Improved generation of recombinant baculovirus genomes in Escherichia coli

Abstract

An improved method for the generation of recombinant baculoviruses by Tn7-mediated transposition is described. The method is based on the modified donor vector (pBVboost) and an improved selection scheme of the baculovirus bacmids in Escherichia coli with a mutated SacB gene. Recombinant bacmids can be generated at a frequency of ∼10⁷/µg of donor vector with a negligible background. This easy-to-use and efficient pBVboost system provides the basis for a high-throughput generation of recombinant baculoviruses as well as a more convenient way to produce single viruses. The introduced selection scheme is also useful for the construction of other vectors by transposition in E.coli.

INTRODUCTION

Since 1983, when the baculovirus expression vector was introduced (1,2), the baculovirus expression vector system (BEVS) has become a useful tool for the expression of recombinant proteins in insect cells (3), and more recently, in vertebrate cells (4). The system has several unique features which account for its popularity. Expression of a gene in a eukaryotic milieu takes advantage of the pathways that facilitate folding, post-transcriptional modifications and assembly of the protein. High expression levels (up to 1 g/l ≌ 10⁹ cells) can be achieved and the system is safe to use. The capsid structure of the baculoviruses allows packaging and expression of very large genes and the vectors are not dependent on helper viruses. The availability of cell lines suitable for suspension cultures in serum-free conditions renders BEVS relatively easy for upscaling (3,5).

Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is the prototype baculovirus and the virus most commonly used in BEVS. Due to the large size of the AcMNPV genome (134 kb) a homologous recombination procedure was originally adopted to insert foreign genes into the baculovirus genome (2). In practice, the target gene is subcloned into a transfer vector containing a suitable promoter, flanked by baculovirus DNA derived from a non-essential locus, such as the polyhedrin gene of AcMNPV. The viral DNA and transfer plasmid are then co-transfected into insect cells. If the target gene is inserted into the polyhedrin locus, an altered plaque morphology of the recombinant viruses can be used for the identification of recombinant viruses. Viral identification may be facilitated by the introduction of a lacZ cassette into the system which enables detection of the recombinant viruses by color (3). However, typically only 0.1–1% of the resulting progeny is recombinant. Several techniques have been developed to further facilitate and speed up the construction of recombinant baculoviruses (6,7). Linearization of the AcMNPV genome reduces the background of wild-type viruses and, as a result, 10–25% of the progeny viruses are recombinant (8). To obtain an even higher proportion of recombinants (85–99%), AcMNPV genome has been further modified by removing an essential gene (ORF 1629) (9). Infective viruses will only be reconstituted by recombination with the transfer vector carrying the gene of interest, whereby an intact ORF 1629 will be restored in the genome. To ease the manipulation of the ORF1629 deleted baculovirus genome, Je et al. (10) have recently described a bacmid form of this virus which can be maintained in Escherichia coli. Generation of recombinant baculovirus by direct cloning into a baculovirus genome is also possible (11,12) as well as the use of the Cre-loxP system (13).

In order to avoid laborious and time-consuming plaque purification processes the genetic material can be introduced into the baculovirus genome by homologous recombination in the yeast Saccharomyces cerevisae (14). This method is rapid (pure recombinant virus within 10–12 days) and it ensures that there is no parental virus background, but it suffers from the need for experience in yeast culturing and the incompatibility of traditional transfer vectors with the system. An even faster approach (pure recombinant virus within 7–10 days) for generation of recombinant baculoviruses uses site-specific transposition with Tn7 to insert foreign genes into bacmid DNA (virus genome) propagated in E.coli cells. The E.coli clones containing recombinant bacmids are selected by color (β-galactosidase) and the DNA purified from a single white colony is used to transfect insect cells (15). This system is compatible for simultaneous isolation of multiple recombinant viruses but suffers from the relative low percentage of recombinant colonies (baculovirus genomes) obtained upon transformation of a special strain of E.coli (DH10Bac). We have thus developed an improved transposition-based system for the generation of recombinant baculoviruses which results in negligible background of parent virus genomes (blue colonies) upon transformation. The improved selection scheme makes this method truly compatible for high-throughput baculovirus preparation and is suitable for rapid isolation of multiple recombinant viral genomes which is vital for the generation of baculovirus expression libraries.

