InsectDirect™ System

InsectDirect Home   •  How does use of the       InsectDirect System benefit me?   •  What does the InsectDirect System offer?      •  Insect Cell Expression Vectors      •  Insect GeneJuice® Transfection Reagent      •  Insect RoboPop™         Ni-NTA His•Bind® Purification Kit         •  A brief protocol for the RoboPop kit         •  Comparison of traditional baculovirus             method and InsectDirect System method         •  Small- and large-scale Sf9 suspension             culture transfections

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.

Baculovirus versus InsectDirect™, 10-ml scale

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