Baculovirus transfer vector

Sf9 cell culture at 1-2 x 10 cells/ml 1 pig linearized BaculoGold DNA (BD Blosclences) Recombinant Baculovirus transfer vector containing the Insert... 

Mix 0.5 xg linearized BaculoGold DNA with 2-5 xg recombinant Baculovirus transfer vector containing the insert and allow the mixture to sit at room temperature for 5 min. 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Cloning the Gene of Interest the into Baculovirus Transfer Vector... 

Alternative bnearized AcMNPV-baculovirus DNA for the generation of recombinant baculoviruses using the transfer vector set pVLl 392/1393 are the BacPAK6 (Takara Clontech) and the Bac-N-Blue DNA (Invitrogen, Thermo Fisher Scientific). 

The protocols described in this article will allow the reader to prepare recombinant baculoviruses for foreign gene expression in insect cells, starting with purified virus DNA and the appropriate recombinant transfer vector. 

The methods described below outline (1) the construction of a representative transfer vector in E. coli, (2) co-transfection of insect cells with this transfer vector and bacmid, (3) propagation of recombinant baculovirus, (4) induction of protein expression, (5) the extraction of the protein from insect cells, (6) the printing of a protein microarray, (7) the assay of a protein microarray for protein kinase activity, and (8) the assay of a protein microarray for cytochrome P450 turnover activity. 

Condreay JP, Kost TA (2003), Virus-based vectors for gene expression in mammalian cells Baculovirus, In Makrides SC (Ed.), Gene Transfer and Expression in Mammalian Cells, Elsevier Science BV, Amsterdam, pp. 137-150. 

Numerous shuttle vectors are available that facilitate convenient transfer from bacteria or mammalian cells. Proteins such as HIV Gp 160, carcinoembryonic antigen, and a form of influenza vaccine have successfully been expressed in baculovirus systems [9]. Potential disadvantages of this system include inappropriate posttranslational modifications, use of insect rather than mammalian cells, and characterization of both the baculovirus and SF9 insect cells used. 

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