MATERIALS AND METHODS

Bacterial strains, plasmids, cell lines and viral DNA

Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA, USA) was used for propagation of plasmids. DH10Bac cells and pFastbac1 donor vector were obtained from Invitrogen. HighQ-1 Transpose Ad 294 cells and pCR259 donor vector were from QBiogene (Carlsbad, CA, USA). pDNR-LIB vector containing SacB gene was purchased from BD Biosciences Clontech (Palo Alto, CA, USA).

Construction of modified donor vector pBVboost

The modified donor vector was constructed by replacing the ampicillin (Amp) resistance gene in pFastbac1 vector with Bacillus subtilis levansucrase gene (SacB) from the pDNR-LIB vector. In practice, pFastbac1 vector was cut by a BspHI restriction enzyme and the linear vector backbone was purified by gel electrophoresis. The SacB expression cassette was obtained from pDNR-LIB by polymerase chain reaction (PCR) with the primers: DNR5′, 5′-GTTATTCATGAGATCTGTCAATGCCAATAGGATATC-3′ (sequence for nucleotides 1263–1282 of pDNR-LIB in bold; BspHI and BglII sites underlined); DNR3′, 5′-TTAGGTCATGAACATATACCTGCCGTTCACT-3′ (sequence for nucleotides 3149–3179 of pDNR-LIB in bold; BspHI site underlined). The PCR was performed essentially as described (16) except that annealing was carried out at 58°C and EXT DNA polymerase (Finnzymes, Helsinki, Finland) was used for amplification. The amplified fragment was digested with BspHI and purified as previously described (16). The purified PCR product was cloned into a BspHI-digested pFastbac1 vector (Invitrogen) for the orientation shown in Figure 1. The resulting plasmid was named pBVboost. The SacB#3 sequence was confirmed by DNA sequencing (ALF; Amersham Pharmacia Biotech, Uppsala, Sweden).

Figure 1.

Plasmid map of the pBVboost donor vector. The insect cell expression cassette is composed of a multiple cloning site (MCS, unique restriction enzymes shown) flanked by the polyhedrin promoter (pPolh) and simian virus 40 polyadenylation site (SV40 pA). Tn7L and Tn7R, left and right ends of the Tn7 cassette; SacB#3, mutated levansucrase gene; ori, the ColE1 origin of replication; GENT, gentamycin gene.

Generation of the attTn7 blocked E.coli strain DH10BacΔTn7

In order to block the cryptic attTn7 site in the DH10Bac genome, pBVboost was cut by BseRI/AvrII. The excised gentamycin (Gent) resistance was substituted with the Amp resistance cassette (ARC) from the pFastbac1. The ARC was obtained by PCR with the primers: DH10Bacinttn7destroybyamp5′, 5′-AAATATGAGGAGTTACAATTGCTAATTAATTAATTCGGGGAAATGTGCGCGGAA-3′ (sequence for nucleotides 471–490 of pFastbac1 in bold; BseRI site underlined); DH10Bacinttn7destroybyamp3′, 5′-CTTGGTCCTAGGATTACCAATGCTTAATCAGTG-3′ (sequence for nucleotides 1430–1449 of pFastbac1 in bold; AvrII site underlined). The PCR was performed as described above. The amplified fragment was digested with BseRI/AvrII and purified as above (16). The purified PCR product was cloned into a BseRI/AvrII-digested pBVboost. The resulting plasmid was named pBVboostΔamp. The sequence of ARC was confirmed by sequencing (ALF). DH10Bac cells were transformed by pBVboostΔamp. Single blue colonies were picked from LB plates containing 50 µg/ml kanamycin sulphate (Kan), 10 µg/ml tetracycline (Tet), 50 µg/ml Amp, 50 µg/ml X-Gal, 1 mM IPTG and 10% sucrose in 5 ml of LB medium. On the next day, the colonies were screened for the presence of intact bacmids by PCR as described (17). Colonies resulting in 325 bp bands (sign of the intact bacmid) in gel electrophoresis were further studied for the absence of donor plasmid by running samples of purified plasmid DNA (Wizard minipreps; Promega, Madison, WI, USA) in gel. The resulting clones were preserved at –70°C as E.coli DH10BacΔTn7.

