This chapter will begin with an introduction to the basic baculovirus-insect expression system, Beijing Information, and then focus on recent advances that have greatly facilitated the use of the system by the average researcher.
Operation method
Arbovirus-Insect Cell Expression System Move i. baculovirus expression vectors The simplest classical baculovirus expression vector is a recombinant baculovirus whose genome contains a segment of exogenous nucleic acid sequence, usually dDNA encoding the target protein, transcribed under the control of a polycomb protein promoter. This chimeric gene consists of a polycomb protein promoter and an exogenous protein-coding sequence that is located in the polycomb seat of the viral genome, replacing the nonessential wild-type polycomb gene. In the laboratory, the recombinant virus is capable of infecting insect cells or larvae (caterpillars), which causes high levels of transcription of the exogenous cDNA at very late stages of infection. The transcribed mRNA is translated to produce the target protein. Polycomb protein promoters always seem to be able to drive extremely high levels of exogenous gene expression at the transcriptional level, and in many cases large quantities of exogenous proteins can be obtained, just as it was originally hypothesized that baculoviruses would be able to make large quantities of polycomb proteins. Indeed, the potential to express recombinant proteins at high levels is a major advantage of the baculovirus-insect cell system. In this context, high level is very broadly defined as producing greater than or equal to 100 mg of recombinant protein per liter of baculovirus-infected insect cell culture or 4 g of insect cells (typically at a cell density of 1X106 cells/mL). Furthermore, proteins expressed at high levels in baculovirus-insect cell systems rarely form inclusion bodies, which are often formed in bacterial expression systems. It is clear to any researcher involved in the production of recombinant proteins that the yields and solubility levels that can be achieved for proteins in any expression system depend greatly on the proteins themselves under study. Therefore, from 25 years of experience with the baculovirus-insect cell expression system, it is concluded that the system is more likely to result in high expression of both nuclear and cytoplasmic proteins than of secreted proteins. Expression levels of the former are lower than those of the latter, typically only a few milligrams to a dozen milligrams per liter (Jarvis, 1997). Another advantage of the baculovirus-insect cell system is the ability to process eukaryotic proteins, including the ability to modify proteins by phosphorylation or glycosylation. However, there are limitations to this view, and it is now well recognized that protein processing in the lepidopteran insect cell host of baculoviruses is different from that of higher eukaryotic cells. An additional nuisance is that baculovirus infection has a negative effect on host protein processing (Azzouzetal., 2000; JarvisandSummers, 1989). Clearly, these are factors to focus on for any researcher interested in producing recombinant proteins that require eukaryotic modifications, especially since such modifications are known to affect function directly or indirectly. Finally, it is noted that the baculovirus-insect cell system has been shown to be very useful for the production of multiproteinsubunitcomplexes (multiproteinsubunitcomplexe) [cf. Berger et al. (2004); Kost et al. (2005) for reviews]. The ability of the system to produce viral particles composed of multiple components, such that they are excellent vaccine candidates, demonstrates the power of the system in this important application area. For example, the baculovirus-insect cell system has been used to produce viral particles composed of poliovirus, bluetonguevirus, adeno-associatedvirus, hepatitisCvirus and papillomavirus. Virus-like particles are composed of a variety of proteins of adeno-associatedvirus, hepatitisCvirus and papillomavirus origin. The virus-like particles can be produced by constructing multiple recombinant viruses that express a single protein, or by constructing a single virus that encodes multiple recombinant proteins to infect insect cells. The first recombinant baculovirus vectors were constructed for the expression of chimeric genes consisting of a polycomb protein promoter and an exogenous coding sequence using basic homologous recombination principles, as described earlier. The detailed techniques of this method can be found in the first edition of this book (Bradley, 1990), as well as in the major papers published by the original authors and the excellent technical manuals prepared by them (O'reillyetal., 1992; SummersandSmith, 1987). Therefore, as with the above', this section will not be praised here. However, a brief introduction is important for background purposes. The common approach involves. (i) construction of bacterial transfer plasmids with chimeric genes that have flanking sequences derived from polycomb regions of the viral genome (Fig. I4.1); and (ii) Co-transfection of cultured insect cells using a