[9]

MAMMALIAN P450 EXPRESSION WITH BACULOVIRUS

93

[9] E x p r e s s i o n of M a m m a l i a n C y t o c h r o m e P 4 S 0 Using Baculovirus By FRANK J. GONZALEZ, SHIOKO KIMURA, SHINJI TAMURA, and HARRY V. GELBOIN Introduction and Principles

A number of systems have been used to express mammalian P450s including yeast, 1 COS cells, 2 retrovirus, 3 Epstein-Barr virus vectors, 4 SV40-based stable expression: and vaccinia virus: Each system has advantages and disadvantages. However, no system described to date can easily and reproducibly make large enough quantities required for physical measurements of P450. Yeast have been used successfully to produce up to 0.5 nmol P450/mg microsomal protein7 but these high levels of expression have not been universally achieved by other laboratories using cDNAs expressing other forms of P450. Although bacterial expression systems have been extensively used to study P450 from Pseudomonas putida, s to date successful expression of mammalian P450s in bacteria has not been described. This may be due to the fact that eukaryotic P450s require an intracellular membrane environment that bacteria lack. The phospholipid bilayer may serve as a template for efficient and accurate folding of the enzyme and uptake of its critical cofactor heme. The eukaryotic system yielding the highest level of expression was designed using baculovirus. 9 The baculovirus system has been described in detail.l° Briefly, baculovirus are a group of over 500 species of virus that infect only insect cells. The virus used for c D N A expression is a nuclear polyhedrosis virus (NPv) that, in its secreted form, is contained 1 K. Oeda, T. Sakaki, and H. Ohkawa, DNA 4, 203 (1985). 2 M. X. Zuber, E. R. Simpson, and M. R. Waterman, Science 234, 1258 (1986). 3 N. Battula, J. Biol. Chem. 264, 2991 (1989). 4 C. L. Crespi, R. Langenback, K. Rudo, Y.-T. Chen, and R. L. Davies, Carcinogenesis 10, 295 (1989). 5 j. Dohmer, S. Dogra, T. Friedberg, S. Monier, M. Adesnik, H. Glatt, and F. Oesch, Proc. Natl. Acad. Sci. U.S.A. 85, 5769 (1988). 6 N. Battula, J. Sagara, and H. V. Gelboin, Proc. Natl. Acad. Sci. U.S.A. 84, 4073 (1987). 7 T. Sakaki, K. Oeda, M. Miyoshi, and H. Ohkawa, J. Biochem. (Tokyo) 98, 167 (1985). s p. B. Unger, I. C. Gunsalus, and S. G. Sligar, J. Biol. Chem. 261, 1158 (1986). 9 G. E. Smith, M. J. Fraser, and M. D. Summers, J. Virol. 46, 584 (1983). l0 M. D. Summers and G. E. Smith, " A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Bulletin No. 1555. Texas Agriculture Experiment Station.

METHODS IN ENZYMOLOGY,VOL, 206

94

HETEROLOGOUSEXPRESSION

[9]

within a capsule of protein called a polyhedrin. This structure, which is made up of a 29-kDa polyhedrin protein, is a means to protect the virus during its natural life cycle in insects but is dispensable when the virus is propagated in cell culture. Therefore, the gene encoding the polyhedrin protein can be used as a site for insertion of the cDNA. Because the baculovirus used for cDNA expression, AcMNPV, has a genome of 130,000 base pairs (bp), the cDNA cannot be directly inserted by standard enzymatic procedures. The cDNA is first cloned into an insertion vector behind the polyhedrin gene promoter. This vector also contains portions of the structural gene for the polyhedrin protein. These sequences allow the plasmid to integrate into the polyhedrin gene of the large viral genome. This integration event, which occurs at a frequency of 0.1 to 5%, inactivates the viral polyhedrin gene, thus allowing a visual screening of cells to determine whether the virus is wild type or recombinant. Cells infected with wild-type virus will contain polyhedrin particles and are classified as occlusion positive, whereas cells lacking expression of the polyhedrin gene, owing to insertional inactivation, will be occlusion negative. Practically, however, occlusion-negative cells are very difficult to detect by eye. The recombinant virus-containing cells are usually identified by plaque hybridization, a time-consuming procedure. Several rounds of plaque purification are required before a homogeneous preparation of virus is obtained using plaque hybridization. The recent development of a vector having the bacterial fl-galactosidase gene will allow screening for recombinant viruses based on metabolism of 5-bromo-4-chloro-3-indolylfl-o-galactopyranoside (X-Gal) and formation of blue pigment.ll The pure virus can then be used to infect cells to analyze for recombinant protein production. The reported levels of expression of foreign proteins using baculovirus range from 1 to 500 mg/liter of infected cultured cells) ° Since insect cells process and modify proteins in a manner similar to cells in higher eukaryotes proteins can be made that are fully functional and either glycosylated and secreted or inserted into their appropriate intracellular compartment. 12 A cDNA can be expressed by inserting it in-frame with the polyhedrin gene ATG codon using insertion vectors pAc700, pAc701, and pAc702. The vectors pVL1392 and pVLl393 can be used to express cDNAs contalning complete reading frames. These plasmids have small polylinker regions for convenient cDNA cloning. Finally, as noted above, the newest n j. Vialard, M. Lalumiere, T. Vernet, D. Briedis, G. Alkhatib, D. Henning, D. Levin, and C. Richardson, J. Virol. 64, 37 (1990). 12 D. L. Jarvis, C. Oker-Blom, and M. D. Summers, J. Cell. Biochem. 42, 181 (1990).