Preparation of electro-competent DH10Bac or DH10BacΔTn7 cells

In order to prepare electro-competent cells, single colonies from LB plates (Kan and Tet for DH10Bac or Kan, Tet and Amp for DH10BacΔTn7 cells at the above concentrations) were inoculated into 10 ml of Super broth (SB; 30 g of tryptone, 20 g of yeast extract, 10 g of 3-N-morpholinopropane sulfonic acid in 1 l of water, pH 7.0) with the appropriate antibiotics. Suspensions were cultivated overnight at 37°C on a shaker. One liter of SB with 5 ml of 2 M glucose was then inoculated with 5 ml of overnight culture until the optical density of the new culture reached 0.8–0.9 (∼2–4 h) at 600 nm. The culture was then chilled on ice for 15 min and centrifuged at 1500 g for 15 min at 4°C. Cells were washed with 800, 500, 300, 200 and 100 ml of ice-cold water/10% glycerol and centrifuged as above. Finally, cells were suspended in a total volume of 3–4 ml of 10% glycerol and preserved in 40 µl aliquots at –70°C.

Transposition of mini-Tn7 cassettes from the donor vectors into bacmids and production of recombinant baculoviruses

Transposition was performed by electro-transforming 40 µl of DH10Bac or DH10BacΔTn7 cells with 0.1–1 ng of pFastbac1 or pBVboost donor vector. Electro-transformation was performed as described (18) using a Bio-Rad Gene Pulser II system (Hercules, CA, USA). The cells were allowed to recover 4 h post-transformation at 37°C with vigorous shaking. Aliquots corresponding to 10–100 pg of donor vector (resulting in 100–1000 colonies) were plated on LB plates supplemented with 7 µg/ml Gent and 10 µg/ml Tet with and without 10% sucrose. Colonies were studied for the presence of recombinant baculovirus genomes by PCR as described above. The recombinant viruses were generated according to the protocol provided by the Bac-To-Bac system (Invitrogen).

Generation of recombinant adenovirus genomes in E.coli by transposition of mini-Tn7 cassettes from the donor vectors into admids

Transposition with the original pCR259 donor vector of the Transpose-Ad system was performed according to the Transpose-Ad manual (QBiogene). To test the compatibility and efficacy of the pBVboost system selection scheme for admid generation, HighQ-1 Transpose Ad 294 cells were used in place of DH10BacΔTn7 cells and the transposition was performed according to the BVboost system.

RESULTS AND DISCUSSION

The site-specific transposition of an expression cassette into a baculovirus genome to generate recombinant baculoviruses (bacmids) in E.coli has become a popular procedure (15). This method is rapid since it eliminates the need for multiple rounds of plaque purification. Insertion of the mini-Tn7 from the donor plasmid into the mini-attTn7 attachment site on the bacmid disrupts expression of the lacZα peptide. Thus, colonies containing the recombinant bacmids are white while blue colonies harbor the unaltered bacmids. However, the original system suffers from the poor selection scheme and the identification of recombinants in E.coli is hampered by colony sectoring and multiple colony morphologies (19). This makes the system incompatible for the high-throughput virus generation required, e.g. for high diversity library construction.

We hypothesized that the system might be improved by incorporating a lethal gene into the donor plasmid. The lethal gene product would kill cells still harboring the donor vector while the combined selection pressure as a result of the successful transposition of the expression cassette from the donor plasmid into the bacmid would rescue only recombinant bacmids. Thus, we constructed a donor vector pBVboost which carries the SacB gene from Bacillus amyloliquefaciens (20). SacB encodes levansucrase, which catalyzes the hydrolysis of sucrose to generate the lethal product levan. Levan will kill cells in the presence of sucrose. Interestingly, only a point mutated form of SacB, SacB#3, proved to be useful together with our optimized use of antibiotics. The reason for this is not known but it probably reflects the need to balance the lethal effect of levan in the presence of sucrose with the use of the combined antibiotics. This was supported by the fact that the use of the intact SacB gene in the donor vector resulted only in a low amount of recombinant bacmids.

The transposition efficacy in the DH10Bac or DH10BacΔTn7 (in which the chromosomal attTn7 site is occupied) cells was studied using the original pFastbac1 or pBVboost donor vectors and the results were compared. As expected, the use of pBVboost resulted in a significant increase in the efficacy to generate recombinant bacmids in the presence of sucrose (Table 1). An over 6-fold increase in the transposition efficacy (white colonies) was detected in favor of pBVboost in DH10Bac cells. Furthermore, the transformation of DH10BacΔTn7 with pBVboost resulted typically in 100% white colonies as compared with only 23% in the pFastbac1 plates. The presence of recombinant bacmids in the morphologically white colonies was proved by PCR (Fig. 2). Notably, the use of the DH10BacΔTn7 strain also yielded a significant increase in the recombinant bacmids with pFastbac1. This is in agreement with the results of Leusch et al. (19) that the chromosomal attTn7 site of the DH10Bac must be occupied in order to avoid transposition of the mini-Tn7 from the donor plasmid into the chromosomal site.

