将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中
1Center for Biological Systems Analysis,University of Freiburg, 2Faculty of Biology,University of Freiburg, 3Freiburg Institute for Advanced Studies (FRIAS),University of Freiburg, 4BIOSS Centre for Biological Signalling Studies,University of Freiburg, 5IMTEK Department of Microsystems Engineering,University of Freiburg, 6Cluster of Excellence livMatS at FIT – Freiburg Center for Interactive Materials and Bioinspired Technologies,University of Freiburg
Summary
在有机溶剂和水性溶剂的界面上，定制的两栖乳素样蛋白质组装成复杂的超分子结构，如由环境参数触发的囊泡、纤维和焦质。所述装配协议产生具有可调特性的蛋白质膜型隔间 （PMBC），能够封装各种货物。
Abstract
定制蛋白质积木是超分子结构（如最小细胞、药物输送车辆和酶支架）组装的多功能候选物。由于其生物相容性和遗传性，弹性蛋白（ELP）是生物技术和生物医学应用的理想构建基块。然而，以蛋白质为基础的超分子结构的组装具有独特的物理化学特性和良好的封装潜力仍然具有挑战性。
在这里，我们提供两种高效的协议，引导两栖ELPs自组装成超分子蛋白结构，如球形角质、纤维和稳定囊泡。提交的装配协议基于具有适应性物理化学特性的ElP生成蛋白膜基隔间（PMBC）。PMBC 演示相分离行为并揭示方法依赖膜融合，并能够封装化学多样化的荧光货物分子。由此产生的PMBC作为药物配方和输送平台、人工细胞和分段反应空间具有很高的应用潜力。
Introduction
用于生物技术应用的超分子结构的组装变得越来越重要。,2,3,4,5对于具有所需物理化学特性的功能性结构（如锥形、囊泡和纤维）的组装，了解和控制这些部件的物理化学和构象特性非常重要。由于在自然界中发现的分子的分子精度，超分子结构的构建基块越来越多地基于脂质、核酸或蛋白质。与合成聚合物相比，蛋白质积木能够精确控制基因水平上的新兴超分子结构6。单个蛋白质构建基块的主要氨基酸（aa）序列本质上编码了从分子到宏观水平的信息，以及最终超分子结构7的三维形状和物理特性。
报告用于组装不同超分子结构的方法通常涉及两栖蛋白，如温度敏感弹性蛋白（ELP）5,8,9， 重组烯烃10和人工蛋白质两栖动物11.温度触发方法导致云母的组装4,10,12纤维13床单14和囊泡9,15,16.有机溶剂在形成基于动态蛋白的囊泡方面应用了方法8,11,14.到目前为止，用于囊泡形成的应用协议通常缺乏对微米大小的装配体的装配控制16,17或装配产量有限5.此外，一些报告的基于ELP的囊泡有削弱封装电位12或久而久之的稳定性有限9.针对这些缺点，所提出的协议使微数和亚微米大小的超分子结构能够自我组装，具有明显的物理化学特性、良好的封装电位和长期稳定。定制的两栖ELP组装成超分子结构，范围从球形锥形和高度有序的扭曲纤维束到单层囊泡，具体取决于应用的协议和相关环境条件。大型孔径蛋白膜基隔间（PMBC）揭示了所有主要表型，如膜融合和相分离行为，类似于脂质体。PMBC 可有效封装化学多样化的荧光货运分子，这些分子可以使用简单的异位荧光显微镜进行监测。本研究中使用的重复ELP域是蛋白质超分子结构的有吸引力的构建基块18.ELP 五角苷酸重复单元 （VPGVG） 已知可以耐受除第四位置的 proline（瓦林、V）之外的不同 aa，同时保持其结构和功能特性19.含有独特亲水性和疏水域的两栖ELP的设计通过在VPGXG重复中插入具有明显疏水性、极性和电荷的aaguest残留物（X）来实现20.两栖ELP域，其中配备了疏水性苯丙氨酸 （F） 或异硫氨酸 （I），而亲水域含有带电谷氨酸 （E） 或精氨酸 （R） 作为客人残留物。可在补充信息和参考中找到符合条件的两栖ELP构造和相应的 aa 序列列表8,21.所有构建基块，其中配备了小荧光染料或荧光蛋白，通过荧光显微镜进行可视化。mEGFP和其他荧光蛋白是N终端融合到ELP两栖爱好者的亲水域。有机染料通过无铜菌株结合，促进烷基苯-azide环增剂（SPAAC）与共同翻译引入的不天然氨基酸（UAA）。UAA的共同翻译合并para-阿齐多芬丙氨酸 （pAzF）22允许对亲水ELP域进行N端修改。通过这种方式，绿色荧光染料BDP-FL-PEG4-DBCO （BDP） 或任何具有紧绷环状素的小荧光分子可用作荧光探针。成功加入UAA pAzF并通过SPAAC对染料进行环化，可通过LC-MS/MS轻松确认，因为相应的试性肽具有有效的电化8.由于荧光蛋白与大多数有机溶剂不相容，因此应用这种小型有机染料拓宽了装配协议的溶剂选择范围。下面介绍了我们实验室开发的两种最有效的超分子结构装配协议。THF 膨胀方法仅与有机染料改性两栖 ELP 兼容。相反，1-丁醇（BuOH）挤出方法与许多蛋白质兼容，如mEGFP，因为所述方法完全保存了这些融合蛋白的荧光。此外，使用 BuOH 挤出方法，小分子的封装和车辆融合行为效果最佳。
Protocol
1. 两栖弹性蛋白（ELPs）的设计与克隆 克隆和设计构造，如其他地方描述的8，,20。质粒可应要求提供。 2. 蛋白质表达、纯化和制备 F20E20-mEGFP 和 F20E20-mCherry 的表达式 接种主要表达文化，从一夜的战前文化到OD600的0.3。在37°C下孵育，在无菌400 mL LB介质中孵育200rpm，并在2升烧瓶中补充抗生素。 <…
Representative Results
囊泡生产协议开发图 1概述了两种不同的囊泡制备方法。左侧的 THF 膨胀方法由连续三个步骤组成，并根据温度产生不同的超分子总成。在图1中，外荧显微镜图像显示由BDP-R20F20组装的囊泡和从BDP-R40F20组装的纤维结构。在右侧图示的BuOH方法专门导致ELP囊泡的形成，与THF膨胀方法相比，产生约两个数量级的囊泡。原理…
