Centre for Molecular Nanoscience, School of Chemistry, University of Leeds, Leeds LS2 9JT, UK.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Jul 15;878(22):1939-45. doi: 10.1016/j.jchromb.2010.05.019. Epub 2010 May 24.
Desthiobiotin-tagged lentiviral vectors have been metabolically produced by DBL producer cells in a 7,8-diaminopelargonic acid (7-DAPA) dependent manner for envelope independent, single-step affinity purification. 7-DAPA, which has little or no affinity for avidin/streptavidin, was synthesised and verified by NMR spectroscopy and mass spectrometry. By expressing the biotin acceptor, biotin ligase and desthiobiotin synthase bioD, DBL cells converted exogenous 7-DAPA into membrane-bound desthiobiotin. Desthiobiotin on the DBL cell surface was visualised by confocal microscopy and the desthiobiotin density was quantified by HABA-avidin assay. Desthiobiotin was then spontaneously incorporated onto the surface of lentiviral vectors produced by the DBL cells. It has been demonstrated by flow cytometry that the desthiobiotinylated lentiviruses were captured from the crude 7-DAPA-containing viral supernatant by Streptavidin Magnespheres and eluted by biotin solution efficiently whilst retaining infectivity. The practical, high yielding virus purification using Pierce monomeric avidin coated columns indicates a highly efficient biotin-dependent recovery of infectious lentiviruses at 68%. The recovered lentiviral vectors had a high purity and the majority were eluted within 45 min. This 7-DAPA mediated desthiobiotinylation technology can be applied in scalable production of viral vectors for clinical gene therapy.
带有去硫生物素标签的慢病毒载体通过 7,8-二氨基辛酸（7-DAPA）依赖性代谢方式由 DBL 生产细胞产生，用于非包膜依赖的、一步单链亲和纯化。7-DAPA 对亲和素/链霉亲和素有很小或没有亲和力，通过 NMR 光谱和质谱学进行了合成和验证。通过表达生物素接受体、生物素连接酶和去硫生物素合酶 bioD，DBL 细胞将外源性 7-DAPA 转化为膜结合的去硫生物素。通过共聚焦显微镜观察到 DBL 细胞表面上的去硫生物素，并通过 HABA-亲和素测定法定量去硫生物素密度。然后，去硫生物素自发地掺入由 DBL 细胞产生的慢病毒载体表面。通过流式细胞术证明，通过 Streptavidin Magnespheres 从含有粗 7-DAPA 的病毒上清液中捕获了去硫生物素化的慢病毒，并通过生物素溶液有效洗脱，同时保持感染性。使用 Pierce 单体亲和素涂覆柱的实用、高产病毒纯化表明，在 68%的情况下，生物素依赖性回收感染性慢病毒的效率非常高。回收的慢病毒载体具有高纯度，大多数在 45 分钟内洗脱。这种 7-DAPA 介导的去硫生物素化技术可应用于临床基因治疗中病毒载体的规模化生产。