mixture of the transfer plasmid and genomic DNA extracted from purified wild-type AcMNPV (Fig. 14.2). In the cotransfected insect cells, the transfer plasmid and the AcMNPV genome produce recombinant viral DNA molecules by homologous recombination that occurs between the flanking sequences of the target chimeric genes on the transfer plasmid and the upstream and downstream sequences of the viral genome polycomb protein genes. In order to knockout the polycomb protein gene and simultaneously knock-in the chimeric gene encoding the target protein, this must be a doublecrossover recombination. Of course, the incidence of such recombination is relatively low, estimated to be around 0.1% (Smithetal., 1983a). Therefore, it is necessary to isolate the few recombinant viruses from the large number of progeny viruses produced by cotransfection. Separation can be readily accomplished by the baculovirus vacuoles assay, but the researcher has to be able to recognize vacuoles formed by recombinant viruses from those produced by a large number of parental viruses. Initially, this can be done by simple visual screening, as the progeny from the parent virus forms polycomb-positive plaques, whereas the recombinant progeny lacks genes to express polycomb proteins and therefore shows polycomb-negative plaques. Trained researchers are able to identify polycomb-negative vacuoles by observing experimental plates under a dissecting microscope. Therefore, having this ability to recognize them is critical to the success of the screening. For many years, many researchers did not have the ability to recognize polycomb-negative baculovirus empty spots, and this is a very serious problem that limits the application of the baculovirus-insect cell system as a tool for the production of expressed recombinant proteins. Beginning around 2010, researchers began to overcome the technical limitations of baculovirus vector isolation and, through a number of modifications to the basic methodology described above, improved the system in other ways as well. These improvements can be divided into two categories: one for the transfer plasmid and another for the parental baculovirus genome. They are described below. Improvements to the transfer plasmid were made for two different purposes. The most important purpose was to make it simple to identify recombinant baculovirus vacuoles by visual screening, which was once difficult for the reasons described above. One usual method of such improvement is to elicit chimeric marker genes under the control of a baculovirus promoter. For example, the /3-galactosidase protein of Escherichia coli is easier to recognize by the naked eye than the polycomb-negative empty spot phenotype (Vialardetal., 1990). The introduction of marker genes into the transfer plasmid is smart, but the potential problems with that improved method should also be noted (O'reillyetal., 1992). The Bowman marker gene can signal not only the desired double-exchange homologous recombination, but also the singlecrossover recombination that occurs between the transfer plasmid and the viral DNA. Single-exchange recombination has a high probability of occurring, and the genome of the resulting recombinant virus contains the entire sequence of the transfer plasmid, including the bacterial replicon, with some randomization of integration sites. These recombinants are genetically unstable, and newly acquired exogenous genes may be lost in 1 or 2 rounds of viral replication. Despite their limitations, transfer plasmids that introduce marker genes into recombinant viral genomes are widely used, and they are especially useful for researchers who are well aware of the potential pitfalls of single-switch recombination. They simply use the marker gene to perform an initial screen, followed by a further screen to identify the integration site of the exogenous gene in the genome, and finally to determine whether double-exchange recombination has transferred the target gene into the recombinant baculovirus vector. The second purpose of the modification of the transfer plasmid was to facilitate the expression and purification of the recombinant protein in the baculovirus-insect cell system. There are too many such modifications to discuss here, but three common ones include introducing the coding sequence of the signal peptide; introducing a purification tag (e.g., His6) at the N- or C-terminus of the protein; and replacing the polycomb protein promoter with another baculovirus promoter, or replacing the polycomb protein promoter with multiple promoter elements to allow simultaneous expression of multiple recombinant proteins during baculovirus infection. This allows for the simultaneous expression of multiple recombinant proteins during baculovirus infection. A recent publication that lists almost all baculovirus transfer plasmids with these two types of modifications and briefly describes their functional features is a good source of information in this area (Possee and King, 2007). However, one class of improvements that is not listed but deserves further attention are the immediate^early) transfer plasmids, in which the polycomb promoter