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Expression of Protein Complex Comprising the Human Prorenin and (Pro)Renin Receptor in Silkworm Larvae Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmids for Improving Biological Function

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Abstract

Three forms of recombinant protein complexes comprising the human prorenin (hPro) and (pro)renin receptor (hPRR) (hPRR/prorenin) were successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids. They were localized in the fat body cells and formed a prorenin-bound hPRR complex. The expressed levels of hPro and hPRR were similar judging from Western blotting. The hPRR/prorenin complex containing 40 μg of hPRR (yield, 43%) and 30 μg of hPro (yield, 34%) was purified from 15 silkworm larvae by a series of purification using anti-FLAG and Strep-Tactin affinity chromatography. The renin activity of the purified hPRR/prorenin complex was 3.8-fold that of the mixture of hPRR and hPro expressed individually in vitro judging from the renin assay. These results show that the unstable transmembrane protein, hPRR, was coexpressed stably with ligand, hPro, and formed a stable protein, hPRR/prorenin complex that showed a high catalytic active form.

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References

  1. Wallrabe, H., Elangovan, M., Burchard, A., Periasamy, A., & Barroso, M. (2003). Confocal FRET microscopy to measure clustering of ligand–receptor complexes in endocytic membranes. Biophysical Journal, 85, 559–571.

    Article  CAS  Google Scholar 

  2. Fenge, C., Jansson, I., Fröberg, T., Jönsson, M., Lüllau, E., Sygowski, L., et al. (2002). Process development for functional membrane receptor production in mammalian cells. Cytotechnology, 38, 109–117.

    Article  CAS  Google Scholar 

  3. Kato, T., Park, E. Y., Murata, T., & Usui, T. (2004). Efficient production of human (1, 3-N-acetylglucosaminyltransferase 2 fused with green fluorescent protein in insect cell. Biochemical Engineering Journal, 19, 15–23.

    Article  CAS  Google Scholar 

  4. Massotte, D. (2003). G protein-coupled receptor overexpression with the baculovirus–insect cell system: A tool for structural and functional studies. Biochimica et Biophysica Acta, 1610, 77–89.

    Article  CAS  Google Scholar 

  5. Motohashi, T., Shimojima, T., Fukagawa, T., Maenaka, K., & Park, E. Y. (2005). Efficient large-scale protein production of larvae and pupae of silkworm nuclear polyhedrovirus (BmNPV) bacmid system. Biochemical and Biophysical Research Communications, 326, 564–569.

    Article  CAS  Google Scholar 

  6. Suzuki, F., Hayakawa, M., Nakagawa, T., Nasir, U. M., Ebihara, A., Iwasawa, A., et al. (2003). Human prorenin has “gate and handle” regions for its non-proteolytic activation. The Journal of Biological Chemistry, 278, 22217–22222.

    Article  CAS  Google Scholar 

  7. Nguyen, G., Delarue, F., Burckle, C., Bouzhir, L., Giller, T., & Sraer, J. -D. (2002). Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. The Journal of Clinical Investigation, 109, 1417–1427.

    CAS  Google Scholar 

  8. Burcklé, C. A., Danser, A. H. J., Müller, D. N., Garrelds, I. M., Gasc, J. M., Popova, E., et al. (2006). Elevated blood pressure and heart rate in human renin receptor transgenic rats. Hypertension, 47, 552–556.

    Article  Google Scholar 

  9. Danser, A. H., & Deinum, J. (2005). Renin, prorenin and the putative (pro)renin receptor. Hypertension, 46, 1069–1076.

    Article  CAS  Google Scholar 

  10. Ichihara, A., Hayashi, M., Kaneshiro, Y., Suzuki, F., Nakagawa, T., Tada, Y., et al. (2004). Inhibition of diabetic nephropathy by a decoy peptide corresponding to the “handle” region for nonproteolytic activation of prorenin. The Journal of Clinical Investigation, 114, 1128–1135.

