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Properties of Two Insect Cell Lines Useful for the Baculovirus Expression System in SerumFree Culture
Takeyuki Sugiura* and Egon Amannt Laboratory for Bone Research, Drug Discovery Research Laboratories, Pharma Research and Development Division, Hoechst Japan Limited 3-2, I-Chome, Minamidai Kawagoe-city, Saitama 350- I 1, Japan Received December I, 1995/Accepted March 5, 1996

The properties of Sf9 and Tn5 insect cells were analyzed comparatively under serum-free culture conditions. Sf9 cells in SF90011 medium apparently utilized sucrose as a primary nutrient both before and after virus infection, yielding small amounts of lactate and ammonia. Tn5 cells in Excell 401 medium consumed all the nutrients examined, including sucrose. The productivity of a recombinant glycoprotein, OSF-2, by Tn5 cells, was moderate in both monolayer and spinner cultures, but the ability to secrete it was compromised in the former case. Relative to the Tn5 cultures, Sf9 produced 30-fold more OSF-2 in either culture mode. 0 1996 John Wiley & Sons, Inc. Key words: cell metabolism baculovirus insect cells recombinant protein OSF-2

INTRODUCTION

The baculovirus expression vector system (BEVS) is now widely accepted as an advantageous system to produce large amounts of recombinant proteins due to its high productivity achieved by a strong polyhedrin promoter and the realization of most post-translational processes occurring in mammalian cells.* Thus far, Sf9, a clonal cell line derived from the ovary of Spodopteru frugiperdu, has been the most commonly used host in the BEVS21and, in general, is very efficient in heterologous expression.12However, Sf9 cells possess some limitation in providing a high yield of secreted protein^.'^ To address this issue, BTI-Tn-5B1-4 (Tn5) cells originated from the eggs of Tricoplusiu ni have recently been identified as a superior host to Sf9 cells for the expression of a secretory enzyme, alkaline phosphatase (ALP).2 Moreover, although serum-free culture is evidently desirable for the BEVS in view of the purification process of secreted proteins,16 to our knowledge, few studies have been reported on the detailed metabolism of the

insect cells grown in a serum-free medium, particularly after the recombinant virus infection. Here we present the characterization of the above insect cell lines useful for the BEVS. Sf9 and Tn5 cells were compared in monolayer and suspension cultures and before and after virus infection, in terms of growth, metabolism, and recombinant protein expression under the serum-free culture conditions. The results should be helpful for the establishment of the effective production process with the BEVS. As a model protein, we chose osteoblast-specific factor 2 (OSF-2), a recently discovered protein selectively expressed in bone.20 Because it is secreted, glycosylated, and has a relatively large molecular mass (about 90 kDa), which are potential obstacles to the efficient expression in the BEVS, we were interested in testing the insect cell lines for their ability to produce the OSF-2 molecule in the medium.
MATERIALS AND METHODS Insect Cells, Culture, and Recombinant Virus

Sf9 cells were grown in SF90011 serum-free medium (Gibco, Grand Island, NY). Tn5 cells adapted to suspension culture were kindly provided by Dr. T. Gong (JRH Bioscience, Lenexa, KS) and grown in Excell 401 serumfree medium (JRH Bioscience). Both cells were maintained in a 50-mL spinner flask (Bellco Glass, Vineland, NJ) at 27C and rotated at 100 rpm. The procedures for transfer vector construction and recombinant virus preparation were as described previously. The recombinant virus, AcNPVOSF2, carried mouse OSF-2cDNA in the correct orientation with respect to the polyhedrin promoter.
Kinetics Experiments with the BEVS System

* To whom all correspondence should be addressed. t Current address: Mikrobiologische Diagnostica, Behringwerke
AG, Postfach 1140 D35001 Marburg, Germany.

For dish culture experiments, replicated cultures were prepared by plating 1 X lo6 of Sf9 or Tn5 cells into 35-mm Falcon dishes with 2 mL of SF900II or Excell

