Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture
Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture
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DOI：
10.1016/j.biomaterials.2009.04.046
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发表时间：
2009-09-01
期刊：
影响因子：
14
通讯作者：
Mori, Hajime
中科院分区：
文献类型：
--
作者：
Ijiri, Hiroshi;Coulibaly, Fasseli;Mori, Hajime
某些昆虫病毒产生稳定的感染性微晶体，称为多角体，其功能是在受感染的幼虫死亡后保护病毒。多角体在受感染的细胞内形成，并且包含嵌入病毒蛋白多角体蛋白的晶格中的许多病毒颗粒。我们以前已经证明，N-末端75个氨基酸的Bombx毛利cypovirus（BmCPV）转塔蛋白（VP 3）可以作为一个多角体蛋白识别信号，导致外来蛋白掺入多角体。用VP 3多角体蛋白识别信号标记的外源蛋白通过与昆虫细胞中的多角体蛋白共表达而并入多角体中。我们已经用这种方法将各种各样的外源蛋白包封到多面体中。BmCPV多角体蛋白的原子结构表明，多角体蛋白的N-末端H1 α-螺旋在交联和稳定多角体中起着重要作用。在这里，我们表明，多角体蛋白H I-螺旋也可以作为多角体蛋白识别信号，并可以像VP 3 N-末端序列靶向到多角体的外源蛋白。此外，这两种靶向方法可以一起使用，以产生含有EGFP和Discosoma sp.红色荧光蛋白（DsRed）的多面体。使用双波长共聚焦显微镜对修饰的多面体进行成像，显示两种外源蛋白以相似的水平均匀地掺入多面体中。我们研究了固定在带有HI或VP 3标签的多角体上的成纤维细胞生长因子-2（FGF-2）、FGF-7和表皮生长因子（EGF）的生物学和生理学特性。这两种方法产生的生长因子是功能性的，诱导成纤维细胞和角质形成细胞的生长。结果证明了改性多面体用于包封和稳定生物活性蛋白质的实用性和灵活性。(c)2009爱思唯尔有限公司保留所有权利。
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 Maori 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 H I-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 HI 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. (c) 2009 Elsevier Ltd. All rights reserved.