|pAB-bee™||Cat. #B3, #K24|
|pAB-bee™ plasmid transfer vector is recommended for expression of proteins translocated into the ER. It 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). Signal sequences widely differ in their ability to facilitate protein translocation. Moreover, foreign signal sequences (e.g. mammalian sequences) could be less efficient in insect cells. Therefore, substitution of the foreign protein's own signal sequence for a powerful insect melittin signal sequence often results in more efficient protein translocation, a higher protein expression level and improved biological activity.|
|pAB-bee™ is designed to ensure both the translocation of the melittin signal-foreign protein fusion product and efficient cleavage of the signal peptide in the ER. To this end, a PCR fragment encoding mature foreign protein can be cloned "in-frame" with the signal peptide into pAB-bee™ vector which is double-digested with NotI and any of the EcoRI, XbaI, SmaI, BamI, or AvrII restriction endonucleases.
Notably, the nucleotides containing NotI site encode alanines where the signalase cleavage in the melittin peptide occurs. Therefore, unlike in similar conventional vectors, the addition of amino acids to the N-terminus of a mature protein is minimized and incorporation of any charged, very hydrophobic or bulky amino acids can be avoided. As little as two alanines could be added to the amino terminus of a mature peptide if NotI is used to digest a PCR fragment at its end. EagI restriction endonuclease, which generates the same sticky ends 5'-GGCC as NotI, can be used instead of NotI. One can add either alanine, glycine, valine, aspartic acid or glutamic acid if EagI is used. This is preferable if the mature protein starts from these or similar amino acids. One can add either alanine isoleucine, methionine, threonine, asparagine, lysine, arginine, serine, glycine, valine, aspartic acid or glutamic acid if EaeI is used instead of Not. The addition could be minimized to just one alanine if class IIS endonucleases, such as BsaI or BspMI are used to digest the PCR fragment. In this case the restriction endonuclease recognition site and 5'-GGCC sticky end at the cleavage site is designed in the forward primer, which is used to generate the PCR fragment. A protein of interest can be provided with carboxy-terminal 8xHis tag. To this end, a gene of interest can be cloned in-frame with the tag using proximal BglII or NotI and distal EcoRI site.
Amino acids on the amino terminus of a mature peptide can influence the signalase cleavage. Therefore, we highly recommend using TargetP program (Emanuelson O. et al., J.Mol.Biol., 300: 1005-10016, 2000) for designing melittin signal peptide-foreign protein junctions. The program is user friendly and is available as a web-server at http://www.cbs.dtu.dk/services/TargetP/.
pAB-bee™ can be also used as a general plasmid transfer vector for expression of proteins without the melittin signal sequence. In this case, a PCR fragment encoding a protein of interest could be cloned into the vector, which was double-digested with BglII and any other restriction endonuclease in the MCS. Thus, the nucleotides encoding mellittin signal sequence are removed. The vector can now function just as any polyhedrin plasmid transfer vector for expression of cytoplasmic proteins or proteins containing their own translocation signals.
Derivatives of pAB-bee™ that facilitate purification of proteins of interest using N-terminal polyhistidine tag (pAB-bee™-8xHis,Cat. #B3H) or C-terminal FLAG-8xHis tag (pAB-bee™-FH, Cat. #B3FH) are available.
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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