|pAB-6xHis-MBP™ vector encodes 6xHis-MBP tag followed by the thrombin site. The vector could be used to provide the tag on either the N- or C-terminus of a fusion protein as it has unique cloning sites on both sides of the tag. The fusion proteins are expressed under control of a very strong polyhedrin promoter. Since the level of expression is high, the fusion proteins are easily detectable as prominent bands after separating crude cell lysates in SDS-PAGE and staining with Coomassie blue.
|In the 6xHis-MBP tag, the 6xHis moiety can be used for popular and inexpensive IMAC affinity purification of the fusion proteins (Donnelly M.I. et al., Protein Expr Purif., 47:446-54, 2006; Tropea J.E. et al., Methods Mol Biol.;363:1-19, 2007), whereas MBP moiety facilitates protein solubility and yield. To this end, it can be placed either on the N- or C- terminus of the fusion proteins (Dyson M.R. et al., BMC Biotechnol., 4:32, 2004, Niiranen L., et al., Protein Expr Purif., 52:210-218, 2007). As a result, the success rate can be dramatically increased for some low yield proteins, such as integral membrane proteins (Korepanova A. et al., Protein Expr Purif., 53:24-30, 2007). MBP can be cleaved from a fusion protein by a protease, such as thrombin (ibid).
|In the pAB-6xHis-MBP™ vector, 6xHis-MBP tag can be provided on the N-terminus of fusion proteins by using any of the SacI, StuI, XbaI, EcoRI, SmaI, NotI unique sites for cloning genes of interest in-frame with the MBP ORF. Thrombin site can be provided if XbaI, EcoRI, SmaI, NotI sites are used. Alternatively, MBP tag or 6xHis-MBP tag could be provided on the C-terminus of the fusion proteins if proximal to the tag, unique BamHI and SpeI sites are used to insert a gene of interest in-frame with the MBP ORF.
If a gene of interest is cloned in lieu of the MBP moiety, the MBP/insert junction could be sequenced using MBPF forward primer
5'-GGTGAAATCATGCCGAACATC-3', which is complementary to the MBP nucleotide sequence in the vicinity of its C-terminus. Insert/vector junction could be sequenced using MR reverse primer 5'-CGTGTCGGGTTTAACATTACGGATT-3' (indicated by the long arrow).
If a gene of interest is cloned upstream of the MBP moiety, the vector/insert junction can be sequenced using the phF primer
5'-AGACGCACAAACTAATATCACAAACTGGA-3' located in the polyhedrin promoter. The insert/MBP junction could be sequenced using MBPR primer MBPR
5'-CTCGAATTTCTTACCGACTTCA-3' located close to the beginning of the MBP nucleotide sequence.
pAB-6xHis-MBP™ vector can be combined with any of the linearized baculovirus DNA vectors, i.e. ProEasy™, ProGreen™, ProFold™-ER1, ProFold™-C2, 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.
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.
Click on PRODUCTS to view the entire list of recombinant baculovirus-related products.