Baculovirus Recombinant Protein Expression Vectors
The baculovirus vector system is widely used for the expression of recombinant proteins in cultured insect cells. It is one of the most versatile and powerful systems for eukaryotic expression of recombinant proteins. This system is particularly advantageous for large-scale preparation of proteins that require expression in eukaryotic host cells. Many eukaryotic proteins undergo posttranslational modifications that can only take place in eukaryotic cells (e.g. glycosylation), or they need a eukaryotic cellular milieu for proper folding (e.g. membrane proteins). In these cases, prokaryotic expression systems are often inadequate and the baculovirus expression system could be a good alternative.
Baculovirus is a double-stranded DNA virus that commonly infects insects, particularly members of the order Lepidoptera (moths, butterflies and skippers). The cloning vector in our baculovirus expression system, pBV, is optimized for use with the baculovirus shuttle vector (known as bacmid) derived from the baculovirus strain AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus), which has a 134 kb genome in its wildtype form.
The gene of interest is first cloned into the pBV vector under the control of a strong promoter. The pBV vector with dual promoters contains both the P10 and polyhedrin (PH) promoters, derived from AcMNPV allowing users to simultaneously express two different genes of interest. This makes the vector particularly suitable for applications requiring the co-expression of two different genes in cells such as studies involving the formation of protein complexes or for following cellular enzymatic reactions. Since both P10 and PH promoters are derived from AcMNPV, in order to reduce possibilities of recombination between duplicated AcMNPV sequences, the P10 promoter is placed in a reverse orientation relative to the PH promoter within the pBV vector.
The entire expression cassette consisting of the two promoters and their downstream genes of interest, along with a gentamicin resistance gene, is flanked by the Tn7 transposon terminal elements, Tn7L and Tn7R. This vector is then transformed into E. coli carrying the bacmid shuttle vector and a helper plasmid. The bacmid is essentially a very large plasmid containing the baculovirus genome modified to carry a lacZ gene and an attTn7 docking site inserted in the lacZ coding region. The helper plasmid expresses the Tn7 transposase. The transposase would then mediate the transposition of the region flanked by Tn7R and Tn7L on the pBV vector, which contains the expression cassette for the two genes of interest and gentamicin resistance, into the attTn7 docking site of the bacmid. Colonies containing recombinant bacmids can be identified by gentamicin selection and blue/white screening (non-recombinant colonies are blue due to lacZ expression whereas recombinant colonies are white due to disruption of lacZ by transposon insertion). Purified bacmid DNA can then be used to transfect insect cells to generate live baculovirus, which can be used to produce the recombinant proteins of interest.
The most commonly used cell line for expressing recombinant proteins from baculovirus vectors is Sf9. This clonal line was derived from ovarian tissue of Spodoptera frugiperda (fall armyworm). This cell line is adaptable to a variety of culture and media conditions, including suspension or monolater culture and serum-free media. Larvae and other Lepidoptera cell lines have also been used extensively, and there are some reports of baculovirus being an effective vector for mammalian cells.
For further information about this vector system, please refer to the papers below.