Protein-liposome conjugates using cysteine-lipids and native chemical ligation
Protein-liposome conjugates using cysteine-lipids and native chemical ligation
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DOI：
10.1021/bc0602782
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发表时间：
2007-03-01
影响因子：
4.7
通讯作者：
Merkx, Maarten
中科院分区：
文献类型：
--
作者：
Reulen, Sanne W. A.;Brusselaars, Wilco W. T.;Merkx, Maarten
脂质体已成为流行的药物递送载体，并且最近还被用作分子成像的造影剂。目前大多数用靶向蛋白质功能化脂质体的方法依赖于蛋白质外部的胺或硫醇基团的反应，这通常导致蛋白质上多个位点的非特异性缀合。在这项研究中，我们提出天然化学连接（NCL）作为一种通用方法，以高度特异性和化学选择性的方式将重组蛋白共价偶联到含有半胱氨酸功能化磷脂的脂质体上。制备了半胱氨酸功能化磷脂 (Cys-PEG-DSPE)，并显示其易于与用作模型蛋白的 EYFP 的 MESNA 硫酯发生反应。使用荧光光谱对 EYFP-脂质体进行表征表明，结合的 EYFP 完全保留了荧光特性，并提供了每个脂质体 120 个蛋白质的下限。 NCL 的一般适用性使用 CNA35 进行了进一步测试，CNA35 是一种最近应用于胶原蛋白荧光成像的胶原蛋白结合蛋白。 CNA35硫酯的NCL产生每个脂质体含有大约100个CNA35拷贝的脂质体。 CNA35 脂质体功能齐全，与胶原蛋白的结合亲和力比 CNA35 高 150 倍。我们的结果表明，NCL 是对现有缀合方法的一个有吸引力的补充，它允许重组蛋白与脂质体和其他基于脂质的组装体直接、共价和高度特异性地偶联。
Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing similar to 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.