- Email: [email protected]
Expression of human activin C protein in insect larvae infected with a recombinant baculovirus
Journal of Virological Methods 72 (1998) 9 – 14
Expression of human activin C protein in insect larvae infected with a recombinant baculovirus Renate Kron, Carsten Schneider, Gertrud Ho¨tten, Rolf Bechtold, Jens Pohl * Biopharm GmbH, Czernyring 22, D-69115 Heidelberg, Germany Received 2 September 1997; received in revised form 25 November 1997; accepted 25 November 1997
Abstract In order to generate dimeric recombinant transforming growth factor-b (TGF-b) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Expression; Baculovirus; Activin; Recombination
1. Introduction Activins and inhibins have been identified initially as gonadal proteins, regulating the secretion of a follicle stimulating hormone (FSH). Biochemical and molecular analyses of the proteins show that they are members of the transforming growth factor-b (TGF-b) superfamily (Massague´ et al., 1994). Genes coding for inhibin and activin * Corresponding author. Tel.: + 49 6221 53830; fax: + 49 6221 538320; e-mail: [email protected]
A, B and AB proteins can be grouped within the inhibin subfamily and are named inhibin a, inhibin bA, inhibin bB. Recently, the authors identified new human (Ho¨tten et al., 1995) and mouse cDNA (Schmitt et al., 1996) inhibin/activin bC subunits. Another member of this family, named activin bD subunit, was identified in Xenopus (Oda et al., 1995) and a further inhibin/activin bE subunit was identified in mouse (Fang et al., 1996). Based on sequence homology, the subunit coding for bC, bD and bE form a subset of related sequences. The activin/inhibin gene products are
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(97)00215-2
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R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
synthesized as preproprotein precursors, which are matured to an 18 kD a subunit or to 14 kD b subunits (Vale et al., 1990). So far, the known activins are homo/heterodimers of the closely related bA and bB subunits. In addition to their effect on FSH production, activins have been shown to regulate growth and differentiation of embryonal carcinoma cells (Schubert and Kimura, 1991); to induce erythropoiesis (Huylebroeck et al., 1990); to stimulate insulin secretion from the pancreas (Hashimoto et al., 1990); and to promote neural cell survival (Lau et al., 1996). In contrast with the known bA and bB subunits, the bC subunit is exclusively expressed in liver tissue (Lau et al., 1996; Schmitt et al., 1996). In initial studies, the bC subunit has been found to form secreted homodimers using a vaccinia virus expression system. However, expression levels have been fairly low (data not shown). To study the activin C biochemically and biologically, large quantities of homogeneous material are necessary. For this purpose, a baculovirus based expression system using insect larvae (Maeda et al., 1995) was chosen. Using a similar system, with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV), the bone morphogenetic protein-2 (BMP-2), another member of the TGF-b superfamily, can be expressed in large amounts in silkworm larvae (Ishida et al., 1994). The expression of dimeric activin C is reported now in different Noctuidae using recombinant Autographa californica nucleopolyhedrovirus (AcMNPV).
2. Materials and methods
2.1. Expression in E. coli To express the activin C protein in bacteria, the same system was used as described for the expression of huGDF5 (Ho¨tten et al., 1994). Essentially, the mature part of the activin C protein was produced in Escherichia coli BL21(DE3)pLysS. In order to simplify purification of activin C, an additional tag containing six histidines was attached to the N-terminus of
the mature protein which facilitates purification with Ni-chelate sepharose (Hochuli et al., 1988). The protein was purified further by reversed phase chromatography.
2.2. Immunological methods A polyclonal antiserum was provided in rabbits using an Escherichia coli derived protein, to detect the expression of recombinant activin C protein. Antibodies were partially purified using protein-A-sepharose and, by affinity with column bound antigen. For detection of proteins under reducing conditions, samples were loaded in the presence of 100 mM DTT. For detection of proteins under non-reducing conditions, the samples were loaded without reducing agents, however, during the transfer to the membranes, the proteins were reduced in the presence of 10 mM DTT in the transfer buffer to increase the sensitivity of the detection system. The bound antibodies were visualized with anti-rabbit IgG coupled with alkaline phosphatase and the Western-Light Protein Detection Kit (Tropix) and compared with a prestained size marker (Gibco, no. 26041-020).
2.3. Generation of recombinant 6accinia 6irus The complete coding sequence of the activin bC gene was cloned in a derivative of the pATA18 plasmid (Stunnenberg et al., 1988), which contains the Escherichia coli xanthine-guanine-phoshoribosyltransferase gene (gpt) (Janknecht et al., 1991). Recombinant vaccinia viruses were constructed and amplified under biosafety conditions according to standard procedures (Ho¨tten et al., 1994). Expression was carried out in NIH3T3 cells (DSM, ACC 59). Cell culture supernatants were collected and viruses removed by filtering through 0.1 mm filters. A total of 500 ml of cell supernatant were precipitated by acetone, solubilized and loaded onto a 15% SDS-PAGE as a positive control. The separated proteins were transferred to PVDF membranes for Western blot analysis as described by the manufacturer (Gelman).
R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
2.4. Generation of recombinant baculo6irus The complete coding sequence of the activin bC gene was subcloned in the pBlueBac III plasmid (Invitrogen) to generate recombinant baculoviruses. Recombination was undertaken in Spodoptera frugiperda (Sf9) (Invitrogen) cells as described in the manufacturers instructions. A stock of recombinant virus was produced (1 × 108 pfu/ml). As a negative control, the pBlueBac III plasmid was recombined without coding sequence (non-coding (N.C.) Virus).
2.5. Infection of insect lar6ae Eggs of silkworm (Bombyx mori, Bombycidae) and cabbage looper (Trichoplusiani, Noctuidae) were kindly provided by B. McCron (Canadian Forest Pest Management Institute Service, Sault Ste. Marie, OT). The larvae were reared on an artificial diet (Katakura Industries, Saitama) (O’Reilly et al., 1994). Manduca sexta (Sphingidae) larvae were kindly provided by Dr Knauf (AgrEvo, Frankfurt), Heliothis 6irescens (tobacco budworm, Noctuidae) larvae, were kindly provided by Dr Harries (BASF AG, Limburgerhof). About 106 pfu of the recombinant virus were injected subcutaneously into the body cavity of every fifth instar larvae with a syringe. Some 3 days after infection, the hemolymph was recovered and stored at − 80°C. Recovery and storage was carried out under argon gas to avoid the auto-catalytic melanization process of the hemolymph triggered by oxygen from the air.
3. Results Initially, activin C was expressed in a bacterial expression system as described for another member of the TGF-b superfamily: huGDF5 (Ho¨tten et al., 1994). This monomeric material was used for the generation of polyclonal antibodies in rabbits and served as a positive control. Furthermore, the homogeneous protein was used to quantify protein levels used in Western blot analysis.
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To produce dimeric activin C protein, a vaccinia virus expression system was used first. However, expression levels remained fairly low and yields of only about 30 mg per liter of cell culture were obtained (data not shown). Therefore, it was decided to use a commercially available baculovirus expression system. Infection of recombinant virus in different commonly used cell lines (Sf9 and Tn 368 (DSM, ACC 177)) resulted in the secretion of a monomeric protein only (data not shown). In order to obtain dimeric protein, it was decided to infect directly insect larvae with the recombinant baculovirus, a method used successfully to express BMP 2, another TGF-b superfamily member (Maeda et al., 1995). These studies had been carried out with recombinant BmNPV which is only able to infect Bombyx mori cells in vitro and in vivo. In contrast with these studies, the authors have been using recombinant AcMNPV. This virus has a broad host range and is therefore able to infect various species. Originally, the AcMNPV was isolated from the fall armyworm (Spodoptera frugiperda, Noctuidae). Therefore, it was decided first to infect different Noctuidae such as Trichoplusia ni and Heliothis 6irescens. In order to increase the amount of hemolymph, larger larvae from other Lepidopteran families were used for the concurrent infections. Manduca sexta (Sphingidae) and Bombyx mori (Bombycidae) larvae, which have also been reported to be semi-permissive to AcMNPV virus were selected. Recombinant with wild-type (wt) baculovirus at different ratios ranging from 1:10, 1:1–10:1 was used for oral infection. During co-infection of cell cultures, wild type and recombinant virus become occluded in polyhedral inclusion bodies (PIBs). However, using these PIBs as inoculum for per os infection of larvae, infection was not observed under the conditions used (data not shown). Therefore, the authors decided to inject the virus directly into the body cavity. Firstly, a time course was established to determine the best time for harvesting of hemolymph. Western blot analysis revealed an optimum of 3 days of infection after inoculation of 106 pfu/larvae. Longer infection periods led to higher mortality of the infected larvae.