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401 medium, respectively. After attachment, they were infected with 0.1 mL of AcNPVOSF2 virus solution at a multiplicity of infection (MOI) of 2 for 1 h. Then, 1.9 mL of the respective fresh medium was replenished to each culture. Cell number was determined daily after the cells were detached by vigorous pipeting. Because both cell lines exhibited a high sensitivity to the mechanical force and, therefore, after cell detachment a significant fraction of the cells were heavily damaged, we measured the number of cells with integrity (i.e., not lysed) by microscopic examination, instead of using the trypan blue dye exclusion test. In parallel, the cells and supernatants were separated by centrifugation and stored at -20C until further analysis. For suspension culture experiments, each insect cell line was inoculated into the Bellco 50-mL glass spinner flask at 1 X lo6 cells/mL with 25 mL of the respective medium. The cultures were infected with the AcNPVOSF2 virus at a MOI of 2 for 1 h, after which 25 mL of the same medium was further supplied. Every day 1-mL aliquots were removed from the cultures for cell counting and storage of samples. For cell growth experiments, Sf9 or Tn5 cells were inoculated at 2 X 105/mL in the spinner flask with 50 mL of the respective serum-free medium. Sample preparation and cell counting were done as described above.
Quantification of Recombinant OSF-2 Produced by BEVS

recombinant one derived from E. coli, we employed the baculoviral-conditioned medium containing a certain amount of OSF-2 as a standard. Hence, the relative amount of OSF-2 was determined from the standard curve. Immunoblotting for OSF-2 was conducted as described previo~sly.'~ The bands corresponding to OSF2 were quantitated by a Bioimage densitometer (Pharmacia, Uppsala, Sweden) for the relative fraction of secreted OSF-2. The secretion rate is defined the ratio of the secreted amount of OSF-2 to its total amount (secretory plus intracellular OSF-2 amount).
Analytical Methods

A Beckman glucose analyzer was used for the measurement of glucose, sucrose, and maltose. Sucrose and maltose were first broken into fructose and glucose by digestion with /3-fructosidase and a-glucosidase. Both enzymes were purchased from Boehringer Mannheim (Mannheim, Germany). Lactate determination was done with a YSI Model 23L lactate analyzer (Yellow Springs Instruments, Yellow Springs, OH). Glutamine, glutamate, and ammonia were quantitated using commercially available kits (Boehringer Mannheim).
RESULTS Growth and Metabolism of Insect Cells in Suspension Culture

For competitive inhibition enzyme-linked immunoadsorbent assay (ELISA), 50 p L of appropriately diluted standard OSF-2-conditioned medium or test samples were preincubated overnight with 200 p L of 1000-folddiluted a-pep2-3 antibodies in TBS (20 mM Trisbuffered saline, pH 7.4, containing 0.5 M NaC1) at 4C. a-pep2-3 antibodies were reactive with the C-terminal amino acid sequence of mouse OSF-2.19 Meanwhile, multiwell plates (Nunc) were coated overnight at 4C with a standard OSF-2-conditioned medium. Then, plates were blocked for 1h with 100 p L of CTB (10 mM Tris-buffered saline, pH 7.2, containing 0.15 M NaCl and 0.25% casein). After washing four times with TTBS (TBS containing 0.02% Tween 20), plates were further incubated 1 h with 50 p L of OSF-2/a-pep2-3 complex. Following intensive washing with TTBS again, 50 p L of 3000-fold-diluted peroxidase labeled swine anti-rabbit immunoglobin G in CTB was added. One hour later, plates were washed thoroughly with TTBS and incubated with a peroxidase substrate (Pierce, Rockford, IL) for color development. The reaction was stopped by the addition of 50 p L of 2N sulfuric acid and the absorbance was read at 492 nm with Beckman immunoreader. The assay was performed in duplicate. Because the only available purified OSF-2 molecule was a denatured

As shown in Figure lA, after a prolonged lag time, Sf9 cells grew with a doubling time of 22 to 24 h. This value is comparable with the published data:,* indicating that, at least during the exponential growth phase, the insect cells were in a good condition. By contrast, Tn5 cells began to proliferate soon after inoculation. The normal growth rate of Tn5 was 13 to 16 h. Tn5 cells adapted to support-free culture still had a tendency to form clumps. SF900II serum-free medium used for Sf9 cells contained a high concentration of glucose, glutamine, and glutamate (Fig. 2A and B). During the culture period, Sf9 cells did not show a prominent consumption of these nutrients. On the contrary, the glucose level gradually increased, probably due to the degradation of sucrose or maltose by the endogenous enzymatic activities of the insect cells.**Sucrose was, indeed, consumed by Sf9 cells, albeit initially at a very slow rate. We additionally detected maltose as another carbohydrate composite of SF900II (the initial concentration was about 0.12 mM). However, a large excess of other sugars such as sucrose and glucose in the medium disturbed the accurate estimation of maltose and, therefore, its level in the Sf9 culture is omitted in Figure 2. In accordance with the slow disappearance of sugars, only a small amount of lactate was generated by Sf9 cells (Fig. 2C). Ammonia

