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BACULOVIRUS SUMMARY Page 1 of 3 PERSPECTIVES ON BACULOVIRUS EXPRESSION SYSTEMS Dr. Bart Frank, Arthritis and Immunology Program Overview In a previous session, advantages of expressing heterologous genes in E. coli were discussed. While high levels of expression in bacterial systems are common, problems of proper folding and lack of posttranslational processing may produce functionally inactive molecules. The baculovirus expression system can be used to circumvent these problems. Baculoviruses infect primarily insects with a narrow host range. Autographa californica, the most commonly used baculovirus for protein expression, infects only 2 lepidopteran (moth) families in nature. Although these viruses may enter other cells types (perhaps by phagocytosis), they are not infectious in them. For example, nucleocapsid proteins are not removed in most human cells. In human hepatic cell lines that do remove these proteins, the virus fails to replicate and express proteins due to the absence of insect transcription factors. Thus, working with baculoviruses is considered safe for humans and contamination of mammalian cell lines in shared biosafety hoods is not a problem. Recombinant DNA guidelines recommend a BL1 biosafety level for most baculovirus expression experiments. Advantages and disadvantages of protein expression in baculovirus There are many advantages to using baculovirus for heterologous gene expression. Heterologous cDNA is expressed well. Proper transcriptional processing of genes with introns occurs but are expressed less efficiently. As with other eukaryotic expression systems, baculovirus expression of heterologous genes permits folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. The insect cytoplasmic environment allows proper folding and S-S bond formation, unlike the reducing environment of the E. coli cytoplasm. Post-translational processing identical to that of mammalian cells has been reported for many proteins. These include proper proteolysis, N- and O-glycosylation, acylation, amidation, carboxymethylation, phosphorylation, and prenylation. Proteins may be secreted from cells or targeted to different subcellular locations. Single polypeptide, dimeric and trimeric proteins have been expressed in baculoviruses. Finally, expression of heterologous proteins is under the control of the strong polyhedrin promoter, allowing levels of expression of up to 30% of the total cell protein. Despite these potential advantages, particular patterns of post-translational processing and expression must be empirically determined for each construct. Differences in proteins expressed by mammalian and baculovirus infected insect cells have been described and overcome in some cases. For example, inefficient secretion from insect cells may be circumvented by the addition of insect secretion signals (ex. honeybee melittin sequence). Improperly folded proteins and proteins that occur as intracellular aggregates may be due to expression late in the infection cycle. In such cases, harvesting cells at earlier times after infection may help. Low levels of expression can often be increased with optimization of time of expression and multiplicity of infection. The complete analysis of carbohydrate structures has been reported for a limited number of glycoproteins. Potential N-linked glycosylation sites are often either fully glycosylated or not glycosylated at all, as opposed to expression of various glycoforms that may occur in mammalian cells. Species-specific or tissue-specific modifications are unlikely to occur. Insect Cell Culture SF-9, SF-21, and High-Five insect cells are commonly used for baculovirus expression. SF-9 and SF-21 are ovarian cell lines from Spodoptera frugiperda. They are grown in Grace's (or a similar) media supplemented with 10% fetal calf serum, lactalbumin, and yeastolate. High-Five cells are egg cells from Trichoplusia ni. These cells are less expensive to maintain since they may be grown without fetal calf http://omrf.ouhsc.edu/~frank/baculsum.html 3/15/2004 BACULOVIRUS SUMMARY Page 2 of 3 serum. They reportedly express higher levels of recombinant proteins, although we have found these differences to be minimal. All three cells lines may be grown at room temperature (optimum = 25 - 27° C), and do not require CO2 incubators. Their doubling time is 18-24 hours. Generalized Outline for Protein Expression in Baculovirus Given the variations of proteins to be expressed, no single protocol will satisfy every need. An outline for baculovirus expression is given below. One should plan 1-2 months to accomplish these goals: 1. Ligate the gene of interest to a bacterial transfer vector. The inserts are typically flanked by portions of viral genes to permit homologous recombination with replication defective, linear, viral DNA. Vectors or inserts may include sequences for protein targeting and purification. Verify direction of the inserts relative to the polyhedrin promoter and purify plasmids for transfections into insect cells. 2. Co-transfect insect cells with the recombinant transfer vector and a linearized viral vector. Electroporation, lipid, and calcium phosphate-mediated transformations work well. Replicative viruses are formed after intracellular homologous recombination between the ends of the viral molecules and portions of the transfer vector that flank the gene of interest. These recombination events insert your gene into the virus and complement defective viral gene(s) to permit viral replication. In some vectors, this may also generate production of marker proteins such as betagalactosidase. Non-recombinant viruses are kept to a minimum with this system. 3. Harvest the transfected cell supernatants. 4. Infect insect cells with dilutions of this supernatant to isolate single virus plaques. Identification of viral plaques can be difficult without the presence of a marker gene product such as betagalactosidase. 5. Infect additional insect cells with viruses from these plaques to amplify the quantity and titer of viral stocks. Protein expression in these cells may be examined in Western blots. 6. Optimize the level of protein expression (MOI, time course of infection) and test for activity in an appropriate bioassay. Summary In summary, heterologous gene expression in baculovirus systems offers several advantages over E. coli. The viruses are considered safe for laboratory personnel. Insect cells infected with the virus recognize and properly process a variety of recognition signals in mammalian, plant and yeast genes, and provide moderate to high yields of biologically active proteins for study. There are numerous books and articles on baculovirus expression. A thorough reference and useful technical manual on this subject is Baculovirus Expression Vectors by D. R. O'Reilly, L. K. Miller, and V. A. Luckow (1992, W. H. Freeman & Co, New York). Invitrogen has established an internet baculovirus discussion group that may be of interest or helpful in troubleshooting. Many companies now sell baculovirus expression vectors or complete expression systems. We have had personal experience and success with products from Invitrogen (http://www.invitrogen.com) and Clontech (http://www.clontech.com/). Detailed procedures from experiments with baculovirus expression in my laboratory can be found in the protocols section of my web page (http://omrf.ouhsc.edu/~frank). http://omrf.ouhsc.edu/~frank/baculsum.html 3/15/2004 BACULOVIRUS SUMMARY Page 3 of 3 Prepared for the November 1998 OMRF Research Technology Forum. Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF Return to Baculovirus Protocol Page http://omrf.ouhsc.edu/~frank/baculsum.html 3/15/2004