is replaced with the promoter of a baculovirus immediate-early gene, such as the iel gene of AcMNPV (Jarvisetal., 1996). The use of promoters from baculovirus early genes (e.g., the iel gene) would seem to violate the previously mentioned principles because these promoters are weaker than the polycomb protein promoters. However, there is some evidence to suggest that recombinant baculovirus expression of exogenous genes under the control of early promoters results in higher quality protein products, even though these early promoters do not initiate the same high level of transcription (ChazenbalkandRapoport,1995;Hill-PerkinsandPossee,1990; Jarvisetal.,1996; Murphyetal.,1990;Rankletal.,1994;SridharetaUW93). Indeed, this approach is particularly applicable to secreted proteins. As mentioned above, these proteins are usually expressed at low levels under the control of polycomb protein promoters. In this case, researchers may be able to capitalize on this benefit of exogenous gene expression earlier in the infection, thus avoiding the negative effects of baculovirus infection on host protein processing pathways, while also obtaining a high level of expressed product. Just like the improvement of the transfer plasmid, the improvement of the parental baculovirus genome has been made to meet a variety of different needs. Initially, the primary goal was to find ways to overcome the inefficiencies in the construction and isolation of recombinant baculovirus vectors, which was the main problem with the original baculovirus-insect cell system. It is now known that the root cause of this problem is the inefficient homologous recombination of transfer plasmids and parental baculovirus DNA in co-transfected insect cell lines. Ultimately, researchers have come up with a number of direct and indirect methods to address this challenge. Kitts and Possee took an important first step toward a solution to the problem by constructing a baculovirus with a linear DNA genome (Kittsetal., 1990). This virus, a recombinant baculovirus obtained by the original homologous recombination method, was mainly characterized by a new DNA sequence at the polycomb protein gene seat that had a Bw36I cleavage site (Fig. 14.3). As a result, this genome was able to be 5 like 361 linearized and used as parental viral DNA mixed with a transfer plasmid to cotransfect insect cells. The most important feature of this method is that the linearized parental viral DNA molecules do not replicate. Thus, linearization largely reduces the number of parental progeny in the cotransfected insect cells. In addition, the linear viral DNA can still be homologously recombined with the transfer plasmid, and the recombined viral DNA molecule re-forms a ring structure and regains its ability to replicate. These factors combined to increase the efficiency of recombinant baculovirus vector production in cotransfected insect cells from 0.1% to 10% to 20%, and also greatly simplified the technique.Kitts and Possee refined the method by constructing a recombinant baculovirus known as BakPAK6, which could be cleaved by Bm36I.The BakPAK6 genome has an E. coli ^cZ gene located in the polycomb protein gene seat, which contains a B361 site, plus a Bsw36I site introduced upstream and downstream of the viral gene, respectively (Fig. 14.4). Importantly, after digestion by &M36I, a portion of or/1629 was deleted. w/1629 is an essential gene of the virus, which is located downstream of the polycomb gene and encodes the nucleocapsidphosphoprotein of the viral nucleocapsid (VialardandRichardson,1993 ). The efficiency of constructing recombinant baculovirus vectors by co-transfection of insect cells using BakPAK6 as parental viral DNA increased to 95%.The success of BakPAK6 stimulated the commercialization and widespread use of a variety of linearized baculovirus DNAs. They were used as starting materials for the construction of recombinant baculovirus vectors. The most prominent of these commercialized baculoviral DNAs include the original BakPAK6 viral DNA commercialized by ClonTech. similarly, there are pre-linearized viral DNAs known as BaculoGold (Pharmingen/BDBiosciences), BacVector by Novagend, Diamond-Bac by Sigma- Aldrich, and BakPAK6 by Sigma- Aldrich. In addition, a slightly modified linearized viral DNA/transfer plasmid system called BaoN-BIue (Invitrogen) recombines and reconstructs the E. coli markers in its genome. Here in this chapter, it is important to emphasize the development of these viral DNA backbones, which effectively solved the major problems in constructing baculovirus expression vectors that existed at the time of publication of the first edition of this book. These linear baculovirus DNAs were widely marketed by commercial companies and were quickly recognized and accepted by biomedical research institutions as improved tools for recombinant baculovirus construction. The availability of these tools and methods (see related section below) made the use of baculovirus expression vectors much easier for the average laboratory researcher with a basic molecular biology background. Ultimately, the baculovirus-insect cell system became more widely