BaculoDirect Baculovirus Expression System

1. BaculoDirect� Baculovirus Expression System BaculoDirect� Expression The Fastest and easiest method for generating recombinant baculovirus Empowered by Gateway� Technology, the BaculoDirect� Baculovirus Expression System is the fastest and easiest method for generating recombinant baculovirus. Typically, baculovirus expression systems utilize tedious site-specific transposition in E. coli or lengthy homologous recombination methods in insect cells to generate recombinant baculovirus. The BaculoDirect� Baculovirus Expression System eliminates these processes, saving you time and getting your baculovirus expression results quicker than ever. It is also the system most suitable for high throughput expression applications. BaculoDirect� is the linearized baculovirus genome engineered to include att sites for recombination with a Gateway� entry clone. Throughout this seminar, we�ll show you how it�s ideal for HTP cloning and expression.Empowered by Gateway� Technology, the BaculoDirect� Baculovirus Expression System is the fastest and easiest method for generating recombinant baculovirus. Typically, baculovirus expression systems utilize tedious site-specific transposition in E. coli or lengthy homologous recombination methods in insect cells to generate recombinant baculovirus. The BaculoDirect� Baculovirus Expression System eliminates these processes, saving you time and getting your baculovirus expression results quicker than ever. It is also the system most suitable for high throughput expression applications. BaculoDirect� is the linearized baculovirus genome engineered to include att sites for recombination with a Gateway� entry clone. Throughout this seminar, we�ll show you how it�s ideal for HTP cloning and expression.

2. Simplified BaculoDirect� Work Flow This is how the BaculoDirect� system works: A Gateway� entry clone and the BaculoDirect� Linear DNA are combined into a tube with LR Clonase� for one hour following the generic LR reaction protocol. A small aliquot of the LR reaction mix is taken and used to transfect insect cells with Cellfectin� Transfection Reagent. Cells lyse releasing recombinant baculovirus. The supernatant/media is collected. At this point, a quick expression test can be performed (SDS-PAGE, western blot) to determine if the protein expresses or not. Also, can continue on to amplify the viral stock and scale-up for high-level expression.This is how the BaculoDirect� system works: A Gateway� entry clone and the BaculoDirect� Linear DNA are combined into a tube with LR Clonase� for one hour following the generic LR reaction protocol. A small aliquot of the LR reaction mix is taken and used to transfect insect cells with Cellfectin� Transfection Reagent. Cells lyse releasing recombinant baculovirus. The supernatant/media is collected. At this point, a quick expression test can be performed (SDS-PAGE, western blot) to determine if the protein expresses or not. Also, can continue on to amplify the viral stock and scale-up for high-level expression.

3. Dissecting the BaculoDirect� Linear DNA This slide lists the important elements in the BaculoDirect� linear DNA. The BaculoDirect� Linear DNA is designed for simple, generation of recombinant baculovirus and expression in insect cells. In addition to attR sites for fast Gateway� cloning, the BaculoDirect� Linear DNA includes: Strong polyhedrin promoter for high-level expression Thymidine knase (TK) gene for negative selection of non-recombinant virus using ganciclovir lacZ gene for quick determination of recombinant virus purity N- or C-terminal V5 epitope tag and polyhistidine (6xHis) sequence for easy detection and purification of recombinant proteins We also offer secreted version of this system with a honeybee melittin (HBM) leader signal sequence Please also note that this DNA is linear so the chance of generating non-recombinant virus is minimized.This slide lists the important elements in the BaculoDirect� linear DNA. The BaculoDirect� Linear DNA is designed for simple, generation of recombinant baculovirus and expression in insect cells. In addition to attR sites for fast Gateway� cloning, the BaculoDirect� Linear DNA includes: Strong polyhedrin promoter for high-level expression Thymidine knase (TK) gene for negative selection of non-recombinant virus using ganciclovir lacZ gene for quick determination of recombinant virus purity N- or C-terminal V5 epitope tag and polyhistidine (6xHis) sequence for easy detection and purification of recombinant proteins We also offer secreted version of this system with a honeybee melittin (HBM) leader signal sequence Please also note that this DNA is linear so the chance of generating non-recombinant virus is minimized.

