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

  ProFold™-PDI Cat. #A7
  ProFold™-PDI baculovirus genomic vector DNA encodes human protein disulfide isomerase (PDI) that facilitates folding of target proteins in the endoplasmic reticulum (rationale). Gene encoding PDI is inserted into baculovirus genome far away from the polyhedrin site (Fig. 1). 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. PDI is expressed very late in infection, at about the same time as the target protein.
  Fig. 1. Schematic representation of ProFold™-PDI DNA. Gene encoding PDI is designated by 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 ProFold™-PDI 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.

ProFold™-PDI 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 the level of synthesis of PDI is elevated to the level comparable with the synthesis of a well expressed target protein, but not in a way that would compromise the target protein synthesis rate (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 chaperone vector Profold™-PDI and conventional vector BacPAK6™ as its folding is not chaperone-dependent. As shown on Fig. 2, target protein (β-gal) is expressed by both vectors to about the same high level, while ProFold™-PDI is also expressing PDI to a comparable level.
             Fig. 2. Protein expression profiles of insect cells infected with BacPAK6™ and ProFold™-PDI –based recombinant baculoviruses that are expressing E.coli β-galactosidase (β-gal) in the polyhedrin site under control of polyhedrin promoter. β-galactosidase was chosen as a target protein for comparison of the general rate of the protein expression in the polyhedrin site between these vectors as its folding and yield is not chaperone-dependent. 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 PDI 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 ProFold™-ER1 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. However, it has to be noted that adding N-terminal tags to the target proteins that start from N-terminal signal sequence is not recommended as this is likely to disrupt their translocation into the ER. Such proteins can be either provided with C-terminal tags or with an N-terminal 8xHis tag that follows strong signal sequence encoded in pAB-bee™-8xHis vector. Up to 3 target proteins can be expressed using pAcAB3 vector that is also compatible with ProFold™-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
ProFold™-PDI, Cat. #A7
  Fig. 3. Schematic representation of plasmid transfer vectors compatible with Profold™-ER1 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 ProFold™-PDI.
Novagen Pharmingen OET Clontech
pTriEx™-1.1 Hygro
pTriEx™-1.1 Neo
pTriEx™-2 Hygro
pTriEx™-2 Neo
pTriEx™-3 Hygro
pTriEx™-3 Neo
pTriEx™-4 Hygro
pTriEx™-4 Neo
pTriEx™-4 Ek/LIC
BaculoGold™ pAcGP67A
BaculoGold™ pAcGP67B
BaculoGold™ pAcGP67C

  Rationale. PDI is a major chaperone participating in the formation of disulfide bonds. PDIs have multiple functions and, though most abundant in the endoplasmic reticulum, they are ubiquitous (Turano, C., et al., J. Cell. Physiol., 193: 154-163, 2002). Initially, PDI was demonstrated to be efficient in oxidative protein folding in vitro (Weissman and Kim, Nature, 365: 185-188, 1993). Subsequently, overexpression of PDI was reported to improve yields of biologically active proteins in yeast and baculovirus expression systems (Robinson A.S. et al., Bio/Technology, 12: 381-384, 1994; Schultz L. et al., Ann. NY Acad. Sci., 721: 148-157, 1994; Shusta, E.V., et al., Nature Biotechnology, 16: 773-777, 1998; Hsu T.A. et al., Protein Expr. Purif., 7: 281-288, 1996; Wang L., et al., J. Biol. Chem., 272: 27644-27651, 1997; Vad et al., J Biotechnol., 116: 251-60, 2005; Xu et al., Metab Eng., 7: 269-79, 2005; Inan et al., Biotechnol Bioeng., 93: 771-8, 2006). PDI can also facilitate folding of target proteins independently of the disulfide bonds formation. For example, secretion of parathyroid hormone that does not contain any cysteine residues was significantly improved in yeast system by co-expression with PDI (Vad et al., J Biotechnol., 116: 251-60, 2005).

A welcome, albeit unexplained effect of PDI in insect cells infected with recombinant baculoviruses, is that PDI improves longevity of such cells. This cytopathic effect is markedly less pronounced in infected cells expressing a high level of PDI, and such cells survive about 24 hours longer than cells infected with a control virus which does not provide for PDI expression. Similarly, PDI overexpression in mammalian cells resulted in increased longevity and time of recombinant protein production (Kitchin and Flickinger, Biotechnol. Progr., 11: 565-574, 1995).

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.

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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.