[9]

MAMMALIAN P450 EXPRESSION WITH BACULOVIRUS

95

vector pJV Nhe111or pBlueBac ~3can be used to express complete cDNAs, and it also has the/3-galactosidase gene for color selection of recombinant viral plaques.

Procedure

A detailed protocol for construction of recombinant baculovirus has been developed by Summers and SmithJ ° These procedures have been used by our laboratory with excellent success. However, we found that direct visual identification of recombinant viral plaques was difficult, although possible using the appropriate conditions and with practice. The recombinant virus could easily be identified using plaque hybridization, but this procedure is rather time-consuming and it was difficult to pick up small areas of cells containing only recombinant virus. Therefore, multiple plaque purifications were required. The use of the novel/3-galactosidase gene-containing insertion vector H should circumvent this problem. However, we have not yet used this system. Under ideal circumstances, when the recombinant plaques can be visually identified, three plaque purifications should suffice. A complete kit (MAXBAC) is now available from Invitrogen Corporation (San Diego, CA). This kit contains all reagents, vectors, virus, and a manual. The manual contains excellent methods for determining viral titer and for plaque purification. Materials. Spodoptera frugiperda (SF9) cells were obtained from the American Type Culture Collection (Rockville, MD; ATCC No. CRL 1711). Grace's insect cell culture medium (Formula No. 86-0050AJ) was purchased for GIBCO Laboratories (Grand Island, NY). pAc373 and baculovirus strain (AcMNPV) were supplied by Dr. Max Summers, Texas A&M University. The AcMNPV, SF9 cells, Grace's medium, and plasmids pVL1392, pVL1393, pAc700, pAc701, pAc702 and pBlueBac can be purchased from Invitrogen Corporation. Prior to beginning construction of a recombinant baculovirus, the manual of methods for baculovirus vectors and insect cell culture procedures ~° should be obtained and read carefully. The following procedures emphasize those points we have found to be especially critical. Growth of SF9 Cells. The SF9 cells are obtained as a frozen cell suspension from the ATCC. The vial should be rapidly thawed at 37° and the cells placed in a sterile tube containing 10 ml of cold fetal calf serum.

13 "Baculovirus Expression System Manual," Version 1.3. Invitrogen Corporation, San Diego, California.

96

HETEROLOGOUS EXPRESSION

[9]

After mixing, the cells are centrifuged down at 500 rpm for 10 min at 4 °. The supernatant is removed and the cells resuspended in 5 ml of cold medium and deposited into a 25-cm 2 T flask. Incubate at 27° for 3 hr until the temperature of the medium equilibrates and cells are attached. Remove the floating cells and media and add 5 ml of fresh medium that had been equilibrated at 27° . We have noted that SF9 cells initially divide very slowly after being thawed. Cells should be kept without replating until full confluency is obtained and some of the cells start floating. These floating cells can be seeded into a new T flask. The cells begin to grow more rapidly with the expected doubling time of 18 to 24 hr after 2 to 3 weeks in culture. However, in our hand, cells maintained on plates have a viability of about 70%. Cells grown in suspension in spinner flasks have viabilities of greater than 95%. Therefore, we begin small spinner flask cultures when enough cells (5 x 105/ml) are obtained from propagation of the original freezer stock in a T flask. The cells reach a density of 3 × 106/ml, and then 80% of the culture medium can be removed and replaced with fresh Grace's medium. Never dilute the confluent cultures more than 5-fold. Diluting the cells more than this results in slow growth. Cells grown in spinner bottles can be transferred to petri dishes the night before transfection or infection experiments and used for construction of recombinant viruses, titering, and expression of recombinant proteins. The cells should not be subcultured for more than 3 months because old cells are sensitive to manipulation required for P450 expression and, in general, give lower yields than cells that have not been propagated for long periods. Therefore, we start new cultures of cells at least once a month. Titering o f Virus. 13 Seed cells at 5 x 106 per 100-ram petri dish. This yields about 50% confluence. Allow the cells to attach to the plate for about 1 hr. Remove the medium and add 3 ml of serial dilutions of virus (prepared in Grace's medium). Rock the plates for 1 hr, then aspirate all of the medium. Add 10 ml of agarose overlay to each plate. Agarose overlay is prepared as follows: Autoclave 2.5% (w/v) low melting agarose in distilled water and cool to 45 °. To this is added an equal volume of medium that has also been equilibrated at 45 °. Dispense 5 ml of 45 ° equilibrated agarose solution into a tube containing 5 ml of medium that had been kept at room temperature. Quickly mix the solution and immediately pipette I0 ml of the agarose solution onto the plates. Make sure the agarose overlay is gently added to the side of the dish and distributed evenly across the surface. (This procedure should first be practiced with empty plates.) Allow the plates to sit in the tissue culture hood for 30 min to 1 hr before transferring to the incubator and incubating for 5 to 6 days. To screen for plaques the plates are placed on a dissecting microscope

