Magneto/optical annexin V, a multimodal protein
Magneto/optical annexin V, a multimodal protein
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DOI：
10.1021/bc049905i
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发表时间：
2004-09-01
影响因子：
4.7
通讯作者：
Josephson, L
中科院分区：
文献类型：
--
作者：
Schellenberger, EA;Sosnovik, D;Josephson, L
多模式蛋白质或用荧光和磁性报告基团标记的蛋白质可用于广泛的应用，包括FACS或荧光显微镜、MRI和/或基于近红外的光学成像，或通过磁性细胞分选来破碎细胞。然而，多模态蛋白质的问题是需要最大化生物活性，这通常通过最小化蛋白质的修饰点的数量来实现，同时连接荧光和磁性标记。在这里，我们描述了磁/光学形式的膜联蛋白V的合成，通过使氨基-CLIO纳米颗粒与Cy5.5和SPDP反应来实现，以产生荧光的巯基反应性纳米颗粒。通过与SATA反应，将单个反应性巯基添加到膜联蛋白V中，这保留了蛋白质结合凋亡Jurkat T细胞的能力。将SATA化的膜联蛋白V与SPDP活化的纳米颗粒反应产生Anx-CLIO-Cy5.5，其是膜联蛋白V的磁/光形式。Anx-CLIO-Cy5.5的结合对凋亡的Jurkat T细胞是特异性的，并且具有3.66 nM的EC 50。这与未修饰的膜联蛋白V与凋亡细胞的相互作用的强度相当，测量为膜联蛋白V（2.4 nM）对FITC-膜联蛋白的置换。我们的缀合策略保留了膜联蛋白V和凋亡细胞之间的相互作用的强度，同时产生探针Anx-CLIO-Cy5.5，其易于通过标准MR成像或NIRF光学方法检测。
Multimodal proteins, or proteins labeled with both fluorescent and magnetic reporter groups, can be used in a wide range of applications including FACS or fluorescence microscopy, MRI and or near-infrared based optical imaging, or to fractionate cells by magnetic cell sorting. A problem with multimodal proteins, however, is the need to maximize bioactivity, often achieved by minimizing the number of modification points of the protein, while attaching fluorescent and magnetic labels. Here we describe the synthesis of a magneto/optical form of annexin V, achieved by reacting the amino-CLIO nanoparticle with Cy5.5 and SPDP, to produce a fluorescent, sulfhydryl reactive nanoparticle. A single reactive sulfhydryl group was added to annexin V by reaction with SATA that preserved the protein's ability to bind apoptotic Jurkat T cells. Reacting SATAylated annexin V with an SPDP activated nanoparticle yielded Anx-CLIO-Cy5.5, a magneto/optical form of annexin V. The binding of Anx-CLIO-Cy5.5 was specific for apoptotic Jurkat T cells and had an EC50 of 3.66 nM. This was comparable to the strength of the interaction of unmodified annexin V with apoptotic cells, measured as the displacement of FITC-annexin by annexin V (2.4 nM). Our conjugation strategy preserves the strength of the interaction between annexin V and apoptotic cells, while yielding a probe, Anx-CLIO-Cy5.5, that is readily detectable by standard MR imaging or NIRF optical methods.