baculoCOMPLETE protein expression kit

Baculoviruses are insect viruses, predominantly infecting insect larvae of the order Lepidoptera (butterflies and moths). A baculovirus expression vector is a recombinant baculovirus that has been genetically modified to contain a foreign gene of interest, which can then be expressed in insect cells under control of a baculovirus gene promoter. The most commonly used baculovirus for foreign gene expression is Autographa californica nucleopolyhedrovirus (AcMNPV). AcMNPV has a circular, double-stranded, super-coiled DNA genome (133894 bp; Accession: NC_001623), packaged in a rod-shaped nucleocapsid. The nucleocapsid can be extended lengthways and thus the DNA genome can accommodate quite large insertions of foreign DNA. The AcMNPV genome forms the basis of the flashBAC DNA provided in this kit. AcMNPV has a bi-phasic life cycle resulting in the  production of two virus phenotypes: budded virus (BV) and occlusion-derived virus (ODV). BVs contain single, rod-shaped nucleocapsids enclosed by an envelope (Figure 1) containing a membrane-fusion protein (GP64). GP64 is acquired when the nucleocapsids bud through the host cell plasma membrane. The BV form of the virus is 1000-fold more infectious for cultured insect cells, compared to the ODV phenotype, and is responsible for cell-to-cell transmission in the early stages of infection. It is the BV form of the virus that delivers the foreign gene into the host insect cell. In the late stages of infection large numbers of occlusion bodies (OB) or polyhedra are formed. These consist of multiple rod shaped nucleocapsids enclosed by an envelope, acquired de novo in the nucleus, and embedded within the para-crystalline matrix of the OB/polyhedra. The major component of the OB matrix is polyhedrin, a protein that is produced by the powerful transcriptional activity of the polyhedrin gene (polh) promoter.

OBs protect the virus and allow them to survive between hosts, within the environment. Most baculovirus expression vectors do not produce polyhedra (see below for details), just the BV form of the virus. This is a useful safety feature because recombinant virus cannot persist in the environment in the absence of polyhedra. 

The baculovirus polyhedrin gene is non-essential for virus replication in insect cells and this has led to the development of the widely-used baculovirus expression vector system, first described by Smith  et al.3. The coding sequences of the polyhedrin gene are replaced by those of a foreign gene, to produce a recombinant baculovirus in which the powerful polyhedrin promoter drives expression of the foreign gene. Hence recombinant baculoviruses are sometimes referred to as polyhedrin/polyhedra-negative viruses. Expression of foreign genes in insect cells using recombinant baculoviruses has become one of the most widely used expression systems, and is often the first choice eukaryotic system. 

The baculovirus expression system has several advantages over bacterial systems: 

Safe to use.  

Can accommodate large or multiple genes

Uses a variety of promoters for early and/or late gene expression  

Uses very efficient gene promoters  

Proteins produced are almost always functional  

Proteins are processed: signal peptide cleavage, nuclear targeting, membrane targeting, secretion, phosphorylation, glycosylation, acylation  

However, it is not without its disadvantages and these lie mainly in the labourintensive and technically demanding steps needed to produce recombinant viruses. The following outlines the development of the baculovirus expression system and the fine-tuning that has been used to improve the system over the years. Generally, the baculovirus genome is considered too large in which to insert the foreign gene directly. Instead the foreign gene is cloned into a transfer vector, which contains sequences that flank the polyhedrin gene in the virus genome. The virus genome and the transfer vector are introduced into the host insect cell and homologous recombination, between the flanking sequences common to both DNA molecules, effects insertion of the foreign gene into the virus genome, resulting in a recombinant virus genome. The genome then replicates to produce recombinant virus (BV phenotype only, as the polyhedrin gene is no longer functional), which can be harvested from the culture medium. In most baculovirus expression systems available that use homologous recombination to transfer the foreign gene into the virus genome, a mixture of recombinant and original parental virus is produced after the initial round of replication. Before using the virus as an expression vector, the recombinant virus has to be separated from the parental virus. Traditionally this has been achieved by plaque-assay or plaque-purification. This process is labourintensive, technically demanding and time-consuming. Many developments have attempted to improve the methods by which recombinant and parental virus may be separated. The frequency of recombination using this system is low (<1%) and recombinant virus plaques can be obscured by parental virus plaques. This problem was partially addressed by the insertion of the  Escherichia coli lacZ gene into the virus genome, in addition to the gene of interest. The recombinant virus plaques could then be stained blue by the addition of X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopranoside) against a background of colourless parent plaques.

However, this did not improve the low recombination efficiency and resulted in the contamination of recombinant protein with β-galactosidase. The efficiency with which recombinant virus could be recovered was improved by the addition of a unique restriction enzyme site (Bsu36I) at the polyhedrin locus (AcRP6-SC). Linearization of the virus genome prior to homologous recombination reduced the infectivity of the virus  DNA but increased the proportion of recombinant virus recovered to 30%. Homologous recombination between the transfer vector and the linear DNA re-circularised the virus genome, restoring infectivity and the production of virus particles.  LacZ was then introduced at the polyhedrin gene locus, replacing the polyhedrin coding region, producing AcRP23.lacZ. A Bsu36I restriction site within lacZ allowed for more efficient restriction of the linear DNA prior to homologous recombination and the presence of  lacZ allowed the selection of colourless recombinant virus plaques against a background of blue parental virus plaques in the presence of X-gal11. Greater than 90% recovery of recombinant virus plaques was achieved by further modifications to produce BacPAK612. BacPAK6 contains the  E. colilacZ gene inserted at the polyhedrin gene locus and Bsu36I restriction enzyme sites in two flanking genes on either side of  lacZ. Digestion with  Bsu36I removes the  lacZ gene and a fragment of an essential gene (ORF 1629)10producing linear virus DNA (BacPAK6) that is unable to replicate within insect cells. Co-transfection of insect cells with BacPAK6 DNA and a transfer vector containing the gene of interest, under the control  of the polyhedrin gene promoter, restores ORF1629 and re-circularises the  virus DNA by allelic replacement. The recombinant baculovirus DNA is then able to replicate in insect cells and in the late phase of infection, virions are assembled and recombinant baculoviruses are produced. However, Bsu36I digestion is never 100% efficient and the final virus population will always contain a mixture of recombinant and parental virus that requires purification by plaque-assay. Despite the fine-tuning and optimisation of the system, a number of steps are still required to produce and isolate recombinant virus. Hence compared to bacterial expression systems, it has not been amenable to high throughput or automated systems.