used and generally recognized as an effective tool for recombinant protein preparation. Meanwhile, Luckow and his colleagues developed a different approach to isolating baculovirus vectors than linearizing the viral genome. Their approach utilized genetic transposition, rather than homologous recombination (LuckowetaL, 1993). This method allowed the researchers to solve the inefficiency of homologous recombination by using a different molecular mechanism to generate recombinant baculovirus DNA. The key to this approach is the construction of a new E. coli strain with a self-replicating plasmid, or bacmid, containing a complete copy of the baculovirus genome (Fig. 14.5). The polycomb protein region of the bacmid contains the E. coli ZacZ gene and the mini-AttTn7 site, which is the attachment site for transposition. This newly constructed E. coli strain also has a helper plasmid encoding a transposase. When the novel E. coli strain is transformed with the baculovirus transfer plasmid, the chimeric gene is transposed to the polycomb region on the baculovirus because the polycomb-driven target gene on the transfer plasmid is flanked by the left and right ends of the Tn7 attachment site. Transposition results in the deletion of the fccZ gene on the bacillus, which allows you to identify bacteria containing recombinant bacilli by performing a blue and white spot screen on screening media. In this way, you can easily isolate recombinant baculovirus DNA encoding the target gene from E. coli clones with white colonies to transform insect cells. the DNA transformation will initiate viral infection and lead to the production of progeny recombinant baculoviruses. This in vivo transposition method produces recombinant baculovirus DNA with 100% efficiency. it was originally commercialized as the Bac-toBac System, developed by LifeTechnologies, Inc. and is now available from Invitrogen, Inc. the bacteriocentric nature of the Bac-toBac System is important because it allows for the use of a bacteriologist who is unfamiliar with virology but is more familiar with bacteriology and the development of a bacteriophage. researchers unfamiliar with virology and more familiar with bacteriology and molecular biology to still work in their own familiar fields. However, one shortcoming of the system is that recombinant baculoviruses retain the bacterial replicon, which makes baculoviruses highly genetically unstable when passed on in insect cells compared to viruses that do not contain this structure [Pijlman et al. (2003) and see below]. More recently, the group of Possee and King have come up with an even smarter idea, which is a cross-hybridization of the linearized baculovirus DNA method and the baculoparticle method (P0sseeetal., 2008). Essentially, they constructed a novel bacillus with a recombinant baculovirus genome that had a bacterial replicon in the polycomb seat and was missing the downstream virulence gene (Figure 14.6). Due to the presence of the bacterial replicon and the missing gene, this bacillus can replicate in E. coli, but not in insect cells. Therefore, parental baculovirus genomic DNA for the construction of recombinant daughter viral vectors can be easily prepared using E. coli carrying this baculovirus. you can simply isolate the viral DNA from E. coli, mix it with the transfer plasmid, and then cotransfect the insect cells. Homologous recombination between the viral and transfer plasmid DNA would simultaneously recover #/2629 by knocking out the bacterial replicon at the polycomb protein locus and inserting the target gene at the same location. Although this method did not improve the efficiency of homologous recombination, it did significantly improve the efficiency of generating recombinant baculoviruses in co-transfected insect cells, since the parental viral DNA is defective and cannot self-replicate. This method has been commercialized by oxfordExpressionTechnologies as flashBAC. one feature of the flashBAC system that is worth re-emphasizing is that, as shown above, bacterial replicons are eliminated from homologous recombination. This has the advantage of eliminating the genetic stability problems associated with the preparation of baculovirus vectors using conventional baculoviral particles (which retain the bacterial replicon) (Pijlmanetal., 2003). Another feature worth re-emphasizing is that the bacilli used in the flashBAC system contain defective baculovirus genomes that do not replicate in insect cells. Obviously, this means that homologous recombination between the parental defective-type virus genome and the transfer plasmid is required to form unassisted zygotic baculoviruses in this system. However, this does not necessarily mean that co-transfected insect cells will only produce recombinant daughter baculoviruses. Due to geneticcomplementation, these cells are also capable of producing progeny viruses derived from defective parental virus genomes. In particular, recombinant viruses produced in co-transfected insect cells are able to provide products which provide an auxiliary function for the cis-assembly of defective viral DNA. At least one round of empty spot purification