AB Vector - The easiest baculovirus expression

  The easiest baculovirus expression
 
  ProEasy™ is devoid of the disadvantages of traditional baculovirus vector technologies
 
  All it involves is mixing vector DNAs with a transfection reagent, applying the mixture to insect cells (all manipulations take several minutes), and harvesting high titer pure virus stocks a few days later.

ProEasy™ is a linearized baculovirus vector DNA which stands apart from other baculovirus vectors. Recombinant baculovirus stocks generated using ProEasy™ vector are stable and provide for as high expression level as plaque-purified BaculoGold™ or BacPAK6™ baculovirus stocks. However, they are much easier to generate. Unlike Bac-to-Bac™ baculovirus stocks, ProEasy™ stocks are stable over many passages and are also easier to generate (Fig.1).

 
  Fig. 1. Comparison of ProEasy™ and traditional baculovirus vectors. Co-transfection of BaculoGold™ or BacPAK6™ linearized baculovirus DNA with plasmid transfer vectors results in a mixture of baculoviruses after transfection, which largely consists of true recombinants expressing a protein of interest. It is often assumed that BaculoGold™/BacPAK™ technology can generate recombinants with efficiency approaching 100%. What is overlooked, is that such recombinant baculovirus stocks commonly contain false-recombinants. Similar to true recombinants, they have the LacZ minus phenotype, and therefore are counted as white recombinant plaques in a plaque assay. However, they do not express a protein of interest (King L.A. and Possee R.D., The baculovirus expression system. A laboratory guide, Chapman & Hall, London, New York, Tokyo, Melbourne, Madras, 1992). The percentage of false-positives varies in different transfection experiments. In our hands, BaculoGold™ or BacPAK6™ post-transfection mixtures typically contained very few (1% or less) non-recombinants (blue plaque phenotype, expression of beta-galactosidase), about 10-60% of false recombinants (white plaque phenotype, no expression of protein of interest or beta-galactosidase), and the rest- true recombinants (white plaque phenotype, expression of protein of interest, no expression of beta-galactosidase). Due to a significant proportion of false recombinants, unpurified BaculoGold™ and Bac-PAK6™ stocks often exhibit a less powerful protein expression level, if compared side-by-side with Bac-to-Bac™ stocks expressing the same protein of interest. An attempt to use such unpurified stocks for protein production inevitably results in further reduction or loss of protein expression, as false recombinants take over the virus stock population. Therefore, purification of such virus stocks by plaque assay or terminal dilution is required to achieve consistently high foreign protein expression levels. Plaque-purified BaculoGold™ or BacPAK6™ virus stocks are reliable over many passages and trouble-free for continuous use in laboratory practice. An alternative Bac-to-Bac™ technology generates practically 100% of true recombinants. However, due to the presence of transposon sequences, Bac-to-Bac™ recombinants reportedly could be unstable, resulting in the deletion of a gene of interest from a recombinant baculovirus and reduction and eventual loss of recombinant protein expression during virus propagation (Pijlman G.P. et al., J. Gen. Virol., 84: 2669-2678, 2003). This effect could be especially pronounced if a target protein is slightly cytotoxic or initially overexpressed. Unstable stocks cannot be reliably remedied, for example by plaque purification.
 
  ProEasy™ linearized baculovirus DNA can be combined with any of the listed below plasmid transfer vectors, i.e. pVL1393, pAcAB3, pAB-bee, pAB-6xHis™, pAB-GST™, pAB-MBP™, and pAB-6xHis-MBP™. These vectors encode various features including tags for affinity purification and for improving yield of soluble target protein. Click on the product name in the table to view the details. If you are not familiar with recombinant baculoviruses, 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 the update.
 
  Relevant products
 
Plasmid transfer vectors                             Click on the product name for technical information!
Cat # Product Specification Unit size Price*
B1 pVL1393 High level of target protein expression 50 μl, 0.5 μg/μl $295.00
B2 pAcAB3 High level expression of up to 3 target proteins using a single recombinant
    baculovirus
50 μl, 0.5 μg/μl $395.00
B3 pAB-bee Provides target proteins with powerful honeybee melittin signal sequence to      facilitate translocation into the ER
Recommended for high level expression of secreted and transmembrane proteins
50 μl, 0.5 μg/μl $495.00
B3H pAB-bee™-8xHis Provides target proteins with powerful honeybee melittin signal sequence to      facilitate translocation into the ER
Recommended for high level expression of secreted and transmembrane proteins
N- or C-terminal 8xHis tag for affinity purification
50 μl, 0.5 μg/μl $495.00
B4 pAB-6xHis™ High level expression of proteins with cleavable N-terminal 6xHis tag 50 μl, 0.5 μg/μl $295.00
B5 pAB-GST™ High level expression of proteins with cleavable N-terminal GST tag 50 μl, 0.5 μg/μl $295.00
B6 pAB-MBP™ High level expression of proteins with cleavable N-terminal MBP tag
MBP tag improves protein solubility and yield
50 μl, 0.5 μg/μl $395.00
B7 pAB-6xHis-MBP™ High level expression of proteins with cleavable N-terminal 6xHis MBP tag
High level expression of proteins with C-terminal MBP tag
MBP tag improves protein solubility and yield
50 μl, 0.5 μg/μl $495.00
 
Linearized baculovirus vector DNA
CsCl purified and digested with Bsu 36.I                  Click on the product name for technical information!
Cat # Product Specification Unit size Price*
A10S ProEasy™ The easiest generation of recombinant baculoviruses
Ideal for HTP applications
High level of target protein expression
25 μl, 2.5 μg
5 transfections
$599.00
 
Controls                                                                    Click on the product name for technical information!
Cat # Product Specification Unit size Price*
C13 NC (negative control) Recombinant baculovirus stock Extracts of cells infected with NC is an ideal negative control as there is no      polyhedrin or target protein expression
NC extracts are run side by side in SDS-PAGE with extracts of cells infected with      recombinant baculoviruses
Recombinant protein is detected as a prominent band which is absent in NC      extracts
1 ml, 108 p.f.u./ml $195.00
C14 GC (green control) Recombinant baculovirus stock Positive control baculovirus in experiments where GFP fluorescence is used to      monitor virus propagation, optimize cultivation conditions, or titrate of virus stocks 1 ml, 108 p.f.u./ml $195.00
C15 GCP (green control plasmid) Plasmid transfer vector DNA Positive control for transfection with linearized GFP- baculovirus DNA. Green      fluorescence of cells is indicative of successful transfection.
50 μl, 0.2 μg/μl $145.00
 
  *Go to PRODUCTS to order online. Alternatively, email an order as pdf attachment to info@abvector.com, or send it by Fax to 1-760-454-2465 or call 1-866-683-2867 to order by phone.
 
  If you have not worked with recombinant baculoviruses, 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.


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