Introduction
The Baculovirus Expression Vector System (BEVS) is a convenient and versatile
eukaryotic system for heterologous gene expression. Baculovirus expression
provides correct folding of recombinant protein as well as disulfide bond
formation, oligomerization and other important post-translational modifications.
Consequently the over-expressed protein exhibits the proper biological
activity and function. The Baculovirus Expression Vector System is based
on the introduction of a foreign gene into a nonessential region of the
viral genome via homologous recombination with a transfer vector containing
the cloned gene; an event that occurs in the cotransfected insect cells.
The production of foreign protein is then achieved by infection of additional
insect cell cultures with the resultant recombinant virus. The Baculovirus
Expression Vector System from BD Biosciences Pharmingen employs a modified
Autographa californica nuclear polyhedrosis virus (AcNPV) genome
- BD BaculoGold™ DNA, and an appropriate transfer vector. The diversity
of AcNPV-based transfer vectors, combined with available S. frugiperda
Sf9 and Sf21 cell lines, establish baculovirus expression
as a preferred system for functional eukaryotic gene expression and the
large-scale production of recombinant proteins. The baculovirus expression
system offers the following advantages over prokaryotic and other eukaryotic
systems:
- High Level of Protein Expression. Yields of up to 100 mg of protein per 109 cells.
- Post-Translational Modifications. Including disulfide bond formation, phosphorylation,
glycosylation, oligomerization and proper folding.
- Relevant Cellular Compartmentalization of Proteins. Secreted, membrane-bound,
cytoplasmic or nuclear.
- Capacity of Large cDNA Inserts. Accommodates genes up to 15 kb.
- Simultaneous Expression of Multiple Genes. With multiple promoter transfer vectors.
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Quick and reliable results using
BD Baculogold™ Baculovirus Expression Vector System |
BD BaculoGold™ Linearized Baculovirus DNA
Unlike the wild-type AcNPV virus, BD BaculoGold™ DNA is an engineered baculoviral DNA
incorporating a lethal deletion. Co-transfection of the BD BaculoGold DNA and a complementing
baculovirus transfer vector restores viability by homologous recombination and rescues the virus
along with the desired recombinant gene. Since only recombinant BD BaculoGold DNA produces
viable virus, use of this improved viral DNA results in recombination efficiencies greater than
99%.
Protein Expression and Purification Kits
In order to facilitate gene expression and protein purification, BD Biosciences Pharmingen has
developed two baculovirus expression and purification kits utilizing affinity purification tags: the
6xHis tag kit and the Glutathione S-transferase (GST) tag kit. Both kits combine the advantages
of rapid expression of functional and soluble recombinant proteins using BD BaculoGold™
baculovirus expression technology with the purification power of the 6xHis and GST affinity
purification systems. Purifications to greater than 90% homogeneity are achieved in a single step
under native conditions.
Insect Cells and Cell Culture Media
A variety of insect cell lines are susceptible to infection with the
AcNPV baculovirus. The cell lines most frequently used are Sf9
and Sf21, originally established from ovarian tissues of Spodoptera
frugiperda larvae. Sf9 and Sf21 cell lines are
available in either our new BD BaculoGold™ Max- XP Serum Free Insect
Cell Medium or TNM-FH fully-supplemented Insect Cell Culture Medium.
BD BaculoGold™ Max-XP Serum-Free Insect Cell Medium
Our latest innovation comes in the form of our new BD BaculoGold™
Max-XP Serum-Free Insect Cell Medium. BD BaculoGold Max-XP medium is produced
using proprietary systems for component analysis, nutrient delivery, and
metabolic pathway feedback. The result is a metabolically enhanced medium
which far out performs the competition. Experiments indicate that the
production of functional recombinant protein in insect cell lines grown
in BD BaculoGold Max- XP medium increases three-fold as compared with
insect cells grown in TNM-FH/10% FBS and other serum-free media. In addition,
BD BaculoGold Max-XP medium is designed to be used in multiple cell lines
and has been shown to optimize growth and recombinant protein production
in Drosophila melanogaster, Tricholplusia ni, Heliothis zea,
in the Spodoptera frugiperda cell lines Sf9 and Sf21,
and more. BD BaculoGold Max-XP medium is ideal for quick, direct adaptation
of your insect cells cultured in serum-supplemented media or in other
serum-free media.