is performed when preparing baculoviruses using the flashBAC system, however the commercial literature from OxfordExpressionSystems states that this is not necessary. Without spot purification, the original recombinant viruses obtained by cotransfection of insect cells will contaminate the defective parental viruses, which will interfere with subsequent vector replication and exogenous gene expression. Probably the simplest way to prepare baculovirus vectors is to use a method that cross-hybridizes Gateway technology with linearized parental viral DNA technology (Hartley, 2003; WalhoutetaL, 2000). This approach involves the recombination of Gateway entry plasmids (entryplasmid) encoding target proteins and linearized parental viral DNA in vitro rather than in vivo. In this system, the parental viral DNA exists in a thread-like form, and the polycomb protein coding sequences in its genome are replaced by the E. coli LacZ gene and the herpessimplexvirus thymidinekinase gene, with the replacement sequences flanked by site-specific recombination sites of phage 1 (attRl and attR2; Figure M.7) (site~specificrecombinationsite). Viruses containing the LacZ gene showed blue empty spots, and the thymidine kinase gene provided a negative selection marker against the parental viral DNA when cultured in insect cells supplemented with a certain nucleoside analog (nucleosideanalog), such as propoxyguanosine (gancyclovir). Linearized viral DNA is mixed with DNA coding for the target protein into a plasmid flanked by site-specific recombination sequences (attLl and attL2) of the A phage, and pure recombinase ^Shen is added to the mixture.! ^! ^-nase) (LRClonase; Invitrogen), which mediates site-specific recombination between the att sites on the entry plasmid and the parental virus. It is important to note that this in vitro reaction is a non-quantitatively efficient recombination reaction, with the product being a mixture of parental baculovirus DNA and recombinant baculovirus DNA. Subsequently, this mixture was transfected into human insect cells and, as noted earlier, cultured in medium containing propoxyguanosine, which prevents replication of the parental virus. After one round of screening, the number of progeny recombinant baculovirus vectors was increased, and they could be detected from a relatively low level of progeny baculovirus background by the empty spot formation assay. In the presence of a galactosidase chromogenic substrate, the recombinants have a white empty spot phenotype and can be easily identified accordingly. This method of obtaining recombinant baculovirus vectors in vitro was originally developed by Franke and his colleagues at LifeTechnologies. It was then commercialized by Harwood and colleagues at Invitrogen as the BaculoDirect system. The system is very fast, efficient and simple to use. However, it should be emphasized that, similar to the flashBAC system, the BaculoDirect system is marketed as "cloning and expression of genes from baculoviruses without the need for vacuolar purification or screening in bacteria," which, as noted earlier, is not good advice, despite the fact that the presence of the thymidine kinase gene product and propoxyguanosine exerts a negative selective pressure on the parental virus. However, insect cells transfected with the in vitro recombinant product will still produce both recombinant zygotes and zygotes of the parental virus. Therefore, researchers using the BaculoDirect system should not believe the manufacturer's claim that recombinant baculoviruses with the target gene can be isolated from parental virus contamination by at least one round of empty-spot purification. In fact, researchers who take this advice and use agar layers containing galactosidase for spot purification usually see at least a few blue spots, which indicates the presence of the parental virus and confirms that spot purification of the recombinant baculovirus is a wise decision. In fairness, the operating manual for the commercially available BaculoDirect system (Invitrogen) includes a method for empty spot purification on medium containing /■ galactosidase, which provides a great convenience. With this knowledge of the in vitro recombination methods described above, a short description of some direct in vitro recombinant DNA methods that have been published in the public domain should also be given. These methods involve the digestion of a modified baculovirus genome and its subsequent direct ligation to exogenous DNA fragments. An example of these methods involves the introduction of two &m36I sites downstream of the polycomb protein promoter on the AcMNPV genome (LuandMiller, 1996). The recognition sequences of these two sites have minor differences. Digestion by the &w36I site produces a single-stranded protruding sequence of 5'-TTA^ (singlestranded5'-TTA-3'overhangingsequence), which can be used by E. coli in the presence of dTTP DNA polymerase IKlenow fragment into a 5~TT-3' single-stranded protrusion. This produces a linear baculovirus DNA molecule to which any DNA fragment can be ligated, provided that it is Ec0RI digested to produce a single-stranded protrusion