Several commercial expression systems for insect cells
are currently available. This tutorial will focus on the Bac-to-Bac
expression system, manufactured by Invitrogen.
This system relies on bacterial cells called DH10Bac, engineered to contain the viral genome. The gene of interest is first cloned into a Donor plasmid and is then transformed into the DH10Bac cells. Inside the cells, the gene of interest is incorporated into the viral genome, becoming an integral part of the baculovirus DNA. Transfection of the viral DNA extracted from the DH10Bac into insect cells will result in the formation and secretion of viable recombinant virus, that can be used for production of the protein of interest.
|1. Cloning of your
gene into a Donor plasmid
You should clone your gene (BHR1) into a Donor plasmid downstream to the polyhedron promoter. (The donor plasmid contains two transposable elements flanking the cloned gene, it also contains the polyhedron promoter and a gene that confers resistance to gentamycin.)
|2. Transposition of
the appropriate fragment from the Donor plasmid into the viral genome
You transform the Donor plasmid into DH10Bac bacteria containing the Baculovirus genome. These bacteria also contain a plasmid encoding for the enzyme Transposase, which catalyses transposition between the transposable elements engineered in the Donor plasmid and those engineered in the LacZ gene within the viral genome.
|3. Isolating recombinant
virus from DH10Bac bacteria
Following transformation you should plate the bacterial cells on selective agar plates and select for white colonies. These colonies should contain your gene, inserted into the LacZ site of the baculovirus genome. In order to clone-purify white colonioes, you should restreak the positive colonies.
|4. Transfection of
viral DNA into SF9 insect cells
After verifying the presence of your gene in the baculovirus genome you should now isolate the viral DNA from the selected DH10Bac white colonies. A simple small scale isolation ("mini-prep") of the viral DNA from a positive white colony will now be used for transfection of insect cells. (In order to select against mixed colonies, you should perform a PCR reaction using primers derived from the LacZ gene flanking the integration sites.) Following transfection, viable virus will start budding into the media, and you should collect the media containing virus three to four days post-transfection.
In order to make sure you are working with a single viral clone and not a mixed population of viruses, you should now perform a plaque purification assay that is done by infecting a monolayer of SF9 cells with serial dilutions of the virus collected in step 4. Overlay of low melting point agarose on the infected cells will enable the formation of single plaques that will be used to propagate a large quantity of homogeneous virus for future experiments. Expanding of the viral stock is done by infecting a large number of SF9 cells with the isolated plaque. This way you can obtain a 10 to 100 fold increase of the viral stock. You can conduct several rounds of viral amplification in order to obtain a high-titer viral stock.
|6. Production of recombinant
protein in SF9 insect cells
Insect cells grown in suspension can now be infected with various amounts of the viral stock obtained in step 5. Cell samples will be analysed for recombinant protein production for several days, in order to determine optimal conditions for protein production.
|7. Protein purification
and activity assay
Isolation of the recombinant protein and characterization of the purified product.
Now is the time to start cloning your BHR1 gene into the Bac to Bac expression system.
Before starting the actual cloning, you should select the essential properties of your vector to suit the specific features of your protein and your specific experimental requirements.
You may examine the site "Anatomy of an Expression Vector" to gain more information about each element in the vector.
vector should contain an origin of replication.
b. The vector should place BHR1 in-frame with a His tag to produce a fusion protein.
c. The vector should contain a sequence encoding RNA polymerase to allow transcription.
d. The vector should contain an early promoter (which induces protein expression at an early stage of viral production)
e. The DNA encoding BHR1 should be added in the right frame to the vector.