Baculovirus Expression Vectors

Transcription

1 Baculovirus Expression Vectors Felicity J. Haines 1, Robert D. Possee 2 and Linda A. King 1 *. 1 School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford, OX3 0BP, UK. 2 NERC Institute of Virology and Environmental Microbiology (CEH, Oxford), Mansfield Road, Oxford, OX1 3SR, UK. *Correspondence to Prof Linda A. King

2 Summary Baculoviruses are insect-specific viruses widely used for the production of many thousands of recombinant proteins, ranging from membrane bound proteins to cytosolic enzymes. The baculovirus expression system was initially developed over 20 years ago and since then has undergone numerous technological improvements to optimise expression of foreign genes within insect cell lines. The expression system is based on the replacement of a very late, non-essential, viral gene, termed polyhedrin, with a gene of interest. The insertion of foreign DNA at the polyhedrin locus within the viral genome results in incorporation of the foreign DNA into progeny virus particles and subsequent high-level expression of the recombinant protein within eukaryotic insect cell lines. More recently, baculovirus vectors have been developed to contain mammalian cell-active promoters and enhancers to permit transient and stable gene expression within higher eukaryotic cell lines, such as human hepatocytes and Chinese hamster ovary cell lines. Additionally, the possible application of in vivo gene delivery utilising this expression system makes baculoviruses an attractive and useful tool for studying the expression and function of gene products within mammalian systems and cell lines. Areas discussed in this review include insect cell culture, several baculovirus expression systems including Bac-to-Bac, flashbac and BacMam technology. Keywords baculovirus expression system gene therapy insect cell mammalian cell protein expression recombinant protein recombinant virus Glossary Bacterial Artifical Chromsome (BAC) an artificial chromosome based on the bacterial fertility (F) plasmid which permits replication of large DNA molecules within bacterial cells Co-transfection introduction of both transfer vector and viral DNA into eukaryotic cells to mediate homologous recombination and ultimately recombinant virus production Homologous recombination the process by which the exchange or replacement of a DNA region occurs between homologous flanking regions of genetic material

3 Introduction Baculoviruses are a diverse group of insect-specific viruses, predominantly infecting insect larvae of the order Lepidoptera. By far the most widely studied member of this family is Autographa californica nucleopolyhedrovirus (AcMNPV) for which the complete genome sequence has been determined. AcMNPV has a circular, double-stranded, super-coiled DNA genome of approximately 130 kilobases packaged in a rod-shaped nucleocapsid. These nucleocapsids can be extended lengthways and thus the virus genome can effectively accommodate large insertions of foreign DNA. Such insertions of foreign genes into the AcMNPV genome has resulted in production of baculovirus expression vectors; recombinant viruses genetically modified to contain a foreign gene of interest, which can be expressed in insect cells under the control of a baculovirus gene promoter. AcMNPV has a bi-phasic replication cycle resulting in the production of two virus phenotypes: budded virus (BV) and occlusion-derived virus (ODV) (Figure 1). BVs consist of a single, rod-shaped nucleocapsid enveloped in a host-derived membrane, enriched in a virally-encoded membrane fusion protein, GP64, which is incorporated into the BV particle during virus budding and release. The budded form of the virus is responsible for the cell-to-cell transmission of the virus both in vivo and in vitro. During the later stages of infection, large numbers of occlusion bodies or polyhedra are formed. The major component of the OB matrix is polyhedrin, a virus-encoded protein produced by the powerful transcriptional activity of the polyhedrin (polh) gene promoter. This protein protects the virus in the environment and allows virus particles to survive outside of their natural host. Insect Cell Culture The traditional cell lines for baculovirus-mediated expression studies are Sf21 and Sf9 cells. Both cell lines were originally derived from the pupal ovarian cells of Spodoptera frugiperda (fall army worm) termed IPLB-Sf-21 cells, with Sf9 cells (IPLB-Sf21-AE) being a clonal isolate of Sf21 cells. High Five cells (BTI-TN-5B1-4), a clonal isolate from Trichoplusia ni (cabbage looper) cell lines, are also used

