|pVL-GFP is a plasmid transfer vector for expressing target proteins in-fusion with the C-terminal GFP tag. The pVL-GFP vector was derived by cloning a PCR fragment encoding the entire ORF of the Aequorea victoria green fluorescent protein into the pVL1393 vector. The pVL1393 plasmid was digested with PstI and BglII restriction endonucleases, and the PCR fragment was digested with PstI and BamHI restriction endonucleases prior to their ligation. The resulting pVL-GFP plasmid contains unique BamHI, XbaI, EcoRI, NotI, and PstI sites which are available for cloning of genes upstream of the GFP ORF.|
|The cloned genes could be sequenced using phF and GFPR primers (indicated by arrows). A flexible linker consisting of small amino acids Ala, Gly, and Ser precedes the GFP ORF in order to separate the target protein from the GFP. The linker could be made shorter or longer by alternative use of EcoRI, NotI or PstI sites. Target protein-GFP synthesis is driven by a very strong polyhedrin promoter, thus providing for strong green fluorescence of cells (GFP reporter). Since the level of expression is high, GFP fusion proteins are also easily detectable as prominent bands after separating crude cell lysates in SDS-PAGE and staining with Coomassie blue. GFP fusion proteins can be purified using GFP-Trap affinity reagents.|
|We recommend combinations of pVL-GFP plasmid transfer vector with linearized baculovirus DNA vectors that do not encode GFP, i.e. ProEasy™, ProFold™-PDI, and ProFold™-0. Click on TECHNOLOGY for more information on baculovirus expression system. Go to BACULOVIRUS TUTORIAL for simple on-line instructions on baculovirus transfection, propagation of recombinant baculoviruses and recombinant baculovirus protein expression studies for technical details.|
|GFP tag is widely used for visualization and isolation of protein complexes, studies of protein-protein interactions, protein trafficking within the cells, and mechanisms of protein degradation (Cristea et al., Mol Cell Proteomics, 4:1933-41, 2005; Michaelson and Philips, Methods Enzymol, 406:296-315, 2006; Hayes et al., Cancer Lett., 206:129-35, 2004 ; Takeuchi et al., EMBO J., 26:123-31, 2007).|
Of particular interest is the ability of the GFP tag to misfold and loose its fluorescence if its fusion partner is misflolded (Waldo G.S. et al., Nature Biotechnol., 17:691-695, 1999). This property was used for selection of target protein mutants with improved solubility (Van den Berg et al., J Biotechnol, 121:291-8, 2006), for finding soluble proteins derived from randomly fragmented cDNAs (Nakayama and Ohara, Biochem Biophys Res Commun, 312:825-30, 2003), and in studies of protein aggregation (Schrodel and de Marco, BMC Biochem, 6:10, 2005).
C-terminal GFP tag can be especially useful for expression of transmembrane proteins that have their C-terminus located at the cytoplasm. If the fusion protein ends up in inclusion bodies, GFP does not fold and is therefore not fluorescent. In contrast, if the fusion is expressed in the cytoplasmic membrane, GFP folds properly and is fluorescent. GFP fluorescence can simplify rapid overexpression screening, detergent extraction, and purification of transmembrane proteins (Drew D. et al., Protein Science, 14: 2011-2017, 2005).
Of significant importance are studies on the aggregation of GFP fusions with proteins related to protein folding diseases. GFP fluorescence facilitates investigation of mechanisms for misfolding of these important drug targets (Krobitsch S. and Lindquist S., Proc Natl Acad Sci U S A, 97:1589-94, 2000; Wang Q. et al., Hum Mol Genet., 14:3673-84, 2005; Pandey N, et al., Exp Neurol., 197:515-20, 2006; Jiang Y. et al., J Biol Chem., 282:7912-20, 2007), as well as simplifies high throughput screening for compounds that inhibit their aggregation (Gazit E, ASC Chemical Biology, 1:417-19, 2006). AB Vector offers GFP-tagged protein folding diseases targets expressed using pVL-GFP vector.
The above plasmid transfer vector is compatible with linearized baculovirus DNA from several other suppliers, e.g. BacMagic™, BacMagic™-2, BacMagic™-3, BacVector™-1000, BacVector™-2000, BacVector™-3000 (Novagen); BaculoGold™, BaculoGold™ bright (BD Pharmingen); flashBACGOLD™, flashBACULTRA™, flashBAC™ (OET); BacPAK6™ (Clontech); Sapphire™ (Orbigen). They are not compatible with Bac-to-Bac® and BaculoDirect™ DNA from Invitrogen. BacMagic™, BacMagic™-2, BacMagic™-3, BacVector™-1000, BacVector™-2000, BacVector™-3000 are trademarks of Novagen; BaculoGold™, BaculoGold™ bright are trademarks of BD Pharmingen; flashBACGOLD™, flashBACULTRA™, flashBAC™ are trademarks of OET; BacPAK6™ is a trademark of Clontech; Sapphire™ is a trademark of Orbigen, Bac-to-Bac® and BaculoDirect™ are trademarks of Invitrogen.
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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