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Western blot analysis of the hemolymph of infected larvae under non-reducing conditions (Fig. 1A) revealed that both Noctuidae, Trichoplusia ni (lane 3) and Heliothis 6irescens (lane 5) produced a protein with an apparent molecular weight of about 20 kD (indicated by a filled arrow head) recognized by the activin-C-specific antiserum. This protein was not expressed in larvae infected with the N.C. recombinant virus (lanes 2 and 4, respectively). The specificity of activin C is shown by the positive control which is superna-
tant from recombinant vaccinia virus infected cells expressing activin C (lane 10). Lane 1 shows the reactivity to the monomeric protein of 15 kD (indicated by an open arrow head) expressed in bacteria. The infection of Manduca sexta larvae resulted only in the expression of monomeric activin C (lane 7) and infection of Bombyx mori did not result in any detectable activin C expression (lane 9). Under reducing conditions (Fig. 1B), the dimeric protein expressed in Trichoplusia ni (lane 3) and Heliothis 6irescens (lane 5) as well as the protein expressed in the vaccinia virus system (lane 10) is reduced to the monomeric 15 kD position, indicating that the dimers molecular weight is underestimated by this analysis, most likely due to a coiled fast migrating form of the dimer. The protein expressed in Manduca sexta larvae remains at the same position on the gel after reduction, confirming its monomeric state. Using bacterial activin C protein, purified to homogeneity, as a standard, the amount expressed in Trichoplusia ni was calculated to be about 30 ng per 4 ml of hemolymph. Since up to 200 ml of hemolymph per animal can be collected, the hemolymph from 20 to 40 larvae contains as much activin C protein as 1 l of cell supernatant from vaccinia virus infected cells.
4. Discussion
Fig. 1. Western blot analysis of activin C produced in insect larvae under non-reducing (A) and reducing (B) conditions. Lanes are: monomeric activin C expressed in bacteria (lane 1); hemolymph of Trichoplusia ni, infected by a non-coding virus (N.C. virus) (lane 2) or activin C expressing virus (lane 3); hemolymph of Heliothis 6irescens, infected by N.C. virus (lane 4) or activin C expressing virus (lane 5); hemolymph of Manduca sexta, infected by N.C. virus (lane 6) or activin C expressing virus (lane 7); hemolymph of Bombyx mori, infected by N.C. virus (lane 8) or activin C expressing virus (lane 9); supernatant of NIH3T3 cells infected by recombinant activin C expressing vaccinia virus (lane 10). Marker sizes are in kD. The filled arrow head indicates the position of the dimeric activin C protein at 20 kD, the open arrow head indicates the position of the monomeric activin C protein at 15 kD.
In order to carry out studies with activin C, large quantities of the protein are required. Most TGF-b proteins are difficult to express in heterologous systems. So far, the TGF-b like proteins BMP-2 and GDF5 have been expressed using a vaccinia virus based expression system. However, using the same system, only low amounts of activin C proteins could be obtained. Therefore, it was decided to use an available commercial baculovirus expression system. Using different insect cell lines such as Sf9 and Tn 368, the authors were only able to obtain secreted monomeric activin C protein. Ishida et al. (1994) reported that using a recombinant Bombyx mori NPV, dimeric BMP-2 protein could be expressed in large quantities after infection of silkworm larvae. Infecting Trichoplusia ni larvae, it was
R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
possible to obtain up to 7.5 mg of dimeric activin C protein per ml hemolymph, corresponding with the amount of protein obtained from 250 ml of cell culture by recombinant vaccinia virus infected cells. Since yield of activin C from all systems tested so far was extremely poor, the use of the baculovirus system in combination with larvae represents a viable alternative. However, the infection of other Lepidopteran species revealed that care should be taken in the selection of the appropriate host using this system. AcMNPV was also found to be semi-permissive to different species. Only Noctuidae were able to process properly and secrete the desired dimeric activin C protein. Recombinant activin C protein was purified and it is now possible to start the identification of the activin C receptor. Knowing the site of receptor expression is a prerequisite for functional studies with activin C. Compared with expensive cell culture systems such as CHO cells, the expression system presented in this study may in certain cases represent the only way to obtain functional proteins in insect cells, due to post-translational problems in insect cell culture.