4 (12) (6) (5) (4) (13) (7) (3) (8) (11) (9) polyhedra (2) (10) 2 infection 1 infection (1) Occlusion derived virion (ODV) Figure 1 Schematic of the bi-phasic AcNPV replication cycle. Polyhedra persist in the environment until ingested by a susceptible larval host. The alkaline environment of the insect midgut dissolves the polyhedra (1) to release occlusion derived virions (ODV). ODV then fuse with midgut epithelial cells (2) and enter the cell. Nucleocapsids (NCs) then travel to the nucleus (3), enter the nucleus most likely via the nuclear pore (4), where the NC is uncoated (5) and viral transcription initiated (6). De novo production of NCs ensues (7) and NCs leave the host cell nucleus (8). NCs are then transported to the plasma membrane where they bud from the host cell acquiring an envelope derived from the host cell membrane, to produce budded virus (BV) (9). BV then attach to further susceptible cells (10), are uncoated within the cell cytoplasm and NCs then traffic to the nucleus (11), following which translation, production of de novo NCs and additional BV occurs (4 10). During the later stages of infection, NCs remaining within the nucleus are occluded within polyhedra (13) which are released from the cell on cell death and host liquefaction (14).

5 for expression of recombinant proteins using the baculovirus expression system and have been found to produce greater levels of recombinant protein for a compared to Sf21 and Sf9 cells in some circumstances. In general, Sf21 or Sf9 cell lines are used for co-transfections, virus amplification and plaque assays whilst T. ni cell lines are employed for protein expression. Insect cell lines are generally considered simple to sustain compared to mammalian cell lines and can be maintained as either suspension cultures, in shake or stirred flasks, or in monolayer cultures in T-flasks or culture dishes. Most insect cell culture medium utilises a phosphate buffering system rather than the carbonate-based buffers commonly used for mammalian cell lines, negating the requirement for CO 2 incubators. The range for growth and infection of most cultured insect cells is between 25ºC to 30ºC, although 28ºC is generally considered the optimal growth temperature. Certain insect cells, for example Sf21 cell lines, require serum supplement. Foetal bovine serum (FBS) is most frequently used as the primary growth supplement in insect cell culture and although promotes cell growth and provides shear force protection important in shake and stirrer cultures, it can also cause excessive foaming, interfere with transfection reagents and is costly. For this reason, a number of commercially-available cell lines have now been adapted and optimised for growth in serum-free media, such as Sf- 900 II SFM and EXPRES-FIVE SFM for Sf9 cell growth. Such media contain amino acids, carbohydrates, vitamins and lipids essential for insect cell growth that reduce the effect of rate-limiting nutritional restrictions or deficiencies present within serum-supplemented media. Serum-free media also tend to support faster cell doubling times and permit cell growth to higher densities than serum-supplemented media which, when used for recombinant virus and/or protein production, ultimately results in higher virus titres or protein yields.

6 Baculovirus expression vector systems Since the application of baculovirus expression systems for the safe and abundant expression of foreign proteins in insect cells in the early 1980 s, this expression system has become one of the most popular methods for the production of large quantities of recombinant proteins within eukaryotic cells. The majority of baculovirus expression systems exploit the polh promoter to drive high level production of foreign proteins. The baculovirus polh gene is non-essential for virus replication in insect cell cultures and therefore can be removed from the virus genome with no detrimental effect to budded virus production. As the baculovirus genome is generally considered too large for direct insertion of foreign gene, the gene of interest is first cloned into a transfer vector containing sequences that flank the polh gene in the viral genome. Virus DNA and transfer vector are co-transfected into the host insect cell and homologous recombination between the flanking sequences common to both DNA molecules occurs. This causes the insertion of the gene of interest into the viral genome at the polh locus, resulting in the production of a recombinant virus genome. The genome then undergoes replication within the host nucleus, generating recombinant BV containing the foreign gene under the control of the strong, late viral polyhedrin promoter. Polyhedra are not produced as the polh gene is no longer functional, having been replaced by the gene of interest. Recombinant baculovirus expression vectors were originally produced using the highly ineffective homologous recombination process between a transfer vector containing the gene of interest and parental virus DNA. Insect cells were co-transfected with the baculovirus and transfer plasmid DNA, producing a mixture of both recombinant and parental viruses, with a recombinant viruses comprising only approximately 0.1% of total virus produced. Isolation of recombinant virus then required plaque purification were recombinant clones were identified by their characteristic occlusion-negative plaque phenotypes.