    CAS  Google Scholar 

  11. Du, D., Kato, T., Nabi, A. N., Suzuki, F., & Park, E. Y. (2008). Expression of functional human (pro)renin receptor in silkworm larvae using BmNPV bacmid. Biotechnology and Applied Biochemistry, 49, 195–202.

    Article  CAS  Google Scholar 

  12. Kato, T., Kageshima, A., Suzuki, F., & Park, E. Y. (2008). Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system. Protein Expression and Purification, 58, 242–248.

    Article  CAS  Google Scholar 

  13. Hiyoshi, M., Kageshima, A., Kato, T., & Park, E. Y. (2007). Construction of cysteine protease deficient Bombyx mori nuclear polyhedrosis bacmid and its application on improved expression of fusion protein. Journal of Virological Methods, 144, 91–97.

    Article  CAS  Google Scholar 

  14. Suzuki, F., Hatano, Y., Nakagawa, T., Terazawa, K., Gotoh, A., Nasir, U. M., et al. (1999). Non-proteolytic activation of human prorenin by anti-prorenin prosegment (pf#1: 1P–15P) antiserum. Bioscience, Biotechnology, and Biochemistry, 63, 550–554.

    Article  CAS  Google Scholar 

  15. Batenburg, W. W., Krop, M., Garrelds, I. M., de Vries, R., de Bruin, R. J., Burcklé, C. A., et al. (2007). Prorenin is the endogenous agonist of the (pro)renin receptor. Binding kinetics of renin and prorenin in rat vascular smooth muscle cells overexpressing the human (pro)renin receptor. Journal of Hypertension, 25, 2441–2453.

    Article  CAS  Google Scholar 

  16. Kaneshiro, Y., Ichihara, A., Takemitsu, T., Sakoda, M., Suzuki, F., Nakagawa, T., et al. (2006). Increased expression of cyclooxygenase-2 in the renal cortex of human prorenin receptor gene-transgenic rats. Kidney International, 70, 641–646.

    Article  CAS  Google Scholar 

  17. Huang, Y., Wongamorntham, S., Kasting, J., McQuillan, D., Owens, R. T., Yu, L., et al. (2006). Renin increases mesangial cell transforming growth factor-β1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms. Kidney International, 69, 105–113.

    Article  CAS  Google Scholar 

  18. Du, D., Kato, T., Suzuki F., & Park, E. Y. (2009). Binding affinity of full-length and extracellular domains of recombinant human (pro)renin receptors to human renin when expressed in the fat body and hemolymph of silkworm larvae. Journal of Bioscience and Bioengineering, in press.

  19. Mendrola, J. M., Berger, M. B., King, M. C., & Lemmon, M. A. (2002). The single transmembrane domains of ErbB receptors self-associate in cell membranes. The Journal of Biological Chemistry, 277, 4704–4712.

    Article  CAS  Google Scholar 

  20. Chiang, W. C., & Knowles, A. F. (2008). Transmembrane domain interactions affects the stability of the extracellular domain of the human NTPDase 2. Archives of Biochemistry and Biophysics, 472, 89–99.

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan.

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Authors and Affiliations

  1. Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Dongning Du & Enoch Y. Park

  2. Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan

    Tatsuya Kato & Enoch Y. Park

  3. Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Fumiaki Suzuki

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  1. Dongning Du
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  2. Tatsuya Kato
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  3. Fumiaki Suzuki
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  4. Enoch Y. Park
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Correspondence to Enoch Y. Park.

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Du, D., Kato, T., Suzuki, F. et al. Expression of Protein Complex Comprising the Human Prorenin and (Pro)Renin Receptor in Silkworm Larvae Using Bombyx mori Nucleopolyhedrovirus (BmNPV) Bacmids for Improving Biological Function. Mol Biotechnol 43, 154–161 (2009). https://doi.org/10.1007/s12033-009-9183-7

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  • DOI: https://doi.org/10.1007/s12033-009-9183-7

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