ProFold™-ER1 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 molecular chaperones 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™-ER1 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™-ER1 is also expressing molecular chaperones PDI and Calreticulin to comparable levels.
Simultaneous high level expression of four recombinant proteins (target protein, PDI, Calreticulin and GFP) rather than one, as compared to any other commercially available vector, is achieved by separating genes encoding these proteins far away from each other on the baculovirus genome to avoid interference of strong promoters that control their expression.
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 either 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.
 |
|
| Novagen | Pharmingen | OET | Clontech | ||
|---|---|---|---|---|---|
|
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 |
Both PDI and Calreticulin assist protein folding in the ER (Williams, J. Cell Science, 119: 615-623, 2005; Appenzeller-Herzog and Ellgaard, Biochim Biophys Acta., 1783: 535-48, 2008). Calreticulin and closely related Calnexin are lectin chaperones that interact with folding glycoproteins bearing monoglycosylated oligosaccharides. They can also directly bind polypeptide chains and facilitate folding of non-glycosylated proteins in the ER. In the baculovirus system, chaperone-assisted folding involving either of these chaperones was demonstrated to be efficient in improving production of biologically active secreted and transmembrane target proteins (Chang et al., J Biol Chem., 272(46): 28925-32, 1997; Tate et al., J Biol Chem., 274: 17551-8, 1999; Higgins et al., Biochim Biophys Acta., 1610: 124-32, 2003; Fourneau et al., J Immunol Methods., 285: 253-64, 2004; Culina et al., J Biol Chem., 279: 54210-5, 2004). For example, co-expression of human Calreticulin with lipoprotein lipase in insect cells resulted in a 9-fold increase in its enzymatic activity (Zhang L. et al., J. Biol. Chem., 278: 29344-51, 2003).
Proteins that are translocated into the ER often contain multiple cysteine residues linked by disulfide bonds. PDI is a major chaperone participating in the formation of disulfide bonds. PDIs have multiple functions and, though most abundant in the ER, 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 soluble or secreted protein in yeast and insect 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.
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.