7 To improve the efficacy of recombinant virus production, a unique Bsu361 restriction enzyme site was engineered into the polh locus of baculovirus DNA to permit linearization of the viral genome prior to co-transfection, giving rise to a higher frequency of recombinant virus production. Further improvements employed multiple Bsu361 sites, with digestion of the viral DNA resulting in a partial deletion within an essential gene, ORF1629. The deletion within this gene prevents replication of parental virus, increasing the yield of recombinant virus to more than 90%. Insertion of the Escherichia coli (E. coli) lacz gene into the polh locus, replacing the polh gene, produced the commercially available BacPAK6 (Figure 2). A Bsu361 restriction enzyme site is located within the lacz gene along with 2 additional sites situated in the two flanking genes either side of lacz. Digestion of BacPAK6 with Bsu361 removes the lacz gene and a fragment of ORF1629 (Figure 2B), resulting in linear virus DNA incapable of replicating within insect cells. Co-transfection of insect cells with BacPAK6 DNA and a transfer vector containing the gene of interest restores the deletion in ORF1629 and recircularises the virus DNA by allelic replacement (Figure 2C). This restoration of the essential virus gene permits replication within insect cells, followed by assembly of nucleocapsids within the nucleus and ultimately production of recombinant viruses. Although the additional Bsu361 sites in BacPAK6 lead to an increased proportion of recombinants compared to previous baculovirus expression systems by reducing parental background, restriction enzyme digestion of virus DNA is never 100% efficient and co-transfection still results in the mixture of both parental and recombinant virus. Isolation of solely recombinant virus requires plaque purification. The presence of lacz allows the selection of colourless, recombinant virus plaques against a background of parental, blue plaques in the presence of X-gal (5-bromo-4-chloro-3-inolyl β-d-galactopranoside). Efforts to remove the requirement for plaque purification and increase both the speed and ease of recombinant virus production have resulted in the development of a number of unique baculovirus expression systems that

8 (A) lef2 lacz ORF1629 Bsu361 Bsu361 Bsu361 Digestion of viral DNA with Bsu361 (B) lef2 Deletion ORF1629 Co-transfection with transfer vector containing GOI and BacPAK6 viral DNA (C) lef2 Gene of interest Restored ORF1629 Isolation of recombinant virus by plaque assay (D) Figure 2. Schematic of recombinant virus production using BacPAK6 DNA. (A) The commercially available BacPAK6 baculovirus DNA contains the Escherichia coli (E. coli) lacz gene inserted at the polyhedrin (polh) locus. A Bsu361 restriction enzyme site is located within the lacz gene in addition to 2 BSU36q sites found in the flanking lef2 and ORF1629 coding regions. (B) Restriction enzyme digestion of the viral DNA results in the removal of the lacz gene and partial deletion of the ORF1629 coding region, a gene essential in viral replication, resulting in linear DNA incapable of replication within insect cells. (C) Cotransfection of insect cells with linear BacPAK6 DNA and a transfer vector containing a gene of interest results in insertion of the foreign gene into the virus DNA and restoration of the ORF1629 deletion, circularising the DNA and permitting virus replication within insect cells and recombinant virus production. (D) Isolation of recombinant virus is achieved via plaque assay.

