Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

Escherichia coli β-Galactosidase Production by Baculovirus-insect Cell System Toshio Hara, Koichi Nonaka, Nobuyuki Etou, Seiya Ogata & Takeshi Kawarabata To cite this article: Toshio Hara, Koichi Nonaka, Nobuyuki Etou, Seiya Ogata & Takeshi Kawarabata (1992) Escherichia coli β-Galactosidase Production by Baculovirus-insect Cell System, Bioscience, Biotechnology, and Biochemistry, 56:7, 1124-1125, DOI: 10.1271/ bbb.56.1124 To link to this article: http://dx.doi.org/10.1271/bbb.56.1124

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Biosci. Biotech. Biochem., 56 (7), 1124-1125, 1992

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Escherichia coli fl-Galactosidase Production by Baculovirus-insect Cell System Toshio HARA, Koichi NONAKA, Nobuyuki ETOU, Seiya OGATA, and Takeshi KAWARABATA*

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Microbial Genetics Division, Institute of Genetic Resources, and Kyushu University, Hakozaki, Fukuoka 812, Japan Received October 25, 1991

Baculoviruses, causative agents of fatal diseases in insects, are not only of interest because of their application as control agents of insect pests in agriculture, I) but have increased importance since they are used as expression vectors for the production of heterologous proteins of proand eukaryotic origin. 2 ) Therefore, baculovirus-insect cell systems have shown great promise recently for the production of biologically active proteins of medical, pharmaceutical, and veterinary importance. 3 ) The cells used to support Autographa californica nuclear polyhedrosis virus (AcNPV) replication are either from the cell line IPLB-Sf21-AEII 4 ) derived from cultured ovaries of Spodoptera jurgiperda or a clonal isolate (Sf9)5) derived from this line. Nowadays, the Sf9 cells are mainly used as a host. Development of the AcNPV transfer vectors has been described 6 • 7 ) and reviewed.1.8) Most of the recent efforts to maximize gene expression in this system have focused on optimizing the placement of the foreign gene with respect to transcriptional and translational signals of the polyhedrin gene. 9 ) In general, culture medium composition has considerable influence on insect cell growth and product formation, but there are only a few publications except those of Mitsuhashi I 0) and Broussard and Summers. I I) TNM-FH and Grace's media are routinely used in many laboratories for the propagation and manipulation of S. jrugiperda cells. However, TNM-FH medium was developed to establish primary cultures of Trichoplusiani cell. 12) In this study, we examined the expression level of the E. coli J1-galactosidase gene in various Spodoptera cells to achieve a suitable and efficient insect cell expression system. Four in.sect cell lines were assessed: a fall armyworm, S. jrugiperda (Sf) designated IPLB-Sf21 AEII 4 ) and a clonal isolate (Sf9),5) S. littoralis designated CLS79, and S. litura designated S1224. Cells were cultured in IPL-41 medium 13) with 10% fetal bovine serum (FBS) (Gibco Labs., New York, U.S.A.). Cultures grew as monolayers in stationary 25-cm 2 TC flasks (Falcon R, Becton Dickinson Labware, New Jersey, U.S.A.) maintained at 27°C. Cells were passaged by flushing the monolayer off the surface of the TC flask with a Pasteur pipette. Wild-type A. californica nuclear polyhedrosis virus (AcNPV) and recombinant virus encoding a fusion J1-galactosidase under the control of the polyhedrin promoter (Ac360-501J1-galt) were used in this study. The fusion protein contains II amino acids of the polyhedrin protein and lacks 7 amino acids of the native E. coli J1-galactosidase. Infection began when medium was aspirated off the monolayer culture, and virus stock solution was added. The virus added at a multiplicity of infection (MOl) of 20 per cell was allowed to absorb for 1hr at room temperature. The inoculum was removed and replaced with appropriate medium, and incubation was done at 27°C for the indicated periods. The cellular lysates prepared by sonication at 3 days postinfection (pj.) were put on the gels. The total protein content of infected cells was analyzed by SDS-polyacrylamide gel electrophoresis (Fig. I). Coomassie blue staining of total infected-cell

Fig. 2.