For pharmaceutical research, the high cell density and optimum protein production obtained with
the BD BaculoGold Max-XP medium make it ideal for large-scale industrial use. In addition, the
composition of the BD BaculoGold Max-XP medium is precisely defined, which facilitates the
purification of recombinant proteins and isolation of virus.
In summary, this metabolically designed serum-free medium enhances the growth of many types
of insect cells, amplifies recombinant protein production, and augments baculovirus yield.
Transfer Vectors
The system provides an array of versatile transfer vectors. Polyhedrin
locus-based and p10 locus-based transfer vectors are available in the
following conformations: with the multiple cloning site in opposite orientations,
with single promoter or multiple promoters for expression of 2, 3, or
4 proteins simultaneously. Vectors are also available in three translational
reading frames for expression of proteins which are singularly- or multiply-tagged
with 6xHis, GST or a signal peptide for protein secretion. Additionally,
vectors are available for easy visualization of GFP-, BFP-, or YFP-tagged
proteins. Please find all currently available products in our online
catalog.
BD Creator™ BacPAK9 Shuttle Vectors from BD Biosciences Clontech
Easy preparation of baculoviral shuttle constructs via Cre-loxP recombination
Do you need to express your protein in a baculoviral system, use vectors that eliminate
complicated subcloning procedures and let you proceed directly to expression in the shortest time
possible? The new pLP-BacPAK9 and pLP-BacPAK9-6xHN Vectors from BD Biosciences
Clontech do just that. These BacPAK9 Shuttle Vectors are BD Creator™ Acceptor Vectors that
provide efficient subcloning and compatibility with our BD BaculoGold™ or BD BacPAK™
Baculovirus Expression Vector Systems from BD Biosciences.
BD BacPAK™ Baculovirus Rapid Titer Kit from BD Biosciences Clontech
The BD BacPAK™ Baculovirus Rapid Titer Kit provides the quickest method for determining titers
of baculovirus stocks, typically the most time consuming part of baculovirus expression protocols.
The kit uses a standard immunological assay to accurately determine baculovirus titers within 48
hours, whereas other methods, such as plaque and end-point dilution assays, require 4–8 days.
Custom Baculovirus Protein Expression Services
BD Biosciences is pleased to offer an extended array of Customized Reagents & Services to
support your experiments from discovery research through to clinical trials. You are no longer
restricted by the boundaries of regular products in our catalog. Simply choose the customized
reagent or service you need.
For detailed information about our Custom Service Program, please contact
us at 1.888.259.0187.
Baculovirus Expression and Purification
- Cloning of a Gene of Interest into a Baculovirus Transfer Vector.
- Cutting the gene of interest out of a customer-supplied vector and subcloning
it into a baculovirus transfer vector.
- Verification of the orientation of the subcloned insert by restriction mapping.
- Large-Scale Amplification of the Recombinant Baculovirus Transfer Vector.
- Generation, identification and purification of recombinant baculovirus. Co-transfection of the recombinant transfer vector with BD BaculoGold™ DNA. Isolation and plaque-purification of ten baculovirus recombinants.
- Amplification of Recombinant Baculovirus for High-Titer Stock. Low-titer stock solution from a single recombinant will be used to generate 500ml of high-titer stock solution. Plaque assay to determine the titer.
- Large-Scale Expression of Proteins from Recombinant Baculoviruses. One to ten litres of insect cells (1x109 cells/l) will be infected with the high titer virus stock solution.
- Protein Purification (on request, only in combination with other services). GST-fusion protein purification over glutathione affinity column. 6xHis-fusion protein purification over Ni-NTA affinity column. Immunoaffinity chromatography (if antibody available).
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