9 involve various mechanisms to transfer foreign genes into the viral genome and shall be reviewed here. Bac-to-Bac The Bac-to-Bac baculovirus expression system (Invitrogen) provides a rapid and efficient method to generate recombinant baculoviruses (Figure 3). The system is based on in vivo bacterial site-specific transposition of an expression cassette into a baculovirus shuttle vector, also known as a bacmid, propagated in Escherichia coli. The bacmid contains a low-copy number mini-f replicon, a kanamycin resistance gene and the laczα gene. Located at the N-terminus of the laczα gene is an attachment site for the bacterial transposon Tn7 that does not interrupt the reading frame of the laczα peptide. The bacmid is propagated in E. coli cells as a large plasmid resistant to kanamycin that demonstrates blue selection in the presence of X- gal and isopropyl-beta-d-thiogalactopyranoside (IPTG). Prior to the production of a recombinant baculovirus using the Bac-to-Bac system, the gene of interest is cloned into a donor plasmid containing either a polh or p10 promoter region (Figure 3A). The expression cassette is flanked by the left and right arms of the Tn7 transposon and contains a gentamicin resistance gene and an SV40 polyadenylation signal to form a mini Tn7. The recombinant transfer vector is then transformed into E. coli cells containing the bacmid, and a helper plasmid encoding a transposase (Figure 3B). The mini Tn7 element from the recombinant transfer vector is transposed to the mini-atttn7 attachment site on the bacmid DNA aided in trans by helper plasmid-encoded transposase. Insertion of the gene of interest into the bacmid disrupts the laczα peptide resulting in white recombinant colonies when exposed to X-gal and IPTG compared to blue parental colonies containing the unaltered bacmid DNA. Having created the recombinant bacmid, the viral DNA must then be extracted from bacterial cells (Figure 3D) followed by transfection into insect cells

10 (A) Polyhedrin promoter (B) (C) Tn7R GOI Tn7L Helper Recombinant donor plasmid Transformation Donor plasmid Bacmid Transposition Antibiotic selection Polyhedrin promoter GOI Helper Competent DH10BAC E. coli cells E. Coli containing recombinant bacmid (F) Extraction of recombinant bacmid DNA (E) Recombinant budded virus particles (D) Determine viral titre via plaque assay Transfection of insect cells with recombinant bacmid DNA Infection of insect cells with P1 stock P1 virus stock (2ml) Recombinant bacmid DNA (G) P1 virus stock (2 10ml) Figure 3. Schematic representation of recombinant virus generation using the Bac-to-Bac system. (A) The gene of interest is first cloned into a suitable donor plasmid containing a polyhedrin (polh) promoter and a mini-tn7 element. (B) The recombinant donor plasmid is then transformed into Escherichia coli (E. coli) cells which contain a bacmid with a mini-atttn7 target site and a helper plasmid. The mini-tn7 element on the recombinant donor plasmid facilitates transposition of the gene of interest (GOI) into the target site on the bacmid along with transposition proteins provided by the helper plasmid, resulting in insertion of the GOI into the bacmid DNA (C). Recombinant virus DNA is then extracted and purified (D) following which insect cells are transfected with the recombinant bacmid DNA (E) to produce a P1 virus stock. (F) The virus titre of is determined via plaque assay and further insect cell cultures infected with the P1 stock to create a P2 stock of between 2 and 10ml (G).