Acknowledgements We thank B. Scheffner, E. Hufnagel, S. Nees, S. Scheuermann and J. Jaschke for excellent technical assistance and M. Paulista for continuous support. Part of this study was supported by the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie, Grant 0311373.
References Fang, J., Yin, W., Smiley, E., Wang, S.Q., Bonadio, J., 1996. Molecular cloning of the mouse activin beta E subunit gene. Biochem. Biophys. Res. Commun. 228, 669–674. Hashimoto, M., Kondo, S., Sakurai, T., Etoh, Y., Shibai, H., Muramatsu, M., 1990. Activin/EDF as an inhibitor of neural differentiation. Biochem. Biophys. Res. Commun. 173, 193 – 200.
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Ho¨tten, G., Neidhardt, H., Jacobowsky, B., Pohl, J., 1994. Cloning and expression of recombinant human growth/differentiation factor 5. Biochem. Biophys. Res. Commun. 204 (2), 646 – 652. Ho¨tten, G., Neidhardt, H., Schneider, C., Pohl, J., 1995. Cloning of a new member of the TGF-b family: a putative new activin bC chain. Biochem. Biophys. Res. Commun. 206 (2), 608 – 613. Hochuli, E., Bannwarth, W., Do¨bli, H., Gentz, R., Stu¨ber, D., 1988. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Biotechnology 6, 1321 – 1325. Huylebroeck, D., Nimmen, K.V., Waheed, A., von-Figura, K., Marmenout, A., Fransen, L., De-Waele, P., Jaspar, J.M., Franchimont, P., Stunnenberg, H., Heuverswijn, Hv., 1990. Expression and processing of the activin-A/erythroid differentiation factor precursor: a member of the transforming growth factor-beta superfamily. Mol. Endocrinol. 4, 1153 – 1165. Ishida, N., Tsujimoto, M., Kanaya, T., Shimamura, A., Tsuruoka, N., Kodama, S., Katayama, T., Oikawa, S., Matsui, M., Nakanishi, T., Kobayashi, J., Nakazato, H., 1994. Expression and characterisation of human bone morphogenetic protein-2 in silkworm larvae infected with recombinant Bombyx mori nuclear polyhedrosis virus. J. Biochem. 115, 279 – 285. Janknecht, R., de Matrynoff, G., Lou, J., Hipskind, R.A., Nordheim, A., Stunnenberg, H.G., 1991. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc. Natl. Acad. Sci. USA 88, 8972 – 8976. Lau, A.L., Nishimori, K., Matzuk, M.M., 1996. Structural analysis of the mouse activin beta C gene. Biochim. Biophys. Acta 1307, 145 – 148. Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y., Furusawa, M., 1995. Production of human a-interferon in silkworm using a baculovirus vector. Nature 315, 592 – 594. Massague´, J., Attisano, L., Wrana, J.L., 1994. The TGF-b family and its composite receptors. Trends Cell. Biol. 4, 172 – 178. Oda, S., Nishimatsu, S., Murakami, K., Ueno, N., 1995. Molecular cloning and functional analysis of a new activin beta subunit: a dorsal mesoderm-inducing activity in Xenopus. Biochem. Biophys. Res. Commun. 210 (2), 581 – 588. O’Reilly, D.R., Miller, L.K., Luckow, V.A. (Eds.), 1994. Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, London, pp. 257 – 269. Schmitt, J., Ho¨tten, G., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., Pohl, J., Schrewe, H., 1996. Structure, chromosomal localization and expression analysis of the mouse inhibin/activin bC (Inhbc) gene. Genomics 32, 358 – 366. Schubert, D., Kimura, H., 1991. Substratum-growth factor collaborations are required for the mitogenic activities of activin and FGF on embryonal carcinoma cells. J. Cell. Biol. 114, 841 – 846.
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Stunnenberg, H.G., Lange, D., Philipson, L., van Miltenburg, R.T., van der Vliet, P.Ch., 1988. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus. Nucleic Acids Res. 16 (6), 2431 – 2444.
Vale, W., Hsueh, A., Rivier, C., Yu, J., 1990. Peptide growth factors and their receptors. In: Sporn, M.B., Roberts, A.B. (Eds.), Handbook of Experimental Pharmacology, vol. 95. Springer, Berlin, pp. 2196 – 2203.
