|
|
SERVICE |
DESCRIPTION |
TIME |
| 1
|
Cloning of a gene
of interest into a Baculoviral expression vector |
-
Amplification/isolation of the gene of interest out of a customer-supplied
construct and subcloning it into a baculoviral expression vector
-
Verification of the authenticity of the subcloned gene by restriction enzyme
digest and sequencing
-
Maxi-prep of the recombinant vector DNA
|
1-2 weeks |
| 2
|
Generation,
identification and
plaque purification
of baculoviral
colonies expressing
target proteins |
-
Transfection of insect cells (SF9) with the recombinant transfer vector (gene
of interest cloned in the vector) and baculoviral DNA.
-
Isolation and plaque-purification of ten baculoviral recombinant clones.
-
Test for expression of the recombinant protein by western blot (customer must
provide appropriate antibodies) if required.
|
3 weeks |
| 3
|
Amplification of
recombinant
baculovirus for
high titre stock |
-
Generation of 500 ml of high-titre viral stock from low-titre stock solution
(approximately 10 ml is required) of a single recombinant
-
A few rounds of amplification of virus may need if the titre of initial virus
is too low.
-
Determination of the titre by end-point dilution assay.
|
1 week |
| 4
|
Large - Scale
Culture of
recombinant
baculovirus |
-
One litre of insect cells will be infected with high-titre virus stocks (25-30
ml of high titre virus required).
|
1 week |
| 4
|
Purification |
-
Protein purification using a Ni-NTA (for 6xHIS-tagged proteins) or glutathione
(for GST-tagged proteins) column at either native or denatured condition.
|
1 week |
| 5
|
Total |
|
8 weeks |
* If customer does not provide the gene, there will be additional charges for
both cloning and sequencing and it may need 1-2 weeks to amplify the gene from
library and to confirm the sequences.
** We do not guarantee the yield of purified protein since it changes from
protein to protein. More large-scale culture may be needed to obtain a certain
amount of protein for antibody production.