11 (Figure 3E). The recombinant virus DNA undergoes transcription and translation, resulting in the production of recombinant budded virus particles which are then harvested and amplified through repeated rounds of infection to generate a high-titre baculovirus working stock in large volume. Using such site-specific transposition of genes into bacmid DNA has a number of advantages over using homologous recombination within insect cells for the production of recombinant baculoviruses. Primarily, the viral DNA isolated from selected bacterial colonies is not mixed with parental DNA, eliminating the need to isolate recombinant from non-recombinant virus and multiple rounds of plaque-purification. The substantially reduces the time taken to isolate and purify recombinant virus from between 4 to 6 weeks to between 7 and 19 days. Secondly, the Bac-to-Bac system easily permits simultaneous production and isolation of several recombinant viruses by one person. In addition to this, the Bac-to-Bac expression system is compatible with the Invitrogen Gateway cloning vectors, a system comprising a multitude of plasmids permitting both single and multiple insertions of genes into a single baculovirus genome. BaculoDirect TM The BaculoDirect TM baculovirus expression system (Invitrogen) uses the Gateway technology to permit the direct transfer of a gene of interest into the baculovirus genome, without the need for production of recombinant bacmid DNA. Gateway technology is a universal cloning method which provides a rapid cloning step, allowing the efficient transfer of DNA sequences into multiple vector systems. The technology is based on the site-specific recombination properties of the bacteriophage, lambda, which facilitates the integration of lambda into the E. coli chromosome (Figure 4A). This integration is mediated by a mixture of enzymes, the lambda recombinase, termed integrase, and the E. coli-encoded integration host factor (IHF) and occurs via intermolecular DNA recombination at specific attachment (att) sites.

12 During lambda integration into the E. coli chromosome, recombination occurs between attb and attp sites, present in the E. coli chromosomal and lambda phage DNA respectively, to give rise to attl and attr sites which flank the integrated phage DNA. This reaction can also be reversed utilising another lambda-encoded enzyme, excisionase, and recombination of attl and attr sites, resulting in the excision of the lambda DNA from the E. coli chromosome, recreating the attb site in E. coli and the attp site present in lambda phage. The Gateway system mimics the lambda integration system in an in vitro environment and combined with the BaculoDirect TM system, provides a suitable system for high-throughput production of multiple recombinant viruses (Figure 4B). Firstly, the gene of interest must be cloned into a suitable vector, termed an entry clone, with the foreign DNA flanked by two recombination sites, attl1 and attl2. BaculoDirect TM Linear DNA has been modified to contain attr1 and attr2 recombination sites located at the polh locus, along with a Herpes simplex virus thymidine kinase gene (HSV1 tk) and a lacz gene located between attr sites. To create a recombinant baculovirus, it is necessary to perform the LR recombination reaction utilising an LR clonase solution, an enzyme mix comprised of integrase, IHF and excisionase, to transfer the gene of interest into the baculovirus DNA. The attr sites present within the virus DNA undergo recombination with the attl sites within the entry clone, resulting in the integration of the foreign gene into the virus DNA to create an attb-containing expression virus. Having performed the LR reaction, baculovirus DNA is then used to directly transfect insect cells. Purification of recombinant viruses ensues following addition of medium containing the selective nucleoside analog, ganciclovir. Ganciclovir is enzymatically phosphorylated by HSV1 tk present in nonrecombinant genomes, and results in the incorporation of this nucleoside into DNA, thereby inhibiting their DNA replication. Recombinant viruses that have lost the counter-selectable marker via homologous recombination are capable

13 (1) attp Phage λ attb E. coli (2) Recombination mediated by phage integrase protein and host integration factor (3) Integration attl Excision attr Integrated prophage Figure 4A. Schematic representation of lambda bacteriophage site-specific transposition in Escherichia coli. Following infection of Escherichia coli (E. coli) cells, the lambda phage DNA integrates into the host genome between integration sites attp and attb found in the phage and E. coli DNA respectively (1). The integration reaction is mediated by both phage (intergase) and host (host integration factor) proteins (2) and results in the production of two new sites, termed attl and attr and the integration of phage DNA into the host genome (3). The integration reaction is reversible mediated by the proteins integrase, host integration factor and excisionase, resulting in the removal of the phage DNA from the I genome, recreating the attb and attp sites in E. coli and phage DNA, respectively.

14 (1) attl1 GOI attl2 attr1 polh locus attr2 Entry clone + BaculoDirect TM Linear DNA (2) attb1 attb2 Recombinant baculovirus DNA containing GOI at polh locus Transfection of insect cells Ganciclovir selection (3) P1 virus stock (2ml) Infection of insect cells with P1 stock Ganciclovir selection (4) P2 virus stock (2ml) Figure 4B. Schematic representation of recombinant baculovirus production using the BaculoDirectTM system. Following successful production of an entry clone containing a gene of interest (GOI), an LR reaction between the entry clone and the BaculoDirectTM Linear DNA (1) generates recombinant baculovirus DNA (2) containing the GOI at the polyhedrin (polh) locus. Insect cells are then transfected with the recombinant baculovirus DNA and recombinant budded virus selected via the addition of ganciclovir, resulting in the production of a 2ml P1 recombinant virus stock (3). This P1 virus stock is then used to infect further insect cell cultures in the presence of the selective compound, ganciclovir, to produce a high titre, 2ml P2 virus stock (4).

15 of replication within insect cells, promoting the amplification of recombinant viruses and production of a low-titre, P1 viral stock. Screening of P1 stocks can then be undertaken to ensure recombinant protein expression and the absence of parental background. Alternatively, the P1 stock can be used to directly infect insect cells in the presence of ganciclovir to produce a larger volume P2 working stock. The high-throughout application of BaculoDirect TM for the production of multiple recombinant viruses appears to offer significant time savings compared to other systems such as Bac-to-Bac. However, this system is currently restricted to the production of recombinant single gene baculoviruses preventing co-expression of multiple proteins. In addition, the necessity to selectively isolate recombinant from parental virus via the addition of ganciclovir is detrimental to total cell numbers and therefore consequently negative to final viral titres, ultimately requiring multiple virus amplification steps before a high-titre working virus stock is produced. flashbac A new platform technology for the production of recombinant baculoviruses has recently emerged that has significant advantages over the other vector systems described above. The flashbac expression system (Oxford Expression Technologies, Oxford, UK) combines the advantages of the traditional homologous recombination system within insect cells with those of the more recent bacterial-based systems. FlashBAC has been developed to remove the requirement for isolation of recombinant from parental virus via plaque purification, resulting in a one-step procedure for recombinant virus production and ultimately significantly reducing the time taken to produce a working high-titre recombinant virus stock. The flashbac expression system is based around a modified AcMNPV genome that contains a bacterial artificial chromosome (BAC) at the polh locus replacing the polh gene. The presence of the BAC within the virus

16 (A) chia flashbac DNA lef2 BAC lef2 Gene ORF1629 Co-transfection of insect cells (B) chia Recombinant virus DNA lef2 Gene ORF1629 (C) P1 virus stock (2ml) (D) P2 virus stock (50ml) Figure 5. Schematic representation of recombinant virus production using the flashbac system. The flashbac expression system relies on a modified baculovirus genome containing a bacterial artificial chromosome (BAC) at the polyhedrin (polh) locus and a partial deletion of the essential ORF1629 viral gene (A). Homologous recombination within insect cells between the flashbac DNA and a transfer vector containing a gene of interest (GOI) flanked by lef2 and the complete ORF1629 gene coding regions, results in the insertion of the GOI at the polh locus and restoration of ORF1629 gene (B). The deletion within ORF1629 results in a P1 stock containing only recombinant baculovirus production with no parental background (C). This P1 stock is then used to infect a larger insect cell culture resulting in the production of a high-titre, highvolume (50ml) P2 working recombinant virus stock (D)