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Figure 1. (A) Growth curve of Sf9 or Tn5 cells grown in the spinner flask with 50 mL of SF90011 or Excell 401, respectively. (B) Cell density of Sf9 or Tn5 cells after virus infection. The insect cells were infected with AcNPVOSF2 on day 0.
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was found to be present in the original serum-free medium, which might be generated during shipment or, less likely, storage. In any case, neither reduction nor further accumulation of the metabolite was observed in the Sf9 culture. Taken together, despite the satisfactory growth in the SF90011medium, Sf9 cells were seemingly inert in the nutrient catabolism with a delayed accumulation of toxic metabolites." Unlike SF90011, the concentrations of the examined compositions in Excell 401 were similar to those of IPL41 documented in the literature,'* reflecting the accuracy of our analysis. While growing, Tn5 cells consumed a considerable amount of glucose and maltose and a small but substantial amount of sucrose (Fig. 2A). Lactate accumulated gradually as those sugars disappeared (Fig. 2C). Similarly,a remarkable amount of ammoniawas produced in proportion to the utilization of glutamine and glutamate (Fig. 2C). The profile of the Tn5 cell metabolism in serumfree Excell 401 closely resembled that of mammalian cells," except for the actual sucrose utilization.

Figure 2. Nutrient concentrationsin Sf9 and Tn5 cell cultures during the growth phase. Sugars (A), amino acids (B), and metabolites (C).

Growth, Metabolism, and Production of Insect Cells after Virus Infection

To further characterize Sf9 and Tn5 cells as a host in the BEVS, the insect cell lines grown in either dish or

spinner culture were infected with the recombinant virus harboring OSF-2 cDNA. To allow for a direct comparison between two culture methods, the conditions for viral infection (initial cell density, MOI, infection time) were identical for both cultures. The growth pattern of Sf9 and Tn5 cells after virus infection is depicted in Figure 1B. Throughout the infection phase, Sf9 cells grew only moderately regardless of the culture mode. On the other hand, Tn5 cells cultured in the dish as monolayer continued to propagate, which is characteristic of this cell line.24By contrast, Tn5 cells in suspension culture reached only a slightly higher cell density like Sf9 cells. As was found for the noninfected Sf9 culture, the glucose level in both Sf9 cultures did not drop substantially after virus infection (Fig. 3A). Sf9 cells consumed sucrose to a much higher degree than Tn5 cells (Fig. 3B). The sucrose consumption became more remarkable after virus infection, suggesting the increased energy requirement for the virus repli~ation.2~ Glutamine and glutamate concentrations increased or occasionally

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Figure 3. Carbohydrates and lactate concentrations in Sf9 and Tn5 cell cultures after virus infection. Glucose (A), sucrose (B), maltose (C), and lactate (D).

decreased in both Sf9 cultures as reported previously (Fig. 4A and B).' This implicates the presence of a complex metabolic pathway(s) for these amino acids in the insect cells. Again, no ammonia production was observed for both Sf9 cultures. The final concentrations

of all the carbohydrates and amino acids examined were similar for the two Sf9 cultures; however, more lactate production was found for Sf9 cells cultured in suspension (Fig. 3D). Glucose metabolism of Tn5 cells proceeded similarly in dish and spinner cultures in spite of the different growth patterns noticed for two cultures (Fig. 3A). Likewise, Tn5 consumed glutamine and glutamate in a simi) . As in the case lar way in both culture modes (Fig. 4 of the growth phase, a slight decline in the sucrose concentration was observed in both Tn5 cultures (Fig. 3B). Maltose disappeared at a more rapid rate in suspension culture than in monolayer culture (Fig. 3C). As was also seen for Sf9 cells, both byproducts, lactate and ammonia, were accumulated by Tn5 cells more prominently in suspension culture (Figs. 3D and 4C). OSF-2 was quantitated with a competitive-inhibition ELISA system (Fig. 5A) and the capability of the insect cells lines to secrete OSF-2 was examined by Western blotting, followed by densitometric analysis (Fig. 5B). In Sf9 cultures, OSF-2 was first detected in the medium on day 2 postinfection and dramatically increased between days 2 and 4 (Fig. 5A). The final productivities of the two Sf9 culture modes were comparable. The overall blotting pattern was in good agreement with the

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Figure 4. Amino acids and ammonia concentrations in Sf9 and Tn5 cell cultures after virus infection. Glutamine (A), glutamate (B), and ammonia (C).

Figure 5. The productivity of recombinant OSF-2 (A) and its secretion rate (B) in Sf9 and Tn5 cultures. The productivity of OSF-2 was measured using a competitive ELISA and is expressed relative to the value of the Sf9 cell dish culture at 4 days after virus infection. The ratio of the secreted OSF-2 was determined by Western blotting, followed by the densitometric analysis.

