Structure heterogeneity and host nucleic acids contamination are two major problems for virus-like particles (VLPs) produced by various host cells. In this study, an in vitro optimized disassembly-purification-reassembly process was developed to obtain uniform and nucleic acid free hepatitis B core (HBc) based VLPs from E. coli fermentation. The process started with ammonium sulfate precipitation of all heterogeneous HBc structures after cell disintegration. Then, dissolution and disassembly of pellets into basic subunits were carried out under the optimized disassembly condition. All contaminants, including host nucleic acids and proteins, were efficiently removed with affinity chromatography. The purified subunits reassembled into VLPs by final removal of the chaotropic agent. Two uniform and nucleic acid free HBc-based VLPs, truncated HBc₁₄₉ and chimeric HBc₁₈₃-MAGE3 I, were successfully prepared. It was found that disassembly degree of HBc-based VLPs had a great influence on the protein yield, nucleic acid removal and reassembly efficiency. 4 M urea was optimal because lower concentration would not disassemble the particles completely while higher concentration would further denature the subunits into disordered aggregate and could not be purified and reassembled efficiently. For removal of strong binding nucleic acids such as in the case of HBc₁₈₃-MAGE3 I, benzonase nuclease was added to the disassembly buffer before affinity purification. Through the optimized downstream process, uniform and nucleic acid free HBc₁₄₉ VLPs and HBc₁₈₃-MAGE3 I VLPs were obtained with purities above 90% and yields of 55.2 and 43.0 mg/L, respectively. This study would be a reference for efficient preparation of other VLPs.

结构异质性和宿主核酸污染是各种宿主细胞产生的病毒样颗粒（VLP）的两个主要问题。在这项研究中，开发了体外优化的拆卸-纯化-重组方法，以从大肠杆菌中获得基于统一且无核酸的乙肝核心（HBc）VLP 。发酵。该过程以细胞分解后所有异质HBc结构的硫酸铵沉淀开始。然后，在优化的分解条件下，将颗粒溶解并分解成基本亚基。亲和色谱可有效去除所有污染物，包括宿主核酸和蛋白质。通过最终除去离液剂将纯化的亚基重新组装成VLP。两个均一且无核酸的HBc型VLP，截短的HBc ₁₄₉和嵌合的HBc ₁₈₃-MAGE3 I，已成功准备。发现基于HBc的VLP的分解程度对蛋白质产量，核酸去除和重组效率有很大影响。4 M尿素是最佳的，因为较低的浓度不会完全分解颗粒，而较高的浓度会进一步使亚基变性为无序聚集体，并且无法有效纯化和重新组装。为了去除强结合核酸，例如在HBc ₁₈₃ -MAGE3 I的情况下，在亲和纯化之前将苯甲酸酶核酸酶添加到拆卸缓冲液中。通过优化的下游过程，获得统一且无核酸的HBc ₁₄₉ VLP和HBc ₁₈₃获得的-MAGE3 I VLP的纯度高于90％，产率分别为55.2和43.0 mg / L。该研究将为有效制备其他VLP提供参考。