17 genome allows the virus genome to be maintained and propagated within bacterial cells from which the circular DNA is then isolated and purified. Additionally, the modified genome contains a deletion in the essential gene ORF1629 which renders the virus inactive and unable to replicate within insect cells (Figure 5A). Homologous recombination between flashbac DNA and a suitable transfer vector containing the gene of interest, following transfection into insect cells, restores the deletion within, and thus the function of, the essential ORF1629 gene (Figure 5B). Simultaneously, the gene of interest is inserted into the viral genome at the polh locus under the control of the polh promoter, concomitantly removing the BAC replicon. The recombinant virus genome, with the restored essential gene is then capable of replication within insect cells, producing recombinant budded virus particles that can be harvested from the culture medium of the transfected cells (Figure 5C). The deletion present within ORF1629 of flashbac DNA prevents replication of any nonrecombinant, parental virus, therefore there is no requirement for recombinant virus isolation. Following transfection, virus can be added directly to insect cells to produce a high-titre working recombinant virus stock. The flashbac system is back compatible with all baculovirus transfer vectors based on homologous recombination in insect cells at the polh locus, including vectors using the polh promoter, dual triple and quadruple expression vectors and those that use other gene promoters such as p10, ie1, or gp64. However, vectors such as pfastbac TM which are designed for sitespecific transposition in E. coli systems such as Bac-to-Bac are not compatible with the flashbac system. The flashbac system is unique amongst baculovirus expression systems as it maximises protein secretion and membrane protein targeting. Within the baculovirus genome are a number of auxiliary genes which are non-essential in the replication and production of budded virus particles in vitro. One such gene is chitinase (chia), an enzyme with exo- and endochitinase activity, essential in the host-to-host transmission of the virus. Following infection of a

18 host insect, chitinase works in synergy with another virally encoded protein, termed cathepsin, to break down the host cuticle, ultimately resulting in tissue liquefaction and release of polyhedra to infect more hosts. Confocal and electron microscopy analysis has shown the localisation of chitinase within the endoplasmic reticulum during baculovirus infection. This severely compromises the function efficiency of the secretory pathway, consequently affecting the levels of recombinant protein production. Deletion of chia from flashbac has substantially improved the efficacy of the secretory pathway for producing recombinant proteins in insect cells, enhancing the yield of secreted of membrane-bound proteins in comparison to proteins produced in recombinant viruses that synthesize chitinase. The one-step flashbac technology facilitates the use of robotic systems for the generation of multiple recombinant viruses without the requirement for plaque purification. It has been demonstrated that co-transfection mixtures from multiple recombinant viruses can be used to infect insect cells for automated high-throughout protein screening in 96-well format. Additionally, this process can be performed solely in insect cells, removing the risk of cross-contamination between insect and bacterial cell cultures. Baculovirus-mediated gene delivery in mammalian cells The baculovirus expression system has been extensively used for the expression of recombinant proteins within insect cells for a number of years. More recently, recombinant baculovirus vectors have been developed to permit transient and stable gene delivery into a number of mammalian cell lines. Such vectors contain baculovirus promoters alongside mammalian cellactive expression cassettes to permit amplification of recombinant viruses in insect cells and expression of recombinant proteins in mammalian cells, respectively. Baculovirus-mediated gene delivery into mammalian cells, known as BacMam technology, was first demonstrated in hepatic cell lines and subsequent

19 studies have reported transduction of human cell lines including HeLa, pancreatic β-cells and primary neural cells, Chinese hamster ovary and porcine kidney cell lines. The transduction efficiencies of different cell lines varies considerably, with baby hamster kidney cells having a transduction efficiency of 95% whilst mouse 3t3 cells demonstrate only 10% efficiency. Transduction efficiency can be enhanced via the addition of various compounds such as trichostain A or sodium butyrate that act as histone deacetylase inhibitors, however these drugs have cytotoxic effects on cell cultures. The majority of recombinant viruses used to transducer mammalian cell lines contain hybrid promoters consisting of a chicken β-actin gene promoter and a cytomegalovirus (CMV) immediate early gene enhancer element along with a p10 baculovirus promoter for expression of the gene in insect cell lines. One significant advantage of BacMam technology is successful gene delivery of foreign DNA into mammalian cells is possible by simply adding recombinant baculovirus inoculum to a culture of mammalian cells. The entry mechanism of baculovirus into mammalian cell is poorly understood, however it is thought that the viral surface glycoprotein, GP64, plays a role in viral entry and endosomal release. Following viral entry, nucleocapsids induce actin filament formation within mammalian cells, a process also known to occur within insect cells involved in the propulsion of virus particles to the nucleus. Reports have demonstrated an increase in nuclear localisation of nucleocapsids in human hepatocytes following the disruption of the microtubule network. This suggests microtubules constitute a barrier to baculovirus transport towards the nucleus and thus disruption of these filaments may provide a simple method with which to increase nuclear localisation of recombinant nucleocapsids and ultimately increase foreign gene expression within mammalian cell lines. Transduction of mammalian cells with recombinant baculovirus is generally considered non-toxic and has no apparent effect on cell growth, even at high multiplicities of infection. The inability of baculoviruses to replicate within mammalian cells provides an attractive vector for in vivo applications, including gene function studies and

