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Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

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Abstract

To improve recombinant human bone morphogenetic protein-2 (rhBMP-2) yield, cell lines stably expressing hBMP2 were cultured in the presence of polyarginine peptide IND-1 and showed up to 6-fold increase in the yield of mature BMP-2. Repeated addition of IND-1 to cell cultures consistently improved BMP-2 yields over 53 days without affecting cell growth and viability. Investigation of its mechanism of action showed that IND-1 inhibited pro-protein convertase (PC) activity when incubated with cell lysates. However, when intact cells were cultured with IND-1, no change in cellular PC activity was observed. Furthermore, knockdown of furin (a prototypical member of the PCs) in cells did not affect their BMP-2 yields, suggesting furin/PC inhibition is unlikely the mechanism by which IND-1 enhances BMP-2 yields. IND-1 as a medium additive thus enhances BMP-2 production in mammalian cell expression systems.

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References

  • Angliker H, Wikstrom P, Shaw E, Brenner C, Fuller RS (1993) The synthesis of inhibitors for processing proteinases and their action on the Kex2 proteinase of yeast. Biochem J 293(Pt 1):75–81

    PubMed  CAS  Google Scholar 

  • Bessho K, Tagawa T, Murata M (1989) Purification of bone morphogenetic protein derived from bovine bone matrix. Biochem Biophys Res Commun 165:595–601

    Article  PubMed  CAS  Google Scholar 

  • Cameron A, Appel J, Houghten RA, Lindberg I (2000) Polyarginines are potent furin inhibitors. J Biol Chem 275:36741–36749

    Article  PubMed  CAS  Google Scholar 

  • Cui Y, Jean F, Thomas G, Christian JL (1998) BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development. EMBO J 17:4735–4743

    Article  PubMed  CAS  Google Scholar 

  • Fuchs SM, Raines RT (2004) Pathway for polyarginine entry into mammalian cells. Biochemistry 43:2438–2444

    Article  PubMed  CAS  Google Scholar 

  • Fugere M, Appel J, Houghten RA, Lindberg I, Day R (2007) Short polybasic peptide sequences are potent inhibitors of PC5/6 and PC7: use of positional scanning-synthetic peptide combinatorial libraries as a tool for the optimization of inhibitory sequences. Mol Pharmacol 71:323–332

    Article  PubMed  CAS  Google Scholar 

  • Heng S, Paule S, Hardman B, Li Y, Singh H, Rainczuk A, Stephens AN, Nie G (2010) Posttranslational activation of bone morphogenetic protein 2 is mediated by proprotein convertase 6 during decidualization for pregnancy establishment. Endocrinology 151:3909–3917

    Article  PubMed  CAS  Google Scholar 

  • Hess GT, WHt H, Fay NC, Payne CK (2007) Cellular binding, motion, and internalization of synthetic gene delivery polymers. Biochim Biophys Acta 1773:1583–1588

    Article  PubMed  CAS  Google Scholar 

  • Hillger F, Herr G, Rudolph R, Schwarz E (2005) Biophysical comparison of BMP-2, ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by the mature growth factor. J Biol Chem 280:14974–14980

    Article  PubMed  CAS  Google Scholar 

  • Hu ZM, Peel SA, Sandor GK, Clokie CM (2004) The osteoinductive activity of bone morphogenetic protein (BMP) purified by repeated extracts of bovine bone. Growth Factors 22:29–33

    Article  PubMed  CAS  Google Scholar 

  • Huang Y, Li Y, Wang YG, Gu X, Wang Y, Shen BF (2007) An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods 322:28–39

    Article  PubMed  CAS  Google Scholar 

  • Israel DI, Nove J, Kerns KM, Moutsatsos IK, Kaufman RJ (1992) Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells. Growth Factors 7:139–150

    Article  PubMed  CAS  Google Scholar 

  • Kacprzak MM, Peinado JR, Than ME, Appel J, Henrich S, Lipkind G, Houghten RA, Bode W, Lindberg I (2004) Inhibition of furin by polyarginine-containing peptides: nanomolar inhibition by nona-d-arginine. J Biol Chem 279:36788–36794

    Article  PubMed  CAS  Google Scholar 

  • Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T (1994) Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol 127:1755–1766

    Article  PubMed  CAS  Google Scholar 

  • Kibler KV, Miyazato A, Yedavalli VS, Dayton AI, Jacobs BL, Dapolito G, Kim SJ, Jeang KT (2004) Polyarginine inhibits gp160 processing by furin and suppresses productive human immunodeficiency virus type 1 infection. J Biol Chem 279:49055–49063

    Article  PubMed  CAS  Google Scholar 

  • Lu H, Khurana S, Verma N, Manischewitz J, King L, Beigel JH, Golding H (2011) A rapid Flp-In system for expression of secreted H5N1 influenza hemagglutinin vaccine immunogen in mammalian cells. PLoS One 6:e17297

    Article  PubMed  CAS  Google Scholar 

  • Molloy SS, Bresnahan PA, Leppla SH, Klimpel KR, Thomas G (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen. J Biol Chem 267:16396–16402

    PubMed  CAS  Google Scholar 

  • Nelsen SM, Christian JL (2009) Site-specific cleavage of BMP4 by furin, PC6, and PC7. J Biol Chem 284:27157–27166

    Article  PubMed  CAS  Google Scholar 

  • Ornatowski W, Poschet JF, Perkett E, Taylor-Cousar JL, Deretic V (2007) Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A-induced cytotoxicity. J Clin Invest 117:3489–3497

    Article  PubMed  CAS  Google Scholar 

  • Peel SA, Hu ZM, Clokie CM (2003) In search of the ideal bone morphogenetic protein delivery system: in vitro studies on demineralized bone matrix, purified, and recombinant bone morphogenetic protein. J Craniofac Surg 14:284–291

    Article  PubMed  Google Scholar 

  • Reddy KR, Lilie H, Rudolph R, Lange C (2005) l-Arginine increases the solubility of unfolded species of hen egg white lysozyme. Protein Sci 14:929–935

    Article  Google Scholar 

  • Stieneke-Grober A, Vey M, Angliker H, Shaw E, Thomas G, Roberts C, Klenk HD, Garten W (1992) Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J 11:2407–2414

    PubMed  CAS  Google Scholar 

  • Urist MR (1965) Bone: formation by autoinduction. Science 150:893–899

    Article  PubMed  CAS  Google Scholar 

  • Wang EA, Rosen V, D’Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci U S A 87:2220–2224

    Article  PubMed  CAS  Google Scholar 

  • Wiberg FC, Rasmussen SK, Frandsen TP, Rasmussen LK, Tengbjerg K, Coljee VW, Sharon J, Yang CY, Bregenholt S, Nielsen LS (2006) Production of target-specific recombinant human polyclonal antibodies in mammalian cells. Biotechnol Bioeng 94:396–405

    Article  PubMed  CAS  Google Scholar 

  • Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S (1991) Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol 113:681–687

    Article  PubMed  CAS  Google Scholar 

  • Yano K, Hoshino M, Ohta Y, Manaka T, Naka Y, Imai Y, Sebald W, Takaoka K (2009) Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing. J Bone Miner Metab 27:355–363

    Article  PubMed  CAS  Google Scholar 

  • Zhou AJ, Clokie CM, Peel SA (2009) Enhancing production of recombinant BMP-2 in mammalian cell culture. FASEB J 23 (Meeting Abstracts): LB209

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Acknowledgment

This work was supported in part by Oral Surgery Foundation of Canada and McEwen Research Fund (#199-03-1570). A.J.Z. is a recipient of a Natural Sciences and Engineering Council Post-Graduate Scholarship (NSERC-PGS) scholarship. We are grateful to Dr. Michelle Visser for careful scrutiny of several drafts of this manuscript.

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Authors and Affiliations

  1. Oral & Maxillofacial Surgery, Faculty of Dentistry, University of Toronto, 124 Edward Street, Room 412, Toronto, Toronto, ON, M5G 1G6, Canada

    Aileen J. Zhou, Cameron M. L. Clokie & Sean A. F. Peel

  2. Induce Biologics Inc., Toronto, ON, Canada

    Sean A. F. Peel

Authors
  1. Aileen J. Zhou
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  2. Cameron M. L. Clokie
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  3. Sean A. F. Peel
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Corresponding author

Correspondence to Sean A. F. Peel.

Additional information

A patent application has been filed describing methods that enhance the production of recombinant proteins, which includes the methods described in this paper (“Methods for enhancing recombinant protein production” (PCT/CA2008/000998)). This pending patent application has been assigned to Induce Biologics Inc. SAFP and CMLC are shareholders in Induce Biologics Inc. SAFP is an employee of Induce Biologics Inc.

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Supplementary Fig. 1

Mature and proBMP-2 yields of CHO-BMP2 cells incubated with furininhibitor decanoyl-RVKR-chloromethylketone (CMK) for 48 h, measured by ELISA. Data are presented as mean ± SD (**P <0.01, ***P <0.001). (TIFF 11850 kb)

Supplementary Fig. 2

BMP-2 yields of CHO-BMP2 cells treated with IND-1 (80 μM) or D-arg (720μM, equivalent of 80 μM IND-1), measured by BMP-2 ELISA. Data are presented as mean ± SD(***P <0.001). (TIFF 8518 kb)

Supplementary Fig. 3

CHO-BMP2 cells were incubated with polyarginine peptides consisting of seven positively D-arginines (7R) with different net charges, created by inserting negatively charged aspartic acids (D) at the ends or throughout the peptide. The cells were treated with (A) 7R-201(COCH3-RRRRRRR-COOH, net charge +5), (B) 7R-202 (COCH3-DDRRRRRRRDD-COOH, +1),(C) 7R-203 (COCH3-DDDRRRRRRRDDD-COOH, -1), (D) 7R-303 (COCH3-RDRRDRRDRRDCOOH, +1) and (E) 7R-304 (COCH3-DRDRRDRRDRRDD-COOH, -1). After 48 h incubation, secreted BMP-2 in the condition media was measured by ELISA. Data are presented as mean ± SD (***P <0.001). (TIFF 20953 kb)

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Zhou, A.J., Clokie, C.M.L. & Peel, S.A.F. Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells. Biotechnol Lett 34, 221–230 (2012). https://doi.org/10.1007/s10529-011-0781-7

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  • DOI: https://doi.org/10.1007/s10529-011-0781-7

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