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Expression of human activin C protein in insect larvae infected with a recombinant baculovirus Renate Kron, Carsten Schneider, Gertrud Ho¨tten, Rolf Bechtold, Jens Pohl * Biopharm GmbH, Czernyring 22, D-69115 Heidelberg, Germany Received 2 September 1997; received in revised form 25 November 1997; accepted 25 November 1997
Abstract In order to generate dimeric recombinant transforming growth factor-b (TGF-b) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Expression; Baculovirus; Activin; Recombination
1. Introduction Activins and inhibins have been identified initially as gonadal proteins, regulating the secretion of a follicle stimulating hormone (FSH). Biochemical and molecular analyses of the proteins show that they are members of the transforming growth factor-b (TGF-b) superfamily (Massague´ et al., 1994). Genes coding for inhibin and activin * Corresponding author. Tel.: + 49 6221 53830; fax: + 49 6221 538320; e-mail: [email protected]
A, B and AB proteins can be grouped within the inhibin subfamily and are named inhibin a, inhibin bA, inhibin bB. Recently, the authors identified new human (Ho¨tten et al., 1995) and mouse cDNA (Schmitt et al., 1996) inhibin/activin bC subunits. Another member of this family, named activin bD subunit, was identified in Xenopus (Oda et al., 1995) and a further inhibin/activin bE subunit was identified in mouse (Fang et al., 1996). Based on sequence homology, the subunit coding for bC, bD and bE form a subset of related sequences. The activin/inhibin gene products are
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(97)00215-2
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R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
synthesized as preproprotein precursors, which are matured to an 18 kD a subunit or to 14 kD b subunits (Vale et al., 1990). So far, the known activins are homo/heterodimers of the closely related bA and bB subunits. In addition to their effect on FSH production, activins have been shown to regulate growth and differentiation of embryonal carcinoma cells (Schubert and Kimura, 1991); to induce erythropoiesis (Huylebroeck et al., 1990); to stimulate insulin secretion from the pancreas (Hashimoto et al., 1990); and to promote neural cell survival (Lau et al., 1996). In contrast with the known bA and bB subunits, the bC subunit is exclusively expressed in liver tissue (Lau et al., 1996; Schmitt et al., 1996). In initial studies, the bC subunit has been found to form secreted homodimers using a vaccinia virus expression system. However, expression levels have been fairly low (data not shown). To study the activin C biochemically and biologically, large quantities of homogeneous material are necessary. For this purpose, a baculovirus based expression system using insect larvae (Maeda et al., 1995) was chosen. Using a similar system, with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV), the bone morphogenetic protein-2 (BMP-2), another member of the TGF-b superfamily, can be expressed in large amounts in silkworm larvae (Ishida et al., 1994). The expression of dimeric activin C is reported now in different Noctuidae using recombinant Autographa californica nucleopolyhedrovirus (AcMNPV).
2. Materials and methods
2.1. Expression in E. coli To express the activin C protein in bacteria, the same system was used as described for the expression of huGDF5 (Ho¨tten et al., 1994). Essentially, the mature part of the activin C protein was produced in Escherichia coli BL21(DE3)pLysS. In order to simplify purification of activin C, an additional tag containing six histidines was attached to the N-terminus of
the mature protein which facilitates purification with Ni-chelate sepharose (Hochuli et al., 1988). The protein was purified further by reversed phase chromatography.
2.2. Immunological methods A polyclonal antiserum was provided in rabbits using an Escherichia coli derived protein, to detect the expression of recombinant activin C protein. Antibodies were partially purified using protein-A-sepharose and, by affinity with column bound antigen. For detection of proteins under reducing conditions, samples were loaded in the presence of 100 mM DTT. For detection of proteins under non-reducing conditions, the samples were loaded without reducing agents, however, during the transfer to the membranes, the proteins were reduced in the presence of 10 mM DTT in the transfer buffer to increase the sensitivity of the detection system. The bound antibodies were visualized with anti-rabbit IgG coupled with alkaline phosphatase and the Western-Light Protein Detection Kit (Tropix) and compared with a prestained size marker (Gibco, no. 26041-020).
