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| Services >> Proteomics |
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| Baculovirus Expression System |
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Baculovirus Expression System
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Baculoviruses are rod-shaped (baculum in Latin means 'stick') dsDNA viruses found mainly in insects. The most common baculovirus used for expression studies is Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which relies on the lepidopteran species Spodoptera frugiperda and Trichoplusia ni as host insects. AcMNPV particles surround themselves with a protective matrix consisting of the protein polyhedrin, which permits survival in the environment and efficient spread to new hosts. Under the control of the extremely strong promoter pPolh, polyhedrin is expressed at extremely high levels (up to 50% of all cellular protein) at the end of the baculovirus life cycle. The baculovirus expression system makes use of the fact that in cell culture a polyhedrin coat is not essential for virus propagation and thus heterologous proteins can be expressed under the control of the pPolh promoter (see O'Reilly et al. (1997) for further reading). Using the sf9 insert cells, crnbio offer the baculovirus expression services.
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Service Contents
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1. Gene acquiring (optional).
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2. Constructing plasmids (optional).
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3. sf9 cell culture and affection.
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4. Protein purification.
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Available Services are as follows, depending on your research needs, you can choose any starting phase or combinations.
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Phase I: Subcloning into Recombinant Baculovirus Transfer Vector
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Description: |
● Amplification/isolation of the gene of interest out of a customer-supplied vector and subcloning it into recombinant Baculovirus transfer vector.
● Verification of the subcloned gene by restriction digest and sequencing.
● Preparation of the recombinant vector DNA suitable for co-transfection with baculovirus DNA. |
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Required Materials: |
● 5 ug of plasmid containing gene of interest plus complete vector sequence in electronic format. |
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Deliverables: |
● 20 ug of ready-to-use recombinant Baculovirus transfer plasmid containing the gene of interest
● Final sequencing data
● Detailed research report |
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Duration: |
● 2 weeks: If insert > 2kb, one week additional per 1kb |
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Price (USD): |
● $998: If insert > 2kb, $399 additional per 1kb |
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Phase II: Generation and Evaluation of Baculovirus Recombinants
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Description: |
● Cotransfection of recombinant Baculovirus transfer vector containing gene of interest with 3 different linearized Baculovirus DNAs. Recombinant clones will be identified and amplified. The customer will choose the best recombinant baculovirus for the protein expression. |
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Required Materials: |
● 20 ug of ready to use recombinant Baculovirus transfer plasmid containing the gene of interest. |
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Deliverables: |
● 3 cell pellets for evaluation of Baculovirus to be used and 50ml recombinant baculovirus solution
● Detailed research report |
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Duration: |
● 2-3 weeks |
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Price (USD): |
● $1750 |
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Phase III: Amplification of Recombinant Baculovirus Stock
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Description: |
● Amplification of the chosen recombinant Baculovirus and titration of final high-titer stock by end point dilution |
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Required Materials: |
● At least 5 ml of recombinant Baculovirus stock |
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Deliverables: |
● Specified quantity (in ml) of high-titer recombinant Baculovirus stock solution ready to express the protein of interest
● Detailed research report |
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Duration: |
● 2-3 weeks |
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Price (USD): |
● $598 for each 500 ml |
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Phase IV: Baculovirus Expression Optimization Service
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Description: |
● Amplification of the chosen recombinant Baculovirus to a high-titer stock of more than 1 x 108 pfu per ml
● Infection of three insect cell lines (Sf21, Sf9, and T.ni using the high-titer stock and with or without booster virus
● Cells are harvested at two different time points(72hr or 96hr) |
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Required Materials: |
● At least 5-10 ml of recombinant Baculovirus stock. |
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Deliverables: |
● 18 sets of cell pellets and media for evaluation of protein yield
● Detailed research report |
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Duration: |
● 2-3 weeks |
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Price (USD): |
● $1850 |
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Phase V: Protein Production Service
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Description: |
● Amplification of recombinant Baculovirus stock for protein production;
● Expression of recombinant protein under optimized conditions. |
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Required Materials: |
● At least 100 ml of high-titer (>108 pfu) recombinant Baculovirus stock. Optimized protein expression conditions are determined by previous evaluation project. |
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Deliverables: |
● Cell pellets ready for protein purification or in the case of a secreted recombinant protein, the customer will receive the supernatant
● Detailed research report |
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Duration: |
● 2~3 weeks (1~5 Liters)
● 4~5 weeks (5~20 Liters)
● 8~10 weeks (20 and above Liters) |
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Price (USD): |
● $598 for each liter of culture |
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Phase VI: Protein Extraction & Purification
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Description: |
● Cell pellet harvested from 1 liter of insect culture will be lysed with our optimized reagents and protocol.
● Protein purification using appropriate affinity medium for 6xHis or GST-tagged recombinant proteins.
● Purification can be performed either under native or denatured conditions (as desired by the customer).
● SDS-PAGE and Western Blot will be performed for quality testing. |
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Required Materials: |
● Cell pellet ready for extraction |
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Deliverables: |
● Protein solution in standard or customer-specified buffer
● SDS-PAGE or Western Blot data
● Detailed research report |
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Duration: |
● 2-3 weeks |
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Price (USD): |
● $1500/L, inquire the price for larger volume |
- References
 Baculovirus Expression System.pdf- baculovirus dual expression system-based vaccine confers complete protection against lethal challenge with H9N2 avian influenza virus in micepdf
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