of y-AATT-3', which is then processed by the Klenow fragment and dATP to a single-stranded protrusion of V-AAW. The main purpose of this refinement is to give convenience to the construction of baculovirus-based cDNA expression libraries. The other two examples of direct in vitro recombination both involved the introduction of homing endonuclease (HEN) cleavage sites in the baculovirus genome. One of these involved the introduction of an I-Sce-I site into the AcMNFV genome to construct a recombinant named Ac-Omega (Ernstetal., 1994). Genomic DNA isolated from this virus can be digested by I-Sce-I and ligated to DNA fragments treated with the same enzyme. Another one is named Homingbac system, which involves the introduction of a single I-Cew-I site into the genomes of many different baculoviruses (Lihoradovaetal., 2007). Genomic DNA isolated from these viruses linearized by I-Cew-I can be ligated to DNA fragments with matchable protruding ends after treatment with BsfXI. Currently, none of the three vectors for direct cloning described in this paragraph are commercialized, and the method has been poorly applied in literature reports. Therefore, it is difficult to make an assessment of the relative success of these direct cloning methods or whether their baculovirus backbones can be widely used for the preparation of baculovirus expression vectors. In addition to addressing early technical challenges regarding the construction of recombinant baculovirus vectors, some of the improvements made to the parental baculovirus genomes were aimed at enhancing the expression of exogenous proteins on recombinant vectors. The basic approach is to delete baculovirus genes that are (i) known to be unnecessary for viral replication in cultured insect cells and are tentatively defined as "accessory" genes; and (ii) thought to interfere in some way with the production of exogenous proteins or degradation of target proteins. This type of baculovirus DNA was originally developed by Bishop and coworkers as a thread-like viral DNA, and was later commercialized by Novagen as BacVector-2000.In addition to the polycomb protein, this viral DNA lacks five other undisclosed accessory genes. The effect of the deletion of these genes on exogenous protein yields has been unclear because there are no reports of comparative studies of exogenous protein expression using baculovirus DNA with or without the deletion of these genes. Next, two other nonessential genes, one encoding a chitinase (Hawtinetd., 1995) and the other encoding a cathepsin-like protease (Slacketal., 1995), were eliminated in BacVector-2000, and the resulting AcMNPV-based parental viral DNA was termed BacVector-3000 (Nova-gen). Other AcMNPV-based viruses missing the viral chitinase and histone-like protease genes include a commercially available baculum known as BestBac (Expression System) and an uncommercialized modified baculum known as Hachide Buckle-DCC (Kabaetal., 2004). flashBACtm system uses a baculum that similarly lacks functional chitin genes. functional chitin genes. The effect of the absence of chitinase and histone-like protease on the parental virus on the expression of exogenous genes in the progeny of recombinant baculovirus vectors is not entirely clear, but some information is available. One such study showed that an exogenous glycoprotein expressed by AcBacDCC lacking both enzymes was less degradable than that expressed by a baculovirus vector containing both enzymes (Kabaetal., 2004). This seems to be consistent with the expectation that deletion of viral protease genes usually promotes better expression of recombinant and secreted proteins. However, more work needs to be done to determine whether deletion of a particular protease gene broadly affects the production of exogenous proteins in the baculovirus-insect cell system. The potential effects of deleting viral chitinase genes have been relatively rarely observed. It has been shown that viral chitinase resides in the endoplasmic reticulum, where it interferes with secretory pathway protein production by saturating the host protein transport machinery (Kabaetal., 2004). If the above mechanism is true, then deletion of the viral chitinase gene and thus the absence of chitinase may increase the expression level of secreted proteins. However, there are no published studies demonstrating the effect of deleting the chitinase gene alone in AcMNPV-based vectors such as flashBAC. Studies have shown that AcBacDCC deficient in chitinase expresses reduced degradation of exogenous proteins, but the virus is also deficient in histone-like proteases, which complicates interpretation of the results. In addition, there has been a publication showing the effect of excision of either the chitinase or histone-like protease genes of recombinant filamentous worm baculovirus (recombinantsilkwormbaculovirus), either alone or in total, on exogenous protein production [Bombyxmorinucleopolyhedrovirus, ( BmNPV), Leeetal. 2006]. In this system, recombinant BmNPV vectors encoding insect cellulase (Cellulase) expressed in silkworms with deletion of the genes for For more product details, please visit Aladdin Sc