* Institute

of Biological Control, Faculty of Agriculture,

proteins showed that Sf9 and Sf21 cells expressed a considerable amount of J1-galactosidase, but the expression level of CLS79 and SI224 cells was quite low. The J1-galactosidase expression level of Sf21 cells appears to be the highest among Spodoptera cells, and the amount of 120-kDa proteins corresponding to J1-galactosidase expressed by Sf21 cells was densitometrically estimated to be 4.1 mg per 5.0 x 10 5 cells in a milliliter of culture broth, representing more than 36% of total cellular protein (see lane B in Fig. I). When seen under the light microscope, the metamorphosed Sf21 cells were typically observed in recombinant Ac360501 J1-gal-infected cells (Fig. 2B). These cells appeared at 2 days p.i.

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29

Fig. 1. SDSPolyacrylamide Gel Electrophoresis Profiles of Proteins from Various Spodoptera Cells Infected with the Recombinant Baculovirus, Ac360-50 1J1-gal, Expressing E. coli J1-Galactosidase. Spodoptera cells were cultured in IPL-41 medium, infected with Ac360-50 1fJ-gal at a MOl of 20, and incubated at 2TC. Cellular Iysates of 2.3 x 104 cells of Sf9 (lane A, 3 days p.i.), Sfll (lane B, 4 days p.i.), CLS79 (lane C, 3 days p.i.), and SI224 (lane D, 4 days p.i.) were put in each well. Molecular weight standards (lane M) and the authentic E. coli fJ-galactosidase (lane G) were put on as markers. The molecular masses and positions of the standard proteins are indicated to the right (myosin, 200 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 29 kDa).

Appearance of Mock-, Recombinant Ac360-501J1-gal-, and AcNPV-infected Sf21 Cells at 4 Days p.i.

Cells were photographed with an Nikon Optiphot at a final magnification of 600x. Panels include cells which are (A) mock-infected, or which are infected with (B) Ac360-501{1-gal, or (C) AcNPV. Scale bar, 10/lm.

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Fig. 3. Course of [3-Galactosidase Activity Production by Infected Sf21 Cells with Recombinant Baculovirus, Ac360-501[3-gal. The intracellular (closed boxes) and extracellular (open boxes) f3-galactosidase activities per milliliter of broth were measured at the indicated times, p.i. The intracellular enzyme fraction in culture fluid was prepared by harvesting, suspending into water, and disrupting cells with Tomy UD-201 (Tomy Co., Ltd.). The extracellular enzyme activity was measured after removing cells by centrifugation at 1500 rpm for 5 min. The conversion to O-nitrophenyl was measured the absorption at 420 nm so that a faint yellow color developed for I min after the ONPG addition. The reaction was stopped with 500 ml of 1M Na 2 C0 3 • f3-galactosidase activity was defined as follows: Units of f3-gal= 1000 x OD 42o jtime (min)jsample volume (ml)

compared the expression level of E. coli [3-galactosidase usingbaculovirus vector at a MOl of 20 in 23 insect cell lines, and they concluded that the [3-galactosidase activity by S. jrugiperda Sf9 cells (8.5x 10 4 units per 10 6 cells) was the highest among the others, including Sf21 cells. Ogonah et al. 16 ) reported that E. coli [3-galactosidase activity by S. jrugiperda Sf9 cells infected with a different baculovirus (pAcNPV246) at a MOl of 5 was 1.7 x 10 5 units per 106 cells. Therefore, the E. coli [3-galactosidase expression level by Sf21 cells was 4.9 times higher than that of the Sf9 cells reported by Hink et al. 15 ) and was found to be 2.5 times higher than that reported by Ogonah. 16 ) Baculovirus-insect cell systems, as high-level protein expression system, have been developed but only a few published results of recombinant proteins approach the 1 g/liter potential associated with the polyhedrin promoter,S) which is expressed at levels approaching 1.2g/1iters. 2 ) The [3-galactosidase fusion protein was highly expressed by Sf21 cells infected with Ac360-501[3-gal-(lane B in Fig. 1). As shown in Fig. 2B, the Ac360501[3-gal-infected Sf21 cells were metamorphosed compared with the AcNPV-infected cells. Now, it progress to achieve the mass production of E. coli [3-galactosidase in this baculovirus (Ac360-501 [3-gal)-insect cell (Sf21) system.

References 1) 2) 3) 4) 5)

and were a majority in the infected cells at 4 days pj. The diameter of recombinant Ac360-501[3-gal-infected cells presumed to contain the largely fusion is 90-100% larger than that of wild-type AcNPV-infected cells, while that of AcNPV-infected cells is 80-100% larger than that of mock-infected cells. One other case has been reported in which the diameter of Ac101-CAT-infected Sf9 cells becomes 50-100% larger than that of uninfected Sf9 cells. 9 ) The size of both S. jrugiperda cells in diameter differed overall: Sf9 cells, 15.7 ± 2.5 ,urn; Sf21 cells, 21.1 ± 2.3 ,urn. No one has reported such a tremendous Ac360-501[3-gal-infected insect cell before. Figure 3 shows a characteristic time trajectory of intracellular and extracellular enzyme activities produced by infected S. jrugiperda cells per 5.0 x 10 5 cells per milliliter of culture broth. The activity of [3-galactosidase were measured by the O-nitrophenyl-[3-D-galactopyranoside (ONPG) assay.14) The net synthesis of [3-galactosidase in Sf21 cells was already attained at 2 days postinfection, and the intracellular [3-galactosidase activity peaks at 4 days p.i. The total [3-galactosidase activity reached 2.1 x 10 5 units per 5.0 x 10 5 cells. As the time postinfection increases, [3-galactosidease is released into the medium, and much of the synthesized enzyme (above 85%) was detected in the medium at 6 days pj. This phenomenon appears to be due to the cell membranes losing integrity, becoming leaky, and ultimately lysing. Hink et al.!5)

1125

6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16)

M. E. Martignoni, "Chemical and Biological Controls in Forestry," American Chemical Society, Washington, D. C., 1984, pp. 55-67. V. A. Luckow and M. D. Summers, Bio/Technology, 6, 47-55 (1988). 1. Wu, G. King, A. J. Daugulis, P. Faulkner, D. H. Bone, and M. F. A. Goosen, Appl. Microbiol. Biotechnol., 32, 249-255 (1989). J. L. Vaughn, R. H. Goodwin, G. L. Tompkins, and P. McCawley, In Vitro, 13, 213-217 (1977). M. D. Summers and G. E. Smith, "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures: Bulletin No. 1555, "Texas Agricultural Experimental Station, 1987, pp. 10-18. D. W. Miller, P. Safer, and L. K. Miller, Genetic Engr.: Principles and Methods, 8, 277-298 (1986). Y. Matsuura, R. D. Possee, H. A. Overton, and D. H. L. Bishop, J. Gen. Viol., 67, 1515-1529 (1987). L. K. Miller, Ann. Rev. Microbiol., 42, 177-189 (1988). V. A. Luckow and M. D. Summers, Virology, 167, 56-71 (1988). J. Mitsuhashi, Advances in Cell Culture, 3, 133-196 (1982). D. R. Broussard and M. D. Summers, J. Invert. Pathol., 54, 144150 (1989). W. F. Hinh, Nature, 226, 466-467 (1970). E. M. Dougherty, R. M. Weiner, and C. F. Reicherderfer, Appl. Environ. Microbiol., 41, 1166-1172 (1981). L. K. Miller, in "Experiments in Molecular Genetics, "Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1972. W. F. Hink, D. R. Thomsen, D. J. Davidson, A. L. Meyer, and F. J. Castellino, Biotechnol. Prog., 7, 9-14 (1991). O. Ogonah, M. L. Shuler, and R. R. Granados, Biotech. Lett., 13, 265-270 (1991).

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