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outcome of the ELISA, except that the OSF-2 band intensity for the Sf9 suspension culture at 2 days postinfection was too weak to be detected by the densitometer (data not shown). The secreted OSF-2 was found to account for 40% to 45% of the total OSF-2 molecule in the Sf9 cultures (Fig. 5B). Tn5 cells in suspension secreted a detectable level of OSF-2 from day 2 after infection, clearly demonstrating an effective virus infection. Thereafter, the OSF-2 concentration in the medium remained at the initial level for 2 additional days, resulting in about 1/30-fold less productivity relative to the Sf9 cultures (Fig. 5A). On immunoblots, the Tn5 suspension culture displayed a faint OSF-2 band only in the supernatant fractions (Fig. 5B). Tn5 cells grown in monolayer culture showed a weak band of OSF-2, exclusively in the cell lysates (Fig. 5B), of which the intensity was equivalent to that of the Tn5 suspension culture (data not shown). Consistent with the Western blotting analysis, the competitive ELISA revealed no detectable level of OSF-2 in the culture medium from the Tn5 monolayer culture (Fig. 5A). Consequently, the ability of the suspension-adapted Tn5 cells to secrete OSF-2 was somehow compromised in the monolayer culture.

DISCUSSION

In this study, we observed that two major nutrients for mammalian cells, glucose and glutamine, were not consumed progressively by Sf9 cells during their growth in the SF900II medium. This finding does not necessarily mean that these nutrients were nonessential for the propagation of Sf9 cells in SF90011, since the actual uptake of sucrose by Sf9 cells obviously indicates the supply of glucose from this disaccharide.'* Presumably, the lower requirement of Sf9 cells for glucose" was further obscured by its supply from sucrose. We cannot eliminate another possibility that Sf9 cells utilized maltose as well for energy production. Similarly, glutamine was likely to be replaced or supplied by some other amino acids or peptides existing in the medium. It is also conceivable that the adaptation of Sf9 cells to the SF90011 serum-free medium facilitated the economical use of these nutrients. Intriguingly, during the growth phase, Sf9 cells showed little production of lactate and ammonia. The low toxic metabolite production of Sf9 cells has been noted in Excell medium and IPL-41 medium containing s e r ~ m .Therefore, ~ ~ ~ , ~ the ~ less active metabolic production is characteristic of Sf9 cells grown in this series of media probably because of the highly efficient oxidative-phosphorylation pathway of the insect cells; which is quite different than that of mammalian cells.18 The metabolism of Tn5 cells in Excell 401 medium appeared more similar to mammalian cell metabolism. It should be noted that, during both growth and infection phases, Tn5 cells showed a substantial consumption
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of sucrose, albeit at a delayed rate. It has been reported that Sf9 cells were either able2*(and this study) or unable1,4J3 to metabolize sucrose. Other cases have been reported to indicate that insect cells do metabolize sucro~e.~ In * 'light ~ of these results, it seems to hold true that insect cells generally have the ability to catabolize the disaccharide, which might be obscured by the ambient conditions, including distinct medium compositions. In spite of the potential disadvantages of OSF-2 for a preferable expression in the BEVS (a high molecular mass and glycosylation), both Sf9 and Tn5 cells were able to synthesize and transport the OSF-2 molecule into the culture medium. There was no difference in the OSF-2 productivity of Sf9 cells between the suspension and monolayer modes. The secretion rate of OSF2 in both cultures was 40% to 45%, in agreement with the published data for ALP.' By contrast, the productivity of OSF-2 by Tn5 cells cultured in the spinner flask was 30fold lower than that obtained with the Sf9 cultures. The possibility remains that the production level could be enhanced by improving the culture conditions for Tn5 cells; however, based on literature data,6.9J0the employed conditions are unlikely to be severely unfavorable for Tn5 cells. Furthermore, from immunoblotting analysis, the productivity of OSF-2 was roughly estimated to be about 30 ,ug/mL for both Sf9 cultures and 1 to 2 pg/rnl for the Tn5 suspension culture, the latter being a reasonable value for secretory proteins in the BEVS.12 Tn5 cells are, therefore, not a poor producer of OSF-2. Rather, Sf9 cells should be recognized as a superior host for OSF-2 production. The Tn5 dish culture failed to secrete OSF-2 into the medium, although the total OSF-2 productivity was virtually identical to the corresponding suspension culture. Besides, in the suspension culture, Tn5 cells ceased to multiply after virus infection, whereas in the monolayer culture they continued to propagate as reported p r e v i o ~ s l yThese . ~ ~ findings suggest that the adaptation of Tn5 cells to support-free culture entailed some alteration in their cell metabolism, thereby changing the characteristics regarding cell growth and protein secretion. The different culture modes also affected the level of accumulated waste metabolites, as exemplified in both insect cell lines (Figs. 3 and 4). The restricted oxygen supply would be responsible for the increased metabolite production in ~ u s p e n s i o neven , ~ ~ at the small working volume employed (50 mL), at which the oxygen provision is normally not a critical problem. In conclusion, Sf9 cells cultured in SF900II medium utilized nutrients effectively and produced metabolites minimally, suggesting that, in the serum-free batch system, merely an adequate oxygen supply would be sufficient for the economical production of target proteins by Sf9 cells. On the other hand, the behaviors of Tn5 cells in Excell medium closely resembled those of mammalian cells with respect to the cell metabolism. This indicates that the fed-batch culture strategy devised for