20 gene therapy. However, baculovirus is rapidly inactivated by human serum complement, destroying the ability of the recombinant virus to transfer genes in vivo. A number of methods have been developed to alleviate this problem, such including production of baculovirus particles pseudotyped with the vesicular stomatitis virus (VSV)-G protein providing a virus more resistant to complement than unmodified virus. Additionally, the use of baculoviruses to express antigens under the control of mammalian promoters has been shown to elicit and immune response in vivo. Recombinant baculoviruses expressing the gb protein from pseudorabies produced an immune response in mice intramuscularly inoculated with the modified baculovirus. Additionally, recombinant baculoviruses expressing the Influenza haemagglutinin protein elicited immune responses in mice when delivered by intramuscular injection and provided immunity to further infection following subjection to a lethal dose of influenza virus. The possibility of utilising baculoviruses for gene therapy and vaccine applications is still a very new, albeit extremely promising, technology and much research continues in this area to optimise baculoviruses as potential gene therapy vectors. Conclusions and future perspectives Manipulation of the baculovirus genome provides a powerful tool for the expression of recombinant proteins in both insect and mammalian cell lines. Advances in vector design and the various commercially-available baculovirus expression systems permits simple, recombinant virus production, with some systems negating the requirement of virus isolation and purification. This, coupled with the automation and high through-put possibilities of virus production using robotic systems for simultaneous multiple-virus production, has resulted in more laboratories employing baculoviruses as their expression system of choice. One of the main disadvantages of the baculovirus expression system is the dissimilarity of the insect-cell protein processing pathways compared to those of higher eukaryotes, for example the N-glycosylation pathway. The

21 production of transgenic insect cell lines expressing humanised protein glycosylation pathways offers a way to overcome this potential problem, enabling the production recombinant polypeptides which demonstrate greater similarity to their native mammalian proteins. Recombinant baculoviruses have become a widely used system for the production of recombinant proteins within insect cells. The availability of the entire baculovirus sequence has and will continue to enable further manipulation of the virus genome to increase and further optimise recombinant protein expression in both insect and mammalian cell lines. It is hoped that the use of recombinant baculoviruses as gene delivery vectors for higher eukaryotic cell lines will become as routine as the use of such viruses for recombinant protein expression within insect cells and that advances in knowledge and technology will continue to expand the possibilities and applications of the baculovirus expression system. Further Reading Hu, Y. (2005). Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacologica Sinica. 26, Hunt, I. (2005). From gene to protein: a review of new and enabling technologies for multi-parallel protein expression. Protein Expression and Purification. 40, King, L. A. and Possee, R. D. (1992). Baculovirus Expression System A Laboratory Guide. London, UK: Chapman and Hall. Kitts, P. A. and Possee, R. D. (1993). A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques. 14, Kost, T. A. and Condreay, J. P. (2002). Recombinant baculovirus as mammalian cell gene-delivery vectors. Trends in Biotechnology. 20,

22 Kost. T. A., Condreay, J. P. and Jarvis, D. L. (2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nature Biotechnology. 23,