[9l

MAMMALIAN P450 EXPRESSION WITH BACULOVIRUS

97

and illuminated from the side with a high intensity lamp, such as a slide projector. The occlusion-positive plaques should be readily identifiable and can be counted for titering purposes. The occlusion-negative plaques, seen when constructing recombinant viruses, have a markedly different appearance. It is useful to practice plaque identifications using stocks of wild-type and recombinant v i r u s e s . 11 Growth of Virus. Seed cells at 50% confluence as described above and allow 1 hr for them to attach to the plate. Remove the medium and add the virus stock to a volume sufficient to cover cells. Incubate for I hr at 27 °, aspirate the inoculum, and add medium. Continue to incubate for 4-6 days before removing cells and medium. Sediment the cells by low-speed centrifugation and store the supernatant at 4 °. The supernatant should have a titer of about 2-3 x 10s plaque-forming units (pfu)/ml and can be used for further infection and to generate more virus. The virus should not be passaged more than 5 times. Otherwise, the virus stock should be started from a single, well-defined occlusion-positive plaque. Purification of Viral DNA.10 The expected yield of viral DNA is 1/zg/ ml of medium from infected cells. Sediment the virus particles from about 100 ml of culture medium by centrifugation at 25,000 rpm in an SW28 rotor at 4 °. Resuspend the virus pellet in 20 ml of 10 mM Tris-HCl, pH 8.0, 1 mM EDTA by rocking overnight at 4 °. Resediment the virus particles at 28,000 rpm in SW40 rotor or equivalent at 4 °. Resuspend the pellets in 0.5 ml of 50 mM Tris-HCl, pH 8.0, 0.1 M EDTA, 0.5% sodium dodecyl sulfate (SDS), and 100/zg/ml proteinase K. Incubate for 2 hr to overnight at 55 °. Gently extract the solution twice with phenol saturated with 0.1 M TrisHCI, pH 8.0, and once with chloroform. Extractions should be carded out by gently rocking at a speed sufficient to emulsify the phases at room temperature for 10 min followed by centrifugation in a microcentrifuge for 10 min at room temperature. The aqueous phase should be removed with a wide bore pipette to avoid sheafing. The DNA is precipitated with ethanol, washed with 80% ethanol, dried, and resuspended in 10 mM TrisHC1, pH 8.0, 1 mM EDTA. The high molecular weight viral DNA will take I to 2 days to resuspend. This can be facilitated by incubation at 65 ° for several hours. Transfection of SF9 Cells. io SF9 cells are seeded to about 50% confluency on a 60-mm petri dish 1 day before transfection. Add O. 1/xg of viral DNA and 20/zg of plasmid DNA (containing the inserted cDNA) to 0.5 ml of 0.5 M CaC12 in a 5-ml polypropylene culture tube. Insert a sterile 1ml pipette, connected to a tank of nitrogen gas, into the DNA-CaCI 2 solution. Slowly introduce bubbles of N2 gas and then add a solution containing 0.5 ml of 50 mM HEPES, pH 7.1,280 mM NaCI, and 1.5 mM NaH2PO4. This should be slowly added so as to avoid formation of large