2.3. Generation of recombinant 6accinia 6irus The complete coding sequence of the activin bC gene was cloned in a derivative of the pATA18 plasmid (Stunnenberg et al., 1988), which contains the Escherichia coli xanthine-guanine-phoshoribosyltransferase gene (gpt) (Janknecht et al., 1991). Recombinant vaccinia viruses were constructed and amplified under biosafety conditions according to standard procedures (Ho¨tten et al., 1994). Expression was carried out in NIH3T3 cells (DSM, ACC 59). Cell culture supernatants were collected and viruses removed by filtering through 0.1 mm filters. A total of 500 ml of cell supernatant were precipitated by acetone, solubilized and loaded onto a 15% SDS-PAGE as a positive control. The separated proteins were transferred to PVDF membranes for Western blot analysis as described by the manufacturer (Gelman).
R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
2.4. Generation of recombinant baculo6irus The complete coding sequence of the activin bC gene was subcloned in the pBlueBac III plasmid (Invitrogen) to generate recombinant baculoviruses. Recombination was undertaken in Spodoptera frugiperda (Sf9) (Invitrogen) cells as described in the manufacturers instructions. A stock of recombinant virus was produced (1 × 108 pfu/ml). As a negative control, the pBlueBac III plasmid was recombined without coding sequence (non-coding (N.C.) Virus).
2.5. Infection of insect lar6ae Eggs of silkworm (Bombyx mori, Bombycidae) and cabbage looper (Trichoplusiani, Noctuidae) were kindly provided by B. McCron (Canadian Forest Pest Management Institute Service, Sault Ste. Marie, OT). The larvae were reared on an artificial diet (Katakura Industries, Saitama) (O’Reilly et al., 1994). Manduca sexta (Sphingidae) larvae were kindly provided by Dr Knauf (AgrEvo, Frankfurt), Heliothis 6irescens (tobacco budworm, Noctuidae) larvae, were kindly provided by Dr Harries (BASF AG, Limburgerhof). About 106 pfu of the recombinant virus were injected subcutaneously into the body cavity of every fifth instar larvae with a syringe. Some 3 days after infection, the hemolymph was recovered and stored at − 80°C. Recovery and storage was carried out under argon gas to avoid the auto-catalytic melanization process of the hemolymph triggered by oxygen from the air.
3. Results Initially, activin C was expressed in a bacterial expression system as described for another member of the TGF-b superfamily: huGDF5 (Ho¨tten et al., 1994). This monomeric material was used for the generation of polyclonal antibodies in rabbits and served as a positive control. Furthermore, the homogeneous protein was used to quantify protein levels used in Western blot analysis.
11
To produce dimeric activin C protein, a vaccinia virus expression system was used first. However, expression levels remained fairly low and yields of only about 30 mg per liter of cell culture were obtained (data not shown). Therefore, it was decided to use a commercially available baculovirus expression system. Infection of recombinant virus in different commonly used cell lines (Sf9 and Tn 368 (DSM, ACC 177)) resulted in the secretion of a monomeric protein only (data not shown). In order to obtain dimeric protein, it was decided to infect directly insect larvae with the recombinant baculovirus, a method used successfully to express BMP 2, another TGF-b superfamily member (Maeda et al., 1995). These studies had been carried out with recombinant BmNPV which is only able to infect Bombyx mori cells in vitro and in vivo. In contrast with these studies, the authors have been using recombinant AcMNPV. This virus has a broad host range and is therefore able to infect various species. Originally, the AcMNPV was isolated from the fall armyworm (Spodoptera frugiperda, Noctuidae). Therefore, it was decided first to infect different Noctuidae such as Trichoplusia ni and Heliothis 6irescens. In order to increase the amount of hemolymph, larger larvae from other Lepidopteran families were used for the concurrent infections. Manduca sexta (Sphingidae) and Bombyx mori (Bombycidae) larvae, which have also been reported to be semi-permissive to AcMNPV virus were selected. Recombinant with wild-type (wt) baculovirus at different ratios ranging from 1:10, 1:1–10:1 was used for oral infection. During co-infection of cell cultures, wild type and recombinant virus become occluded in polyhedral inclusion bodies (PIBs). However, using these PIBs as inoculum for per os infection of larvae, infection was not observed under the conditions used (data not shown). Therefore, the authors decided to inject the virus directly into the body cavity. Firstly, a time course was established to determine the best time for harvesting of hemolymph. Western blot analysis revealed an optimum of 3 days of infection after inoculation of 106 pfu/larvae. Longer infection periods led to higher mortality of the infected larvae.
