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A cell-free approach to virus-like particle production and artificial post-translational modification.

机译：一种无细胞方法，可生产病毒样颗粒和人工翻译后修饰。

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摘要

Virus-like Particles (VLPs) are produced from the structural proteins of viruses and typically mimic the structure of the virus capsid but do not contain the genome. VLPs have become increasingly attractive for vaccine applications as (1) there is no chance of infection from the vaccine due to the lack of a genome in VLPs, (2) a small dose results in lasting memory immunity, and (3) immunity is rapidly gained due to the highly symmetric VLPs which stimulate the rapid thymus independent B-cell response. In addition, VLPs are also under investigation for drug delivery applications as VLPs are patterned after the virus capsid which has evolved to protect its cargo against extreme extracellular conditions but also deliver it to infect a cell. Also, VLPs can be used as a nano-building block to construct nanoscale devices and materials.;Currently a variety of production technologies are used to produce VLPS. However, each technology has its own limitations, such as costs, production yields, and scalability. In an attempt to create a better VLP production technology, an E. coli-based cell-free protein synthesis (CFPS) system was used. The E. coli-based CFPS has many of the advantages of E. coli-based protein production with which include high protein yields, rapid protein production, and relatively low cost. In addition, the open CFPS environment enables the direct manipulation of, and substrate addition to, the translation environment. This provides the potential to overcome some of the limitations of an E. coli production system such as an inability to perform post-translational modification, low solubility of some mammalian proteins, and an inability to produce some mammalian virus-based VLPs.;For the first time to our knowledge we tested an E. coli-based CFPS system's potential to produce VLPs. The MS2 coat protein VLP and a human Hepatitis B core protein VLP were produced at the highest reported volumetric yields to date and with high assembly efficiencies. Disulfide bond containing VLPs were also produced at high assembly efficiencies with our CFPS system. Over 90% of the disulfide bonds in the human Hepatitis B core protein VLP and the Qbeta bacteriophage VLP were formed by either manipulating the cell-free environment redox potential or by a post-CFPS oxidation incubation. Furthermore, the ability to encapsidate cargo into the MS2 coat protein VLP was demonstrated by simply adding in the cargo to the VLP production environment. The production of complex mammalian virus-based VLPs (such as the Dengue virus VLP which has never been successfully produced in previously in any system) was also tested with limited success. However, continuing work to modify the cell-free environment to more closely mimic eukaryotic systems may enable the production of such VLPS.;In an effort to compensate for this limitation as well as extend VLP applications, the open CFPS environment was modified for the site-specific incorporation of biochemically unique unnatural amino acids. A less soluble alkyne terminal unnatural amino acid p-propargyloxyphenylalanine was incorporated into CFPS produced proteins at yields 2 orders of magnitude higher than with the in vivo system. By incorporating either an alkyne and azido terminal unnatural amino acid using CFPS synthesis, we have demonstrated for the first time a single-step, linker-less, site-specific protein-to-protein covalent conjugation using a copper(I)-ctalyzed azide-alkyne [3+2] cycloaddition. This technology has been extended to VLP applications with high attachment efficiencies of PEG to unnatural amino acid containing VLPs were realized with good VLP recoveries. This technology was also used to control the attachment orientation of a superfolder green fluorescent protein that was attached site-specifically to an MS2 VLP. With the ability to control the decoration of VLPs with proteins, a vaccine against any protein or molecule and thus any disease could potentially be produced. This technology would also be valuable for drug delivery and nanostructure applications as well.
机译：病毒样颗粒（VLP）由病毒的结构蛋白产生，通常模仿病毒衣壳的结构，但不包含基因组。 VLP对于疫苗的应用变得越来越有吸引力，因为（1）由于VLP中缺少基因组，疫苗没有被感染的机会；（2）小剂量可导致持久的记忆免疫力；（3）免疫力迅速由于高度对称的VLP刺激了快速的胸腺非依赖性B细胞反应而获得的VLP。此外，VLP也正在研究用于药物递送的应用，因为VLP是在病毒衣壳之后形成图案的，该衣壳已经进化为保护其货物免受极端细胞外条件的侵害，但也可以将其递送以感染细胞。而且，VLP可用作纳米构造块来构造纳米级设备和材料。；目前，各种生产技术已用于生产VLPS。但是，每种技术都有其自身的局限性，例如成本，产量和可伸缩性。为了创建更好的VLP生产技术，使用了基于大肠杆菌的无细胞蛋白质合成（CFPS）系统。基于大肠杆菌的CFPS具有基于大肠杆菌的蛋白质生产的许多优点，其中包括高蛋白产量，快速蛋白质生产和相对较低的成本。另外，开放式CFPS环境可以直接操纵平移环境，并为平移环境添加底物。这为克服大肠杆菌生产系统的某些局限性提供了潜力，例如无法执行翻译后修饰，某些哺乳动物蛋白的溶解度低以及无法生产某些基于哺乳动物病毒的VLP。据我们所知，我们第一次测试了基于大肠杆菌的CFPS系统生产VLP的潜力。 MS2外壳蛋白VLP和人乙型肝炎核心蛋白VLP以迄今为止报道的最高体积产量和高组装效率生产。我们的CFPS系统还以高组装效率生产了含二硫键的VLP。人类乙型肝炎核心蛋白VLP和Qbeta噬菌体VLP中超过90％的二硫键是通过操纵无细胞环境的氧化还原电位或通过CFPS后氧化孵育而形成的。此外，通过简单地将货物添加到VLP生产环境中就证明了将货物封装到MS2外壳蛋白VLP中的能力。还测试了基于复杂哺乳动物病毒的VLP（例如以前从未在任何系统中成功生产过的登革热病毒VLP）的生产，但效果有限。但是，继续进行工作以将无细胞环境修改为更接近真核生物的系统，可能会产生此类VLPS。为了弥补这一局限性并扩展VLP应用，已针对现场修改了开放CFPS环境生化独特的非天然氨基酸的特异性结合。将溶解度较低的炔烃末端非天然氨基酸对-炔丙基氧基苯基丙氨酸掺入CFPS生产的蛋白质中，其产率比体内系统高2个数量级。通过使用CFPS合成掺入炔烃和叠氮基末端非天然氨基酸，我们首次证明了使用铜（I）催化的叠氮化物的单步，无接头，位点特异性的蛋白质-蛋白质共价结合-炔[3 + 2]环加成。这项技术已扩展到VLP应用，通过高VLP回收率实现了PEG与非天然氨基酸VLP的高附着效率。该技术还用于控制超级文件夹绿色荧光蛋白的附着方向，该蛋白特定于位点附着在MS2 VLP上。由于具有用蛋白质控制VLP修饰的能力，可以针对任何蛋白质或分子以及因此产生任何疾病的疫苗。该技术对于药物递送和纳米结构应用也将是有价值的。

著录项

  • 作者

    Bundy, Bradley Charles.;

  • 作者单位

    Stanford University.;

  • 授予单位 Stanford University.;
  • 学科 Engineering Biomedical.;Engineering Chemical.;Biology Virology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 197 p.
  • 总页数 197
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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