4. BaculoDirect� Saves You Time Time savings: tedious and time-consuming techniques required with other systems eliminated The biggest benefit of the BaculoDirect� Baculovirus expression System is speed. This system makes baculovirus expression more convenient, requiring less than half the hands-on time and overall time necessary for traditional systems.The biggest benefit of the BaculoDirect� Baculovirus expression System is speed. This system makes baculovirus expression more convenient, requiring less than half the hands-on time and overall time necessary for traditional systems.

5. Baculovirus expression systems typically require bacterial transformation and isolation of a large bacmid, as with Bac-to-Bac� system, or cotransfection of a transfer vector and linear baculovirus DNA into insect cells, as with Bac-N-Blue� and BaculoGold� systems. The BaculoDirect� system accelerates your research by eliminating these time-consuming steps. Hands-on time necessary for expression is dramatically reduced. From initial transfection to isolation of your baculovirus stock takes approximately 8 hours of hands-on time. The BaculoDirect� System gives you rapid results, with less effort, and fewer tedious steps. Baculovirus expression systems typically require bacterial transformation and isolation of a large bacmid, as with Bac-to-Bac� system, or cotransfection of a transfer vector and linear baculovirus DNA into insect cells, as with Bac-N-Blue� and BaculoGold� systems. The BaculoDirect� system accelerates your research by eliminating these time-consuming steps. Hands-on time necessary for expression is dramatically reduced. From initial transfection to isolation of your baculovirus stock takes approximately 8 hours of hands-on time. The BaculoDirect� System gives you rapid results, with less effort, and fewer tedious steps.

6. Advanced Selection to Ensure Viral Purity Another revolutionary design of the BaculoDirect� Expression System is its recombinant virus selection mechanism. The BaculoDirect� Linear DNA includes two built-in time-saving checks- the lacZ gene and the thymidine kinase gene- to ensure the isolation of a pure baculovirus stock. During the LR reaction, the TK and lacZ genes are recombined out as by-products. Recombinant virus will no longer contain either gene so recombinant virus can survive selection with ganciclovir and will not stain blue when cells are stained with B-galactosidase. That is to say, selection with ganciclovir will eliminate any non-recombinant parental virus that remains. Therefore, the viral stock you generate will be highly pure recombinant. Staining with B-galactosidase is a quick and easy way to visualize the purity of the viral stock. For expression screening, this eliminates the need to perform a plaque assay. You are able to quickly see pure viral stock. Please note that GFP in this slide is just an example. Another revolutionary design of the BaculoDirect� Expression System is its recombinant virus selection mechanism. The BaculoDirect� Linear DNA includes two built-in time-saving checks- the lacZ gene and the thymidine kinase gene- to ensure the isolation of a pure baculovirus stock. During the LR reaction, the TK and lacZ genes are recombined out as by-products. Recombinant virus will no longer contain either gene so recombinant virus can survive selection with ganciclovir and will not stain blue when cells are stained with B-galactosidase. That is to say, selection with ganciclovir will eliminate any non-recombinant parental virus that remains. Therefore, the viral stock you generate will be highly pure recombinant. Staining with B-galactosidase is a quick and easy way to visualize the purity of the viral stock. For expression screening, this eliminates the need to perform a plaque assay. You are able to quickly see pure viral stock. Please note that GFP in this slide is just an example.

7. High-level Expression with the BaculoDirect� System Several genes were tested and shown to exhibit high level protein expression with the BaculoDirect� system.Several genes were tested and shown to exhibit high level protein expression with the BaculoDirect� system.

8. Expression of Ultimate� ORFs using BaculoDirect� Baculovirus System This is a good example of using the BaculoDirect� Expression System for high throughput expression. Our 96-well format expression protocol has been tested for you to work with large numbers of samples at the same time. Gateway� Technology is one of the key advantages here to make this procedure easy and efficient for you.This is a good example of using the BaculoDirect� Expression System for high throughput expression. Our 96-well format expression protocol has been tested for you to work with large numbers of samples at the same time. Gateway� Technology is one of the key advantages here to make this procedure easy and efficient for you.

9. Here is a summary of the advantages of the Gateway�-engineered BaculoDirect� Baculovirus Expression System.Here is a summary of the advantages of the Gateway�-engineered BaculoDirect� Baculovirus Expression System.

10. BaculoDirect� System Products We offer N-term, C-term, and Secreted BaculoDirect� product sets. In each set, we offer complete expression kits for new customers and support transfection kits for come-back customers.We offer N-term, C-term, and Secreted BaculoDirect� product sets. In each set, we offer complete expression kits for new customers and support transfection kits for come-back customers.