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R. Kron et al. / Journal of Virological Methods 72 (1998) 9–14
Western blot analysis of the hemolymph of infected larvae under non-reducing conditions (Fig. 1A) revealed that both Noctuidae, Trichoplusia ni (lane 3) and Heliothis 6irescens (lane 5) produced a protein with an apparent molecular weight of about 20 kD (indicated by a filled arrow head) recognized by the activin-C-specific antiserum. This protein was not expressed in larvae infected with the N.C. recombinant virus (lanes 2 and 4, respectively). The specificity of activin C is shown by the positive control which is superna-
tant from recombinant vaccinia virus infected cells expressing activin C (lane 10). Lane 1 shows the reactivity to the monomeric protein of 15 kD (indicated by an open arrow head) expressed in bacteria. The infection of Manduca sexta larvae resulted only in the expression of monomeric activin C (lane 7) and infection of Bombyx mori did not result in any detectable activin C expression (lane 9). Under reducing conditions (Fig. 1B), the dimeric protein expressed in Trichoplusia ni (lane 3) and Heliothis 6irescens (lane 5) as well as the protein expressed in the vaccinia virus system (lane 10) is reduced to the monomeric 15 kD position, indicating that the dimers molecular weight is underestimated by this analysis, most likely due to a coiled fast migrating form of the dimer. The protein expressed in Manduca sexta larvae remains at the same position on the gel after reduction, confirming its monomeric state. Using bacterial activin C protein, purified to homogeneity, as a standard, the amount expressed in Trichoplusia ni was calculated to be about 30 ng per 4 ml of hemolymph. Since up to 200 ml of hemolymph per animal can be collected, the hemolymph from 20 to 40 larvae contains as much activin C protein as 1 l of cell supernatant from vaccinia virus infected cells.
4. Discussion
Fig. 1. Western blot analysis of activin C produced in insect larvae under non-reducing (A) and reducing (B) conditions. Lanes are: monomeric activin C expressed in bacteria (lane 1); hemolymph of Trichoplusia ni, infected by a non-coding virus (N.C. virus) (lane 2) or activin C expressing virus (lane 3); hemolymph of Heliothis 6irescens, infected by N.C. virus (lane 4) or activin C expressing virus (lane 5); hemolymph of Manduca sexta, infected by N.C. virus (lane 6) or activin C expressing virus (lane 7); hemolymph of Bombyx mori, infected by N.C. virus (lane 8) or activin C expressing virus (lane 9); supernatant of NIH3T3 cells infected by recombinant activin C expressing vaccinia virus (lane 10). Marker sizes are in kD. The filled arrow head indicates the position of the dimeric activin C protein at 20 kD, the open arrow head indicates the position of the monomeric activin C protein at 15 kD.
In order to carry out studies with activin C, large quantities of the protein are required. Most TGF-b proteins are difficult to express in heterologous systems. So far, the TGF-b like proteins BMP-2 and GDF5 have been expressed using a vaccinia virus based expression system. However, using the same system, only low amounts of activin C proteins could be obtained. Therefore, it was decided to use an available commercial baculovirus expression system. Using different insect cell lines such as Sf9 and Tn 368, the authors were only able to obtain secreted monomeric activin C protein. Ishida et al. (1994) reported that using a recombinant Bombyx mori NPV, dimeric BMP-2 protein could be expressed in large quantities after infection of silkworm larvae. Infecting Trichoplusia ni larvae, it was
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possible to obtain up to 7.5 mg of dimeric activin C protein per ml hemolymph, corresponding with the amount of protein obtained from 250 ml of cell culture by recombinant vaccinia virus infected cells. Since yield of activin C from all systems tested so far was extremely poor, the use of the baculovirus system in combination with larvae represents a viable alternative. However, the infection of other Lepidopteran species revealed that care should be taken in the selection of the appropriate host using this system. AcMNPV was also found to be semi-permissive to different species. Only Noctuidae were able to process properly and secrete the desired dimeric activin C protein. Recombinant activin C protein was purified and it is now possible to start the identification of the activin C receptor. Knowing the site of receptor expression is a prerequisite for functional studies with activin C. Compared with expensive cell culture systems such as CHO cells, the expression system presented in this study may in certain cases represent the only way to obtain functional proteins in insect cells, due to post-translational problems in insect cell culture.
Acknowledgements We thank B. Scheffner, E. Hufnagel, S. Nees, S. Scheuermann and J. Jaschke for excellent technical assistance and M. Paulista for continuous support. Part of this study was supported by the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie, Grant 0311373.
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