98

HETEROLOGOUS EXPRESSION

[9]

precipitates. Allow the precipitates to form for 15 min. Aspirate the medium from the cells and add 2 ml of fresh Grace's medium. Add the DNA-calcium phosphate precipitates slowly to the medium of the cells with gentle swirling. Incubate at 27° for 4 hr, remove the medium, wash the cells once with 5 ml of Grace's medium, and then add 5 ml of Grace's medium. Incubate for 4 to 6 days until plaques develop. Withdraw both the medium and cells and remove the cells and debris by sedimentation. The supernatant contains a mixture of wild-type and recombinant virus. The latter represents from 0.1 to 5% of the total virus. Screening and Isolation o f Recombinant Viruses. Visual screening for identification of recombinant virus as occlusion-negative plaques has been discussed earlier. Several methods for discrimination between wild-type and recombinant plaques have been developed.I°.13 Recombinant virus can also be identified by using the insertion vector pJV NheI (pBlueBac) containing an independently expressed/3-galactosidase gene by including 0.15 mg/ml X-Gal. We have relied on plaque hybridization methods to identify and purify recombinant virus.14 We have found, however, that seeding cells at 50% confluence is critical, and sometimes up to 5 rounds of plaque purification is required before the recombinant virus are homogeneous. Plaque hybridization is described in detail by Summers and Smith. 1° It is essential that the cDNA insert used as a probe be free from vector DNA. This can usually be accomplished by two gel purifications of the cDNA insert. Once you have trained your eyes to distinguish plaques of recombinant from wild-type virus, visual screening should be the shortest way to obtain the recombinant virus. The final plaque picks should be amplified as described above and carefully screened for the presence of wild-type virus. The recombinant virus can also be screened using antibody probes to detect recombinant antigen on filters. It is also possible to screen for recombinant plaques using dilutions into microtiter plates.13'15 In this case, a recombinant plaque is first identified by visualization or by plaque hybridization. A plug of agarose containing the positive plaque is picked up with the back end of a Pasteur pipette and dissolved in 0.5 ml of medium. This is then distributed into 24-well plates containing about I x 105 cells/well. After 4 to 6 days, the cells can be shaken from the bottom of the wells and a portion of each well can be analyzed for the presence of either cDNA sequences or recombinant protein using cDNA probes or antibodies, respectively. Positive wells can 14A. Asseffa, S. Smith, J. Gillette, H. V. Gelboin, and F. J. Gonzalez, Arch. Biochem. Biophys. 274, 481 (1989). 15 j. Pen, G. W. Welling, and S. Welling-Wester, Nucleic Acids Res. 17, 451 (1989).

[9]

MAMMALIAN P450 EXPRESSION WITH BACULOVIRUS

99

then be divided into 96-well plates and the process repeated until single pure recombinants are obtained. Expression o f Mammalian P450. Cytochrome P450 can be expressed at a high level using baculovirus except for the noted deficiency in heme incorporation. 14 This is probably due to the inability of the SF9 cells to synthesize enough heme, de nooo, to accommodate the large amount of P450 apoprotein. This problem can be corrected by addition of hemin to the culture medium. However, we have found that hemin can be toxic to SF9 cells that have been in culture for several months. This can be circumvented by using fresh cells (less than 2 months of passage) and by incubation of the cells with Grace's medium containing 20% (v/v) fetal calf serum instead of 10% (v/v). The hemin is added to the medium at a concentration of 4/zg/ml from a stock of 2 mg/ml dissolved in 0.4 M NaOH and 100% ethanol (1 : 1 solution). To express P450, the virus is used at a multiplicity of infection of 2 pfu/ cell. The media is changed 24 to 48 hr after infection to fresh medium containing 20% fetal calf serum and heroin. The cells are harvested 72 hr after infection, washed twice with phosphate-buffered saline (PBS), and homogenized in 0.1 M potassium phosphate, pH 7.2. For most enzyme assays and spectral analysis, total cell lysate is used. We have achieved levels of expression of up to 0.7 nmol/mg total cell lysate protein of P450 holoenzyme. For spectral measurements, total cell lysate is made in 0.1 M sodium phosphate, pH 7.2, 20% (v/v) glycerol, and 0.2% (w/v) Emulgen 913 (Kao Chemical Co., Tokyo). Insoluble material is removed by centrifugation at 10,000 g for 10 min at 4°. The protein is then divided into sample and reference cuvettes. Carbon monoxide gas is gently bubbled into the sample cuvette for 15 sec, and a few grains of sodium dithionite is dissolved in both sample and reference cuvettes. Difference spectra are then recorded. P450 contents are determined as described. 16

t6 T. Omura and R. Sata, J. Biol. Chem. 2,39, 2379 (1964).

Expression of mammalian cytochrome P450 using baculovirus.

[9] MAMMALIAN P450 EXPRESSION WITH BACULOVIRUS 93 [9] E x p r e s s i o n of M a m m a l i a n C y t o c h r o m e P 4 S 0 Using Baculovirus By FRA...
418KB Sizes 0 Downloads 0 Views