|pAB-bee™-8xHis is a versatile plasmid transfer vector which provides for affinity purification of proteins translocated into the endoplasmic reticulum (ER), such as secreted proteins. Using this vector, polyhistidine tags could be provided at amino, carboxy or both amino and carboxy-termini of the proteins. pAB-bee™-8xHis encodes honeybee melittin signal sequence which can enable efficient translocation of proteins into the ER of Spodoptera frugiperda cells (Tessier D.C. et al. Gene, 98:177-83, 1991). The signal sequence is followed by a signalase cleavage site, polyhistidine tag and multiple cloning sites (MCS) for insertion of genes encoding proteins of interest. Another polyhistidine tag is encoded downstream of the MCS and could be provided on the carboxy-terminus of expressed protein.|
|pAB-bee™-8xHis is designed to ensure both the translocation of the melittin signal-polyhistidine tag-foreign protein fusion product and efficient cleavage of the signal peptide in the ER. You can verify cleavage of the signal peptide in your fusion protein using TargetP program (Emanuelson O. et al., J.Mol.Biol., 300: 1005-10016, 2000). The program is user friendly and is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/. After the cleavage, protein is provided with N-terminal polyhistidine tag to allow its affinity purification. To this end, a PCR fragment encoding mature foreign protein can be cloned "in-frame" with the signal peptide and polyhistidine tag into pAB-bee™-8xHis vector which is double-digested with NotI and any of the EcoRI, XbaI, SmaI, BamI, or AvrII restriction endonucleases.
A protein of interest can be provided with both amino and carboxy-terminal 8xHis tags. To this end, a gene of interest can be cloned in-frame with the tags using proximal NotI and distal EcoRI site.
Alternatively, a protein of interest could be provided only with the carboxy-terminal tag. In this case a PCR fragment encoding protein of interest could be cloned between proximal BglII and distal EcoRi sites.
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.
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