence |
Fusion Tags N-terminal |
C-terminal | Protease cleavage sites† |
Ek/LIC vector option |
| pIEx-1 | hr5/ie1 | No | His•Tag®/S•Tag™ | HSV•Tag® | Tb/Ek | Yes†† |
| pIEx-2 | hr5/ie1 | No | GST•Tag™/His•Tag/S•Tag | HSV•Tag | Tb/Ek | Yes†† |
| pIEx-3 | hr5/ie1 | Yes | GST•Tag/His•Tag/S•Tag | HSV•Tag | Tb/Ek | Yes†† |
| pIEx-4 | hr5/ie1 | No | None | S•Tag/His•Tag | None | No |
| pIEx-5 | hr5/ie1 | Yes | None | S•Tag/His•Tag | None | No |
| pIEx-6 | hr5/ie1 | No | His•Tag | S•Tag | Ek | No |
| pIEx-7 Ek/LIC* | hr5/ie1 | No | His•Tag | S•Tag | Ek | Yes |
| pBiEx-1 | hr5/ie1, T7lac | No | His•Tag/S•Tag | HSV•Tag | Tb/Ek | No |
| pBiEx-3 | hr5/ie1, T7lac | No | None | S•Tag/His•Tag | None | No |
* pIEx-7 is available only as an Ek/LIC vector. The pIEx-7 Ek/LIC Vector Kit includes Linearized Ek/LIC Vector; T4 DNA Polymerase, LIC-qualified; polymerase buffer; DTT, EDTA, and dATP solutions; Nuclease-free Water; competent cells; SOC Medium; and controls.
† Tb: thrombin; Ek: enterokonase
†† pIEx-1, pIEx-2, and pIEx-3 vectors are also available in an Ek/LIC Vector Kit.
Insect GeneJuice® Transfection Reagent
Sf9 cells transfected with pIEx-1/β-gal using Insect GeneJuice® Transfection Reagent Transfected cells were stained for β-galactosidase activity using the X-Gal Solution–based BetaBlue™ Staining Kit.
Comparison of Traditional Baculovirus Method and InsectDirect® System Method
For InsectDirect®, target ORFs were cloned into pIEx-7. Sf9 cells in 10-ml suspension cultures (1 × 106 cells/ml) were transfected with 15 mg of the recombinant plasmids using Insect GeneJuice® Transfection Reagent. For baculovirus expression (BEVS), target ORFs were cloned into pBAC-2cp transfer plasmid. The recombinant transfer plasmid was cotransfected with linearized BacVector™ 3000 DNA. After two rounds of plaque purification, a high-titer viral stock was amplified and titered. Sf9 cells in 10-ml suspension cultures (1 × 106 cells/ml) were infected at an MOI=5. Both the InsectDirect®-transfected and baculovirus-infected cultures were incubated at 28°C with shaking for 72 h. Total culture extracts were prepared by the addition of Insect PopCulture™ Reagent (0.6 ml) followed by the addition of Benzonase® Nuclease (5 ml). Samples were taken at this point to reveal the total cell protein (TCP). Ni-NTA His•Bind® resin (125 ml per culture) was then added to the extracts. After mixing on a rocking platform for 30 min at 4°C, the mixture was centrifuged and the retained resin was washed with 3 ml 1X Ni-NTA Wash Buffer. The target protein was eluted with 250 ml 1X Ni-NTA Elute Buffer.InsectDirect® Comparison to Traditional Baculovirus Method
Lane Sample
- Perfect Protein Markers, 15–150 kDa
- BEVS: Heat Shock Protein 2, Total cell protein
- BEVS: Heat Shock Protein 2, Eluate
- InsectDirect®: Heat Shock Protein 2, Total cell protein
- InsectDirect®: Heat Shock Protein 2, Eluate
- BEVS: Phosphatase 1, Total cell protein
- BEVS: Phosphatase 1, Eluate
- InsectDirect®: Phosphatase 1, Total cell protein
- InsectDirect®: Phosphatase 1, Eluate
- BEVS: Protein Kinase 11, Total cell protein
- BEVS: Protein Kinase 11, Eluate
- InsectDirect®: Protein Kinase 11, Total cell protein
- InsectDirect®: Protein Kinase 11, Eluate
- BEVS: Heat Shock Protein 3, Total cell protein
- BEVS: Heat Shock Protein 3, Eluate
- InsectDirect®: Heat Shock Protein, Total cell protein
- InsectDirect®: Heat Shock Protein 3, Eluate