Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子
Summary
在这里, 我们提出一个程序, 荧光 functionalize 二硫化物 Qβ vip 与 dibromomaleimide。我们描述了 Qβ的表达和纯化, dibromomaleimide 功能分子的合成, dibromomaleimide 和 Qβ的共轭反应。生成的黄色荧光共轭粒子可作为细胞内的荧光探针。
Abstract
最近在生物医学和材料研究中出现的病毒样粒子 (VLPs) 的增加, 可以归因于它们的生物合成、离散大小、遗传可编程性和生物降解能力的简化。虽然他们很容易 bioconjugation 反应, 添加合成配体在其表面上, bioconjugation 方法的范围, 这些水中出生的衣壳是相对有限的。为了促进功能性生物材料研究的方向, 必须考虑非传统的 bioconjugation 反应。本协议中描述的反应使用 dibromomaleimides 在噬菌体 Qβ的基础上引入了一个 vip 的溶剂暴露二硫化物键的新功能。此外, 最终产品是荧光, 它具有生成追踪体外探针使用商业上可用的筛选器集的额外好处。
Introduction
使用纳米病毒衣壳已成为一个令人兴奋的领域, 目的是扩大在生物医学研究领域的应用范围 1, 2, 3.Recombinantly 表达的病毒样粒子 (VLPs) 的结构来源于病毒, 但它们缺乏原始的病毒遗传物质, 使得它们无传染性的蛋白质纳米粒子。由于表面特征是基因编制的, 并且每个衣壳在它之前和之后被表达与它是相同的, 有可能知道氨基酸的无功侧链的位置和数量以原子精确度。在许多情况下, 外部和内部表面都具有多种溶剂暴露的氨基酸残留物, 这可以通过 bioconjugation 反应进行功能化–在分子和合成物之间形成共价键的反应。分子4,5。
Bioconjugation 反应帮助生物分子在相对简单的方式有更多不同的功能。感兴趣的分子, 如治疗药物6, 荧光标记7和聚合物8,9可以预先合成和特征, 然后它们被附着在 VLPs 表面上。在生物医学和生物材料研究中, 一个特别常见的 vip 是基于噬菌体 Qβ的 vip, 正如 recombinantly 所表达的, 它是一个28纳米 icosahedral 病毒衣壳10。Qβ上最常见的反应部位是 lysines, 尽管最近我们已经将 dibromomaleimide 化合物的成功共轭11与二硫化物, 通过 Qβ-贝克反应将 Haddleton 的孔隙线进行了传递。反应的收益率很好, 同样重要的是, 不会失去粒子的热稳定性。同时, 这种反应产生共轭诱导荧光, 可用于跟踪这些微粒对细胞的吸收。在这项工作中, 我们展示了聚乙二醇 (PEG) 在 Qβ表面上的共轭, 通过 Haddleton 反应, 这导致了一个明亮的黄色荧光。这些粒子可以被跟踪, 因为它们被细胞所接受。本协议将帮助研究人员生成基于 Qβ的新型荧光 PEGylated 蛋白质纳米粒子, 尽管它的原理适用于含有溶剂暴露二硫化物的许多其它 VLPs 中的一种。
Protocol
1. 准备 制作溶源性汤 (磅) 琼脂和倒盘12。 用含有 wtQβ涂层蛋白序列的 pET28 质粒转化 BL21 (DE3)。 解冻大肠杆菌BL21 (DE3) 在冰浴中的主管细胞。在离心管中放置50个细胞的 ul。 将质粒的 2 ul 添加到一管中, 轻轻地轻拂管子。然后在冰上孵育30分钟。 热休克细胞四十五年代在水浴那是在确切地42°c。热震后立即将管子放回冰浴…
Representative Results
dibromomaleimide 衍生物可通过 dibromomaleimide 酸酐与主胺15之间的缩合反应合成。另外, 使用 n-羧酸激活 34-dibromomaleimide 的温和合成方法16在这里被开发了与 methoxypolyethylene 乙二醇 (peg) 反应产生 DB peg (图 1)。核磁共振用于识别复合结构 (图 2)。Qβ vip 是一个28纳米 icosahedral 蛋白质纳米粒子, 这是由180?…
Discussion
与较小的蛋白质纯化相比, 纯化噬菌体 Qβ的一个独特步骤是蔗糖梯度离心。在氯仿/正丁醇萃取步骤后, 用5-40% 蔗糖梯度进一步纯化 Qβ。在离心过程中, 粒子根据它们的大小分开。更大的微粒旅行到更高的密度区域, 而小微粒停留在低密度区域。Qβ移动到梯度的下第三, 并保持在那里, 而较小的蛋白质杂质被困在离心管的顶部。Qβ的胶体悬浮在蔗糖梯度中表现出强烈的廷德尔散射, 当 LED 光从下面照射…
Disclosures
The authors have nothing to disclose.
Acknowledgements
J.J.G. 承认美国德州科学基金会 (DMR-1654405) 和德克萨斯癌症预防研究所 (CPRIT) (RP170752s) 的支持。
Materials
| LB Broth (Miller) | EMD Millipore | 1.10285.0500 | |
| Tryptone, Poweder | Research Products International | T60060-1000.0 | |
| Yeast Extract, Poweder | Research Products International | Y20020-1000.0 | |
| Anhydrous magnesium sulfate | P212121 | CI-06808-1KG | |
| Sodium Chloride (Crystalline/Certified ACS) | Fisher Scientific | S271-10 | |
| Potassium Chloride | Fisher Scientific | BP366-500 | |
| Elga PURELAB Flex 3 Water Purification System | Fisher Scientific | 4474524 | |
| Potassium Phosphate Monobasic | Fisher Scientific | BP362-1 | |
| Potassium Phosphate Dibasic Anhydrous | Fisher Scientific | P288-500 | |
| Sucrose | Fisher Scientific | S25590B | |
| Ethanol | Fisher Scientific | BP2818500 | |
| Isopropyl β-D-1-thiogalactopyranoside (IPTG) | Sigma Aldrich | I6758-1G | |
| Fiberlite F10-4×1000 LEX rotor | Fisher Scientific | 096-041053 | |
| Ammonium Sulfate | P212121 | KW-0066-5KG | |
| Chloroform | Alfa Aesar | 32614-M6 | |
| 1-Butanol | Fisher Scientific | A399-4 | |
| SW 28 Ti Rotor, Swinging Bucket, Aluminum | Beckman Coulter | 342204: SW 28 Ti Rotor/ 342217: Bucket Set | |
| Type 70 Ti Rotor, Fixed Angle, Titanium, 8 x 39 mL, | Beckman Coulter | 337922 | |
| Coomassie (Bradford) Protein Assay | Fisher Scientific | PI23200 | |
| TRIS Hydrochloride | Research Products International | T60050-1000.0 | |
| Tetramethylethylenediamine | Alfa Aesar | J63734-AC | |
| Tris(2-carboxyethyl)phosphine hydrochloride | Sigma Aldrich | C4706-2G | |
| 2 3-Dibromomaleimide 97% | Sigma Aldrich | 553603-5G | |
| Polythylene Glycol | Alfa Aesar | 41561-22 | |
| Sodium Phosphate | Fisher Scientific | AC424375000 | |
| Acrylamide/bis-Acrylamide | P212121 | RP-A11310-500.0 | |
| Sodium dodecyl sulfate | Sigma Aldrich | L3771-100G | |
| Ammonium Persulfate | Fisher Scientific | BP179-100 | |
| FV3000 confocal laser scanning microscope | Olympus | FV3000 | |
| Labnet Revolver Adjustable Rotator | Thomas Scientific | 1190P25 | |
| 1000 mL Sorvall High Performance Bottle, PC, with Aluminum Cap | Thermo Scientific | 010-1459 | |
| Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer | Thermo Scientific | 3141-0250 | |
| Nunc Round-bottom tubes; 38 mL; PC | Thermo Scientific | 3117-0380 | |
| 2 L Narrow Mouth Erlenmeyer Flasks with Heavy Duty Rim | Pyrex | 4980-2L | |
| Amicon Ultra-4 Centrifugal Filter Units | Millipore Sigma | UFC801024 | |
| M-110P Microfluidizer Materials Processor | Microfluidics | M-110P | |
| Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes | Thermo Scientific | 3117-0380PK | |
| Bottle, with Cap Assembly, Polycarbonate | Beckman Coulter | 41121703 | |
| Cylinder, Graduated – Polypropylene 250 mL | PolyLab | 80005 | |
| 533LS-E Series Steam Sterilizers | Getinge | 533LS-E | |
| TrueLine, Cell Culture Plate, Treated, PS, 96 Well, with Lid | LabSource | D36-313-CS | |
| Falcon 15 mL Conical Centrifuge Tube | Fisher Scientific | 14-959-53A | |
| Microcentifuge Tube: 1.5mL | Fisher Scientific | 05-408-129 | |
| VWR Os-500 Orbital Shaker | VWR Scientifc Products | 14005-830 | |
| Tetra Handcast systems | Bio-Rad | 1658000FC | |
| Polypropylene, 250 mL | Beckman Coulter | 41121703 | |
| Spectrofluorometer NanoDrop | Thermo Fisher Scientific | 3300 | |
| Long Needle | Hamilton | 7693 | |
| Exel International 5 to 6 cc Syringes Luer Lock | Fisher Scientific | 14-841-46 | |
| P1000 Pipetman | Gilson | F123602 | |
| P200 Pipetman | Gilson | F123601 | |
| P100 Pipetman | Gilson | F123615 | |
| P20 Pipetman | Gilson | F123600 | |
| P10 Pipetman | Gilson | F144802 | |
| Intel Weighing PM-100 Laboratory Classic High Precision Laboratory Balance | Intelligent Weighting Technology | IWT_PM100 | |
| Falcon 50 mL Conical Centrifuge Tube | Fisher Scientific | 14-432-22 | |
| 4–15% Mini-PROTEAN TGX Gel, 10 well, 50 µl | Bio-Rad | 456-1084 |
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Cite This Article
Chen, Z., Detvo, S. T., Pham, E., Gassensmith, J. J. Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry. J. Vis. Exp. (135), e57712, doi:10.3791/57712 (2018).