Table 1. Typical transposition efficacy by the original and the new pBVboost baculovirus generation system.

Donor vector				DH10Bac				DH10BacΔTn7
	No sucrose		Sucrose		No sucrose		Sucrose
	White colonies (%)	c.f.u./µg	White colonies (%)	c.f.u./µg	White colonies (%)	c.f.u./µg	White colonies (%)	c.f.u./µg
pFastbac1	3	2.6 × 107	9	2.3 × 107	18	8.6 × 107	23	8.7 × 107
pBVboost	3	0.8 × 107	61	0.6 × 107	18	2.9 × 107	100	1 × 107

Figure 2.

Verification of the transposition in the white colonies generated by the new pBVboost-based method. PCR analysis of (A) DH10Bac or (B) DH10BacΔTn7 cells transformed by pBVboost. A ∼2300 bp sized band was detected from the 10 randomly picked white colonies (lanes 1–10) indicating the presence of recombinant bacmids. A 325 bp band indicates the presence of parental bacmid. Lanes M, Ladder Mix molecular weight marker (Fermentas AB, Vilnius, Lithuania). Lane +, positive control (blue colony). Lane –, negative control (no template).

A system for the generation of recombinant adenovirus genomes in E.coli (admids) by Tn7-mediated transposition was recently described (21). Transposition efficacy of the system was, however, modest (21) (Table 2). Since the basic idea behind the admid system is the same as with the BVboost system, we studied the compatibility of our improved selection scheme for the admid system. The replacement of the bacmid cells (DH10BacΔTn7) with the admid cells (HighQ-1 Transpose Ad 294) in the BVboost system resulted in an ∼7-fold increase in the number of recombinant admids as compared with the original admid system (Table 2). The presence of the recombinant admids in the morphological white colonies was proved by PCR. The increase in the efficacy is in agreement with the results obtained with the BVboost system (Table 1). The deletion of the cryptic attTn7 site, still present in the genome of HighQ-1 Transpose Ad 294 cells, should further improve the system with up to 100% recombinant admids, analogous to the BVboost system. These results suggest that the presented selection scheme may be generally applicable for the efficient generation of vectors by transposition in E.coli.

Table 2. Compatibility of the BVboost system selection scheme for the transposition-mediated adenovirus generation.

Donor vector	BVboost protocol		Transpose-Ad protocol
	HighQ-1 Transpose Ad 294 cells		HighQ-1 Transpose Ad 294 cells
	White colonies (%)		White colonies (%)
	No sucrose	10% sucrose
pCR259	ND	ND	10–15
pFastbac1	5	10	ND
pBVboost	5	65	ND

ND, not determined.

Cloning of a transgene into pBVboost does not affect the improved selection scheme since we have already produced several recombinant baculoviruses by this new method with similar transposition efficiency as shown above. We have also found the yields and expression characteristics of these viruses to be identical to viruses generated by the original system. Both systems produce high-titer viruses (∼10⁸ p.f.u./ml) capable of expressing large quantities of desired gene products in insect cells or, with a suitable promoter, in mammalian cells (22). However, a striking difference as compared with the original method is that recombinant bacmids can be generated at a frequency of ∼10⁷/µg of donor vector with a negligible background. This frequency may further be improved by optimizing the preparation of competent DH10BacΔTn7 cells and by further optimizing the transformation protocol. An additional advantage of the pBVboost system is that due to the powerful selection scheme there is no need for the color selection (i.e. no need for expensive X-Gal and IPTG reagents in the plates). This makes the system very cost effective. The overview of the BVboost system is presented in Figure 3.

Figure 3.

Overview of the BVboost baculovirus generation system.

In conclusion, the use of the presented new selection scheme bypasses the disadvantages associated with the original transposition-based generation of baculovirus genomes in E.coli while retaining the simple, rapid and convenient virus production. Addition of the lethal gene into the donor plasmid along with an E.coli strain, in which the chromosomal attTn7 is occupied, permits efficient selection of the recombinant bacmids in a cost-effective manner. The improved pBVboost system is compatible with high- throughput applications like expression library screening but also enhances the construction of single recombinant viruses.