Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture

Hiroshi Ijiri, Fasseli Joseph Coulibaly, Gento Nishimura, Daisuke Nakai, Elaine Chiu, Chiemi Takenaka, Keiko D Ikeda, Hiroshi Nakazawa, Norio Hamada, Eiji Kotani, Peter Metcalf, Shin Kawamata, Hajime Mori

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.
Original languageEnglish
Pages (from-to)4297 - 4308
Number of pages12
JournalBiomaterials
Volume30
Issue number26
DOIs
Publication statusPublished - 2009

Cite this

Ijiri, Hiroshi ; Coulibaly, Fasseli Joseph ; Nishimura, Gento ; Nakai, Daisuke ; Chiu, Elaine ; Takenaka, Chiemi ; Ikeda, Keiko D ; Nakazawa, Hiroshi ; Hamada, Norio ; Kotani, Eiji ; Metcalf, Peter ; Kawamata, Shin ; Mori, Hajime. / Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture. In: Biomaterials. 2009 ; Vol. 30, No. 26. pp. 4297 - 4308.
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title = "Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture",
abstract = "Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.",
author = "Hiroshi Ijiri and Coulibaly, {Fasseli Joseph} and Gento Nishimura and Daisuke Nakai and Elaine Chiu and Chiemi Takenaka and Ikeda, {Keiko D} and Hiroshi Nakazawa and Norio Hamada and Eiji Kotani and Peter Metcalf and Shin Kawamata and Hajime Mori",
year = "2009",
doi = "10.1016/j.biomaterials.2009.04.046",
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Ijiri, H, Coulibaly, FJ, Nishimura, G, Nakai, D, Chiu, E, Takenaka, C, Ikeda, KD, Nakazawa, H, Hamada, N, Kotani, E, Metcalf, P, Kawamata, S & Mori, H 2009, 'Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture' Biomaterials, vol. 30, no. 26, pp. 4297 - 4308. https://doi.org/10.1016/j.biomaterials.2009.04.046

Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture. / Ijiri, Hiroshi; Coulibaly, Fasseli Joseph; Nishimura, Gento; Nakai, Daisuke; Chiu, Elaine; Takenaka, Chiemi; Ikeda, Keiko D; Nakazawa, Hiroshi; Hamada, Norio; Kotani, Eiji; Metcalf, Peter; Kawamata, Shin; Mori, Hajime.

In: Biomaterials, Vol. 30, No. 26, 2009, p. 4297 - 4308.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture

AU - Ijiri, Hiroshi

AU - Coulibaly, Fasseli Joseph

AU - Nishimura, Gento

AU - Nakai, Daisuke

AU - Chiu, Elaine

AU - Takenaka, Chiemi

AU - Ikeda, Keiko D

AU - Nakazawa, Hiroshi

AU - Hamada, Norio

AU - Kotani, Eiji

AU - Metcalf, Peter

AU - Kawamata, Shin

AU - Mori, Hajime

PY - 2009

Y1 - 2009

N2 - Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.

AB - Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.

UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19477509

U2 - 10.1016/j.biomaterials.2009.04.046

DO - 10.1016/j.biomaterials.2009.04.046

M3 - Article

VL - 30

SP - 4297

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JO - Biomaterials

JF - Biomaterials

SN - 0142-9612

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ER -