Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag
Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag
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DOI：
10.1016/j.biomaterials.2006.06.025
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发表时间：
2006-11-01
期刊：
影响因子：
14
通讯作者：
Backer, Joseph M.
中科院分区：
文献类型：
--
作者：
Backer, Marina V.;Patel, Vimal;Backer, Joseph M.
开发具有固定化生长因子的组织工程支架需要用于共价连接功能活性蛋白质的简便且可靠的方法。在这里，我们描述了一种新的方法来表达重组蛋白的基础上表达的目的蛋白与15个氨基酸长的融合标签（Cys标签），它利用一个自由的巯基位点特异性的共轭。为了验证这种方法，我们使用常见的巯基定向双功能交联剂将用N末端Cys标签表达的单链血管内皮生长因子（scVEGF）与纤连蛋白（FN）偶联。我们发现，当用作可溶性配体时，FN-scVEGF缀合物保留与游离scVEGF类似的VEGF活性。在FN-scVEGF包被的平板上生长的表达VEGF受体VEGFR-2的细胞显示出与在等量游离scVEGF存在下在FN上生长的细胞所观察到的形态表型相似的形态表型。此外，在相同浓度范围内，无论是固定的还是游离的scVEGF，都观察到293/KDR细胞生长刺激。VEGF受体酪氨酸激酶抑制剂NVP-AAD 777-NX可阻断固定化scVEGF和可溶性scVEGF的作用。这些数据表明，通过Cys-标签的位点特异性固定化提供了一种简便可靠的方法，用于将功能活性生长因子永久沉积在合成或蛋白质支架上，用于先进的组织工程。(c)2006爱思唯尔有限公司版权所有。
Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering. (c) 2006 Elsevier Ltd. All rights reserved.