Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3
Engineering of SV40-based nano-capsules for delivery of heterologous proteins as fusions with the minor capsid proteins VP2/3
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DOI：
10.1016/j.jbiotec.2007.12.006
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发表时间：
2008-03-20
影响因子：
4.1
通讯作者：
Handa, Hiroshi
中科院分区：
文献类型：
--
作者：
Inoue, Takarnasa;Kawano, Masa-Aki;Handa, Hiroshi
SV40的衣壳被认为是用于递送生物活性物质的潜在纳米胶囊。 SV40衣壳由VP1主要衣壳蛋白的72个五聚体和次要外壳蛋白VP2/3的72个拷贝组成。我们之前已经证明，当通过杆状病毒系统在昆虫Sf9细胞中表达时，VP1自组装成病毒样颗粒（VP1-VLP），其在形态上与SV40病毒颗粒没有区别，并且可以很容易地纯化。在这里，我们证明与 VP2/3 融合的异源蛋白可以有效地整合到 VP1-VLP 中。使用EGFP作为模型蛋白，我们优化了该封装系统，发现与VP2/3的C端融合是优选的，并且VP2/3的C端VP1相互作用结构域足以掺入VLP中。封装 EGFP 的 VLP 保留了附着在细胞表面并进入细胞的能力。使用该系统，我们将酵母胞嘧啶脱氨酶 (yCD)（一种将 5-氟胞嘧啶转化为 5-氟尿嘧啶的前药修饰酶）封装到 VLP 中。当 CV-1 细胞受到包裹 yCD 的 VLP 攻击时，它们对 5-氟胞嘧啶诱导的细胞死亡变得敏感。因此，目标蛋白可以通过与 VP2/3 融合而封装在 VP1-VLP 中，并成功递送至细胞。 (C) 2007 Elsevier B.V. 保留所有权利。
The capsid of SV40 is regarded as a potential nano-capsule for delivery of biologically active materials. The SV40 capsid is composed of 72 pentamers of the VP1 major capsid protein and 72 copies of the minor coat proteins VP2/3. We have previously demonstrated that, when expressed in insect Sf9 cells by the baculovirus system, VP1 self-assembles into virus-like particles (VP1-VLPs), which are morphologically indistinguishable from the SV40 virion and can be easily purified. Here, we show that heterologous proteins fused to VP2/3 can be efficiently incorporated into the VP1-VLPs. Using EGFP as a model protein, we have optimized this encapsulation system and found that fusion to the C-terminus of VP2/3 is preferable and that the C-terminal VP1-interaction domain of VP2/3 is sufficient for incorporation into VLPs. The VLPs encapsulating EGFP retain the ability to attach to the cell surface and enter the cells. Using this system, we have encapsulated yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that converts 5-fluorocytosine to 5-fluorouracil, into VLPs. When CV-1 cells are challenged by the yCD-encapsulating VLPs, they become sensitive to 5-fluorocytosine-induced cell death. Therefore, proteins of interest can be encapsulated in VP1-VLPs by fusion to VP2/3 and successfully delivered to cells. (C) 2007 Elsevier B.V. All rights reserved.