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mammalian cells is applicable to the effective cultivation of Tn5 cells without sermum. The productivity of OSF2 by Tn5 cells was moderate, whereas its productivity by Sf9 cells was 30-fold higher, reinforcing the significance of the careful selection of a host cell line for efficient recombinant protein production in the BEVS.
We thank Drs. H. B. Maruyama and T. Matsuishi for their continuing support, H. Takamatsu and Dr. S. Tanaka for establishment of the competitive ELISA, and Dr. M. Kimura for his help on the operation of the Bioimage densitometer. We are also grateful to Dr. T. Gong for his generous gift of support-free adapted Tn5 cells.

References
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10. Meyer, S. L., Lang, D. M., Forbes, M. E., Knight Jr., E., Hirsch, J. D., Trusko, S. P., Scott, R. W. 1994. Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells. J. Neurochem. 62:825-833. 11. Neermann, J., Wagner, R. 1996. Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells. J. Cell. Physiol. 166:152-169. 12. OReilly, D. R., Miller, L. K., Luckow, V. A. 1992. Baculovirus expression vectors: a laboratory manual. W. H. Freeman and Co., New York. 13. Reuveny, S., Kemp, C. W., Eppstein, L., Shiloach, J. 1992. Carbohydrate metabolism in insect cell cultures during cell growth and recombinant protein production. Ann. NY Acad. Sci. 665: 230-237. 14. Smith, G. E., Ju, G., Ericson, B. L., Moschera, J., Lahm, H.-W., Chizzonite, R., Summers, M. D. 1985. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Proc. Natl. Acad. Sci. USA 82: 8404-8408. 15. Stavroulakis, D. A., Kalogerakis, N., Behie, L. A., Iatrou, K. 1991. Kinetic data for the BM-5 insect cell line in repeated-batch suspension cultures. Biotechnol. Bioeng. 3 8 : 116-126. 16. Sugiura, T., Maruyama, H. B. 1992. Production of recombinant protein C in serum-containing and serum-free perfusion culture. Cytotechnology 7 159-164. 17. Sugiura, T., Maruyama, H. B. 1992.Factors influencing expression and post-translational modification of recombinant protein C. J. Biotechnol. 2 2 353-363. 18. Sugirua, T. 1992. Effects of glucose on the production of protein C in mammalian cell culture. Biotechnol. Bioeng. 39: 953-959. 19. Sugiura, T., Takamatsu, H., Kudo, A., Amann, E. 1995. Expression and characterization of murine osteoblast-specific factor 2 (OSF-2) in a baculovirus expression system. Prot. Express. Purif. 6: 305-311. 20. Takeshita, S., Tezuka, K.-I., Kikuno, R., Amann, E. 1993. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem. J. 294 271-278. 21. Vaughn, J. L., Goodwin, R. H., Tompkins, G. J., McCawley, P. 1977. The establishment of two cell lines from the insect Spodopteru frugiperdu (Lepidoptera: Noctuidae). In Vitro 13 213-217. 22. Wang, M.-Y., Vakharia, V., Bentley, W. E. 1993. Expression of epoxide hydrolase in insect cells: a focus on the infected cell. Biotechnol. Bioeng. 42: 240-246. 23. Wang, M.-Y., Kwong, S., Bentley, W. E. 1993. Effects of oxygen/ glucose/glutamine feeding on insect cell baculovirus protein expression: a study on epoxide hydroxylase production. Biotechnol. Prog. 9:355-361. 24. Wu, S.-C., Jarvis, D. L., Dale, B. E., Liao, J. C. 1994.Heterologous protein expression affects the death kinetics of baculovirusinfected insect cell cultures: a quantitative study by use of n-target theory. Biotechnol. Prog. 10 55-59.

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