Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture.
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.
Ijiri
Hiroshi
H
Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.
Coulibaly
Fasséli
F
Nishimura
Gento
G
Nakai
Daisuke
D
Chiu
Elaine
E
Takenaka
Chiemi
C
Ikeda
Keiko
K
Nakazawa
Hiroshi
H
Hamada
Norio
N
Kotani
Eiji
E
Metcalf
Peter
P
Kawamata
Shin
S
Mori
Hajime
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D016207:Cytokines / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D051523:Fibroblast Growth Factor 7 / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D017433:Protein Structure, Secondary; D012087:Reoviridae / Q000737:chemistry*; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism
D016207:Cytokines; D055767:Immobilized Proteins; D008164:Luminescent Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; C413663:fluorescent protein 583; D016222:Fibroblast Growth Factor 2; D051523:Fibroblast Growth Factor 7; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor