从外膜囊泡内蛋白质导演包装<em>大肠杆菌</em>：设计，生产和纯化
Summary
A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.
Abstract
在应用合成生物学技术，外膜囊泡（OMV）进行编程的兴趣日益增加正在导致对OMV在传统的纳米颗粒被证明太难合成了一些非常有趣和独特的应用。迄今为止，所有革兰氏阴性细菌已被证明产生的各种货物，包括小分子，肽，蛋白质和遗传物质的OMV示范包装。根据其不同的货物，OMV有牵连的许多生物过程，从细胞 – 细胞通信的基因转移和取决于所细菌生产的OMV毒力因子递送。直到最近，细菌的OMV成为在广泛的应用中使用通过的技术的发展，以控制和重组蛋白的直接包装成OMV访问。这个协议描述了生产，纯化的方法，以及利用酶包装OMV的提供改进overa所有的生产重组酶，增加灸，提高酶的稳定性。这个协议的成功利用将导致创建了同时产生的重组蛋白，并指示它对于OMV封装通过创造重组蛋白和外膜锚蛋白之间的合成键的细菌菌株的。该协议还详细介绍了从细菌培养以及正确的处理技术和东西隔离OMV适应这个协议使用了其他独特的应用程序，如时要考虑的方法：药物给药，医疗诊断和环境整治。
Introduction
这里介绍的是进行设计，生产和酶加载细菌外膜囊泡（OMV）的纯化方法。 OMV是小，主要是单层，脂蛋白，从30-200纳米1,2的尺寸范围。已研究了迄今为止所有革兰氏阴性和革兰氏阳性菌都从它们的表面3,4-表明要么OMV或胞外囊泡（EV）的释放。由OMV产生的确切机理还没有被完全阐明由于分泌它们以及它们所服务的不同功能的不同的细菌种群。 OMV已经显示小分子，肽和蛋白质运输各种各样的货物，以提供各种复杂的信号，基因易位遗传物质，和毒力功能5,6-。
OMV生物合成的确切机制尚不充分表征，并出现细菌种类之间不同。尽管THIs in issue争议，我们已经开发了通过创建目的蛋白质和高度丰富的蛋白内源性细菌外膜和随后的OMV之间的合成联动增强的重组蛋白的包装效率成OMV的方法。在不存在的合成联动，或人工引入的亲和力，所述重组表达蛋白和监查之间所观察到的包装效率很低7。这个结果是可以预料的OMV中的蛋白质通过在在细菌表面或通过定向包装OMV形成的精确时刻随机机会封装或者发生由机制未充分了解的掺入。一些成功，在这依赖于机缘凑巧封装，但有效的包装周质空间内过度表达只需通过包装蛋白被发现是高度比别人届蛋白依赖的一些蛋白质高效率包装在不包装在所有8-10。通过利用共同合成生物学技术我们试图工程师大肠杆菌 （ 大肠杆菌 ），以同时生产，包装和分泌感兴趣成OMV活性酶绕过关于OMV是如何形成的，以及如何货物由细菌对选定的当前知识限制打包。
对于本申请的目的，选择了分裂蛋白生物结合系统作为选择的合成联动，以促进定向包装入OMV。顾名思义分裂蛋白生物结合系统包括与彼此交互的两个互补亚基域。选择用于此协议的目的，分裂蛋白结构域称为抓间谍者（SC）和SpyTag（ST）的结构域，并且从化脓性链球菌纤连蛋白结合蛋白（FbaB）11而得。这种分裂蛋白系统是使W异常母鸡两个亚单位是接近度之内的异肽键的近端天冬氨酸和赖氨酸的氨基酸残基建立共价键之间自发地形成。异肽键的形成不需要另外伴侣蛋白，催化酶或辅因子，并且可以在室温（RT）和在宽范围的生理相关条件下12容易发生。
作为概念验证，选择从Brevundimonas微小假磷酸三酯酶（PTE）（EC 3.1.8.1）被打包成衍生OMV 13 大肠杆菌 。 PTE包含双核锌/锌活性部位，并具有通过水解反应转化aryldialkylphosphates成dialkylphosphates和芳基醇14分解有机磷酸酯的能力。接触有机磷酸酯通过神经肌肉接头处出不来乙酰胆碱酯酶抑制乙酰胆碱水解损害神经递质适当的功能摹有机磷衍生化合物极其危险的15。长期或显著暴露于有机磷通常会导致不可控制的抽搐，通常通过窒息引起死亡。而PTE呈现朝向对氧磷的最高的催化活性，一种非常有效的杀虫剂，也是能够水解各种其它农药和V / G型化学神经剂16的。为了便于OMV的包装，一个细菌质粒被设计编码包含诱导型启动子，周质定位序列，和一个短的多克隆位点的SC基因序列上游的基因构建体。领导者和SC序列允许建立一个基因开关为目标的PTE-SC融合蛋白为OMV包装周质空间之间的PTE基因的插入。而这里描述的努力集中在PTE中，酶的基因是可互换的，并且可以很容易地与其他基因序列置换到FAC备用酶或蛋白质的ilitate包装。
作为合成连杆的第二部分，一种丰富的外膜蛋白（OmpA的）被选择为呈现ST肽序列。而锚蛋白可以改变的选择，这是至关重要的，该蛋白具有在周质空间内呈现出宽松的域，耐受融合构建而不诱导细胞毒作用，是已知的存在于监查，并且不聚集时，它是重组产生的。的OmpA是已知在细菌外膜和随后的OMV 17被高度表达的37.2 kDa的跨膜孔蛋白的蛋白质。它是在小分子的运输牵连，<2纳米的尺寸，穿过细菌膜18。天然的OmpA有两个结构独特结构域，跨膜β桶基序和已知与肽聚糖19相互作用的periplasmically可溶性C末端部分。在突变的OmpA-ST融合designeð这里的OmpA的C-末端部分被删除和ST被融合到periplasmically面对N-或C-末端。删除的OmpA的周质部分减小外膜，并导致膜去稳定化，导致超囊泡7肽聚糖之间相互作用的次数。基因组的OmpA保持除重组表达OmpA的-ST的构建，以减轻毛膜不稳定。
Protocol
Representative Results
Discussion
这个协议功能表现出代表涉及在其中感兴趣的酶产生和由大肠杆菌包装到OMV封装技术。与许多复杂的技术有多个区域，其中，协议可以被修改以适应不同的独特的应用，其中一些在下面详述使用。而OMV包装和酶包封的机构可以适于特定需要有该协议内的几个步骤，是成功的关键。最初的去除细菌的完整和细胞碎片的是显著的重要性，并会影响在定量或样品分析的所有下游的尝试。接着过滤?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was funded by the Office of Naval Research through Core funds provided to the Naval Research Laboratory.
Materials
| IPTG | Any | Always prepare fresh or aliquot and freeze. | |
| L-arabinose | Any | Can be prepared ahead of time and stored at 4C. | |
| Ampicillin | Any | Add immediately prior to use after media cools sufficiently from being autoclaved. | |
| Chloramphenicol | Any | Add immediately prior to use after media cools sufficiently from being autoclaved. | |
| TB/LB Culture Media | Any | Other growth medias will likely work similarly. | |
| Triton X-100 | Any | One of many potential suitable surfactants. | |
| Baffled culture flasks | Any | The baffles promote higher levels of aeration. | |
| CHES | Fisher Bioreagents | BP318-100 | Optimal buffer used for paraoxon degredation (pH > 8). |
| Paraoxon | Chem Service | N-12816 | Very toxic substance to be handled carefully and disposed of properly. |
| Syringe Filter 0.45 µm | Thermo Scientific | 60183-221 (30 mm) | Filter diameter will depend on volume of sample. Low protein binding membrane is critical. |
| Shaker incubator | New Brunswick | Excella E24 | Precise temperature and mixing is essential for reproducable bacterial growth. |
| Sorvall Culture Centrifuge | Thermo Scientific | RC 5B PLUS | Large volume (500 mL) culture centrifuge capable of 7,000 x g. |
| Sorvall Ultracentrifuge | Thermo Scientific | WX Ultra 90 | Capable of centrifugal forces ≥150,000 x g. |
| Ultracentrifuge Rotor | Thermo Scientific | AH-629 | Ensure the proper rotor and tubes are used and that everything is properly balanced. |
| Ultra-Clear Ultracentrifuge Tubes (25 x 89 mm) | Beckman Coulter | 344058 | Ensure no stress fractures are present prior to use and that tubes are presicely balanced. |
| Spectrophotometer | Tecan | Infinite M1000 | Necessary for enzyme kinetic assays. |
| DLS / particle tracking | NanoSight | LM10 | Necessary for OMV size distribution and concentration determination. |
| BL21(DE3) | NEB | Suitable bacterial expression strain. | |
| pET22 | EMD Millipore | 69744-3 | Other plasmids can be used in place of these. |
| pACYC184 | NEB | Other plasmids can be used in place of these. | |
| Gel Extraction Kit | Qiagen | 28704 | Example kit. |
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