AB Vector - ProFold™-C1 and ProFold™-C2

  ProGreen™ Cat #A1, #K20
 
  ProGreen™ baculovirus genomic vector DNA encodes Aequorea victoria green-fluorescent protein (GFP) Fig. 1. GFP allows monitoring of recombinant virus propagation, optimization of cultivation conditions, and titration of virus stocks by "terminal green dilution." The polyhedrin site is reserved for insertion of target genes under control of strong polyhedrin promoter. Initially, the polyhedrin site was occupied by E.coli β-galactosidase gene. To allow insertion of a target gene, β-galactosidase gene was removed from the baculovirus DNA using Bsu36.I restriction endonuclease which cleaves immediately downstream of the polyhedrin promoter, in the β-galactosidase gene and in the downstream essential gene ORF 1629.
 
  Fig. 1. Schematic representation of ProGreen™ DNA. Genes GFP is designated by green arrow. Polyhedrin promoter is designated by the black triangle. E.coli β-galactosidase gene is originally present in the polyhedrin site. However, in commercially available ProGreen™ vector it is removed alongside with a part of an essential gene to allow substitution of β-galactosidase gene for a gene of interest (target gene), refer to TECHNOLOGY for more details.

ProGreen™ can drive the rate of the target protein synthesis that is close to the maximum achievable in a recombinant baculovirus system for a concrete target protein, while simultaneously expressing GFP to a high level (Fig. 2). E.coli β-galactosidase (β-gal) was expressed as a target protein in the polyhedrin site for to allow adequate comparison of the rate of the target protein synthesis between ProGreen™ and conventional vector BacPAK6™. As shown on Fig. 2, target protein (β-gal) is expressed by both vectors to about the same high level, while ProGreen™ is also expressing GFP.
 
 
 
             Fig. 2. Protein expression profiles of insect cells infected with BacPAK6™ and ProGreen™ –based recombinant baculoviruses that are expressing E.coli β-galactosidase (β-gal) in the polyhedrin site under control of polyhedrin promoter. Spodoptera frigiperda Sf9 cells were infected with recombinant baculoviruses at multiplicity 5 plaque forming units per cell and harvested at 60 h post infection. Cells were lysed in 50 mM Tris-HCl pH 8.0, 150 mM EDTA, 0.5% NP-40 and the lysate was centrifuged at 30,000g for 15 min. β-galactosidase and GFP are highly soluble and are found almost exclusively in the supernatant. The supernatant proteins were separated in 11% SDS-PAGE and stained with Coomassie blue.
 
 
  Methods. Go to Baculovirus tutorial for simple step-by-step hands-on instructions. Refer to TECHNOLOGY for conceptual details. You can use the same methods as for BaculoGold™ (BD Pharmingen) if you are already experienced with these vectors.

Recommended plasmid transfer vectors. As shown on Fig. 3 below, a large selection of plasmids is compatible with ProGreen™ vector to suite various protein expression and purification requirements. We offer vectors that can provide a highly efficient signal sequence (pAB-bee™, pAB-bee™-8xHis, pAB-bee-FH™ plasmid transfer vectors) to facilitate expression of secreted and membrane proteins. A target protein can be expressed with a GST, His, GFP, MBP or dual His/ MBP, or FLAG/His tag or expressed without a tag using the pVL1393 plasmid vector. Up to 3 target proteins can be expressed using pAcAB3 vector that is also compatible with ProGreen™-C1 and -C2 vectors. You can also use any plasmid transfer vectors that are compatible with BacPAK6™ or BaculoGold™ vectors.
 
 






pVL1393, Cat. #B1
pAB-GST™, Cat. #B5
pAB-6xHis™, Cat. #B4
pVL-FH, Cat. #B9
pAB-6xHis-MBP™, Cat. #B7
pAB-MBP™, Cat. #B6
pVL-GFP™, Cat. #B8
pAB-bee, Cat. #B3
pAB-bee™-8xHis, Cat. #B3H
pAB-bee-FH™, Cat. #B3FH
pAB-bee, Cat. #B3
 
 
 
ProGreen™, Cat. #A1
  Fig. 3. Schematic representation of plasmid transfer vectors compatible with ProGreen™ baculovirus vector DNA. All plasmid transfer vectors have the same plasmid “backbone” that allows plasmid propagation in E.coli (designated by blue double line), but they differ in the part downstream of the polyhedrin promoter (designated by black triangle). Refer to TECHNOLOGY for conceptual details. Click on the plasmid name for detailed information on each plasmid. Click on the Cat# for pricing and ordering.

  Table 1. Plasmid transfer vectors from other suppliers compatible with ProGreen™.
 
Novagen Pharmingen OET Clontech
pTriEx™-1.1
pTriEx™-1.1 Hygro
pTriEx™-1.1 Neo
pTriEx™-2
pTriEx™-2 Hygro
pTriEx™-2 Neo
pTriEx™-3
pTriEx™-3 Hygro
pTriEx™-3 Neo
pTriEx™-4
pTriEx™-4 Hygro
pTriEx™-4 Neo
pTriEx™-4 Ek/LIC
pBAC™-1
pBACgus™-1
pBAC™-2cp
pBACgus™-2cp
pBAC™-3
pBACgus™-3
pBAC4x™-1
pBACgus4x™-1
pBAC™-5
pBACgus™-5
pBAC™-6
pBACgus™-6
pBACsurf™-1
BaculoGold™pAcGHLT-A
BaculoGold™pAcGHLT-B
BaculoGold™pAcGHLT-C
BaculoGold™ pAcGP67A
BaculoGold™ pAcGP67B
BaculoGold™ pAcGP67C
pVL1392-XylE
pOET™1-5
pOET™2
pOET™1C_6xHis
pOET™2C_6xHis
pOET™1N_6xHis
pOET™2N/C_6xHis
BacPAK™8
BacPAK™9
BAcPAK™8-gus

  If you have not worked with baculovirus expression system, also referred to as baculovirus expression vector system or BEVS, you can get a quick update at TECHNOLOGY. Go to BACULOVIRUS TUTORIAL for simple on-line instructions on baculovirus transfection, propagation of recombinant baculoviruses and recombinant baculovirus protein expression studies.

Click on PRODUCTS to view the entire list of recombinant baculovirus-related products.

pTriEx™, pBAC™, BacMagic™, BacVector™ are trademarks of Novagen, BaculoGold™ is a trademark of BD Pharmingen, flashBAC™ and pOET™ are trademarks of Oxford Expression Technologies (OET), BacPAK™ is a trademark of Clontech, Sapphire™ is a trademark of Orbigen.


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