Insect Cell Proten Expression

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Expression Vector System for Insect Cell

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Merck:/Freestyle/BI-Bioscience/Genomic-Analysis/InsectCell-cover.jpgInsect Cell Expression
A rapid, powerful, virus-free method for heterologous protein expression in insect cells

How does use of the InsectDirect® System benefit me?
  • Allows protein expression in 48 hours, not 2 to 3 weeks
  • Omits the need for a time-consuming production of recombinant baculovirus
  • Provides an option for HT screening of multiple targets
  • Produces up to 80 µg target protein per 1 ml culture
  • Includes a protocol that can be scaled for larger culture volumes and higher protein yields

A. Traditional baculovirus method
B. InsectDirect® System method
Day 1 Cotransfect insect cells with recombinant
transfer plasmid plus linearized AcNPV vector DNA
Day 1 Transfect Sf9 cells with pIEx recombinant
plasmid using Insect GeneJuice® Transfection Reagent
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Day 4 Choose plaque and replate
Day 3 Proceed with purification
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Day 8 Choose plaque and amplify
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Day 11 Screen for expression; amplify
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Day 14 Titer viral stock; optimize MOI
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Day 18 Infect insect cells and express protein
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Days 20 - 21 Harvest cells and proceed with purification

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.


The pIEx™ series of multisystem expression vectors allows rapid characterization/expression of target genes in both <i>E. coli</i> and insect cells using the same construct.
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The pIEx™ series of multisystem expression vectors allows rapid characterization/expression of tdarget genes in both E. coli and insect cells using the same construct. These vectors feature the same hr5 enhancer and the ie1 promoter as the pIEx vectors to direct expression in insect cells and also allow for expression in E. coli by the tightly controlled T7lac promoter.


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.
Insect GeneJuice® Transfection Reagent is a liposome-based transfection reagent optimized for maximal transfection of Spodoptera insect cells with minimal toxicity. It can be used for both transient and stable transfections in serum-containing or serum-free media. Insect GeneJuice® is ideal for HT or large-scale protein expression when using the pIEx or pBiEx vectors for suspension culture transfection of Sf9 and other insect cells.

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

Comparison of traditional baculovirus method and InsectDirect® System method
Lane Sample
  1. Perfect Protein Markers, 15–150 kDa
  2. BEVS: Heat Shock Protein 2, Total cell protein
  3. BEVS: Heat Shock Protein 2, Eluate
  4. InsectDirect®: Heat Shock Protein 2, Total cell protein
  5. InsectDirect®: Heat Shock Protein 2, Eluate
  6. BEVS: Phosphatase 1, Total cell protein
  7. BEVS: Phosphatase 1, Eluate
  8. InsectDirect®: Phosphatase 1, Total cell protein
  9. InsectDirect®: Phosphatase 1, Eluate
  10. BEVS: Protein Kinase 11, Total cell protein
  11. BEVS: Protein Kinase 11, Eluate
  12. InsectDirect®: Protein Kinase 11, Total cell protein
  13. InsectDirect®: Protein Kinase 11, Eluate
  14. BEVS: Heat Shock Protein 3, Total cell protein
  15. BEVS: Heat Shock Protein 3, Eluate
  16. InsectDirect®: Heat Shock Protein, Total cell protein
  17. InsectDirect®: Heat Shock Protein 3, Eluate