Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation
Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation
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DOI：
10.1016/j.ymthe.2005.09.016
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发表时间：
2006-04-01
影响因子：
12.4
通讯作者：
Darling, D
中科院分区：
文献类型：
--
作者：
Nesbeth, D;Williams, SL;Darling, D
慢病毒载体表面上的非病毒、宿主来源的蛋白质可以对载体的生物学产生深远的影响，因为它们既可以促进感染，又可以提供对补体失活的抵抗力。我们利用这一点来设计“非包膜”病毒相关蛋白的特定翻译后修饰。细菌生物素连接酶 (BirA) 和改良的人 Delta LNGFR 已被引入 HEK293T 细胞，其蛋白质产物定向至内质网腔。然后，BirA 将生物素与已与 Delta LNGFR 融合的受体肽偶联。这导致生物素与细胞表面表达的 Delta LNGFR 的胞外结构域共价连接。来自这些细胞的慢病毒载体在游离生物素存在的情况下用生物素进行代谢标记。这些生物素化的慢病毒载体对链霉亲和素顺磁颗粒具有高亲和力，一旦捕获，很容易在体外操作。用 VSV-G 或双嗜性包膜假型化的慢病毒载体的浓度超过 4500 倍就说明了这一点。这种新的细胞系具有广泛应用于与慢病毒载体生产兼容的包膜假型的潜力。
Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human Delta LNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the Delta LNGFR. This results in the covalent linkage of biotin to the extracellular domain of the Delta LNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.