Purification of HIV-1 gag virus-like particles and separation of other extracellular particles
Purification of HIV-1 gag virus-like particles and separation of other extracellular particles
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DOI：
10.1016/j.chroma.2016.05.053
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发表时间：
2016-07-15
影响因子：
4.1
通讯作者：
Jungbauer, Alois
中科院分区：
文献类型：
--
作者：
Steppert, Petra;Burgstaller, Daniel;Jungbauer, Alois
包膜病毒样颗粒（VLPs）越来越多地被用作疫苗和免疫疗法。通常，非常耗时的密度梯度离心技术用于纯化VLPs。然而，优化大规模VLP生产的进展增加了对快速，成本效益和可扩展的净化工艺的需求。我们开发了一种纯化CHO细胞中产生的HIV-1 gag VLPs的色谱方法。澄清和过滤后的细胞培养上清直接在阴离子交换单体上处理。大多数宿主细胞杂质通过柱，而VLPs被线性或阶梯盐梯度洗脱；DNA的主要部分在VLPs之前被洗脱，直径在100-200 nm范围内的颗粒可以被分离成两个部分。较早洗脱的部分富含与外泌体或微泡相关的细胞外颗粒，而较晚洗脱的部分含有大多数最纯的HIV-1 gag VLPs。苯并酶处理后，含外泌体部分的DNA含量不能降低，表明DNA被包裹。通过蛋白质组学分析，鉴定出许多外泌体标记。我们提出了一种实验室方法，可以作为快速下游加工包膜VLPs的基础。最多2000剂，每剂含有1 × 10(9)个颗粒，可在47分钟内用1 mL的整体石处理。该方法与密度梯度离心相比，生产率提高了220倍。(C) 2016作者。Elsevier B.V.出版
Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200 nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1 x 10(9) particles, could be processed with a 1 mL monolith within 47 min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity. (C) 2016 The Author(s). Published by Elsevier B.V.