Abgent's selection of bacterial and baculovirus
protein expression systems provides a choice for your application.
We will work with your specifications for quantity, bioactivity,
structure, post-translational modifications, and purity to express
proteins that meet your specialized requirements. Project status and data reports are monitored by our exclusive in-house custom service database tracking system.
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| Phase |
Time |
I. Cloning of a gene of interest into a baculoviral expression vector1
- 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.
|
2 weeks |
II. 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 desired.
|
3 weeks |
III. Amplification of recombinant baculovirus for high titer stock
- Generation of 500 ml of high-titer viral stock from low-titer stock solution (approximately 10 ml is required) of a single recombinant.
- A few rounds of amplification of virus may need if the titer of initial virus is too low.
- Determination of the titer by end-point dilution assay.
|
1 week |
IV. Large-scale culture of recombinant baculovirus
- One liter of insect cells will be infected with high-titer virus stocks (25-30 ml of high titer virus required).
|
1 week |
V. Purification2
- 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 |
| Total |
8 weeks |
|