Discussion
在遵循所述协议以组装定义的超分子结构时出现故障，主要导致非特异性聚合体的形成（图2，IV），或导致均匀分布的ELP-两栖动物。协议的关键步骤如下：
对于两栖ELP的高表达率，相对低温20°C是最佳选择。为了成功纯亲性ELP进行亲和纯化，在溶质缓冲液中尿素浓度为4M，经验证可最好地溶解两栖ELP，提高可溶性洗脱分数中的蛋白质产量。如果需要在水…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢BMBF的财政支持和生物系统分析中心（ZBSA）提供的研究设施。我们感谢P.G.舒尔茨，TSRI，拉霍亚，加利福尼亚州，美国提供质粒pEVOL-pAzF。我们感谢弗赖堡大学生物系统分析中心（ZBSA）的生命成像中心（LIC）的工作人员帮助他们提供共聚焦显微镜资源，并在图像记录方面给予出色的支持。
Materials
| 1 µm and 0.2 µm Steril Filter | VWR | ||
| 1,4-Dithiothreitol | Merck | ||
| 1-butanol. >99.5% p.a. | Roth | ||
| 2log DNA ladder | NEB | ||
| 2-Mercaptoethanol | Roth | ||
| 50 mL Falcon tubes | VWR | ||
| 79249 Alkyne Mega Stokes dye | Sigma Aldrich | ||
| Acetic acid glacial | VWR | ||
| Acetonitrile, anhydrous, 99.8% | Sigma-Aldrich | ||
| Ampicillin sodium-salt, 99% | Roth | ||
| BDP-FL-PEG4-DBCO | Jena Bioscience | ||
| Biofuge | Heraeus | ||
| Bottle Top Filter with PES membrane (45 µm, 22 µm) | Thermo Scientific | ||
| Brillant Blue G250 (Coomassie) | Roth | ||
| BspQI | NEB | ||
| Camera DS Qi1 | Nikon | ||
| Centrifuge 5417r | Eppendorf | ||
| Centrifuge 5810r | Eppendorf | ||
| CF-400-Cu square mesh copper grid | EMS | ||
| Chloramphenicol | Roth | ||
| CompactStar CS 4 | VWR | ||
| Dextran, Texas Red, 3000 MW, neutral | Life Technologies | ||
| Digital sonifier | Branson | ||
| Dimethylsulfoxide (DMSO) | Applichem | ||
| Dnase I | Applichem | ||
| EarI | NEB | ||
| EcoRI-HF | NEB | ||
| Environmental shaker incubator ES-20 | Biosan | ||
| Ethanol absolute | Roth | ||
| Ethidium bromide solution | Roth | ||
| Filter supports | Avanti | ||
| Glass plates | Bio-Rad | ||
| Glycerol Proteomics Grade | Amresco | ||
| Glycin | Applichem | ||
| H4-Azido-Phe-OH | Bachhem | ||
| Heat plate MR HeiTec | Heidolph | ||
| HindIII | NEB | ||
| HisTrap FF crude column | GE Life Sciences | Nickel column | |
| Hydrochloride acid fuming, 37%, p.a. | Merck | ||
| Illuminator ix 20 | INTAS | ||
| Illuminator LAS-4000 | Fujifilm | ||
| Imidazole | Merck | ||
| Immersions oil for microscopy | Merck | ||
| Incubators shakers Unimax 1010 | Heidolph | ||
| Inkubator 1000 | Heidolph | ||
| IPTG, >99% | Roth | ||
| Kanamycinsulfate | Roth | ||
| L(+)-Arabinose | Roth | ||
| Laboratory scales Extend ed2202s/224s-OCE | Sartorius | ||
| LB-Medium | Roth | ||
| Lyophilizer Alpha 2-4 LSC | Christ | ||
| Lysozyme, 20000 U/mg | Roth | ||
| Microscope CM 100 | Philips | ||
| Microscope Eclipse TS 100 | Nikon | ||
| Microscopy cover glasses (15 x 15 mm) | VWR | ||
| Microscopy slides | VWR | ||
| Microwave | Studio | ||
| Mini-Extruder Set | Avanti Polar Lipids | ||
| NaCl, >99.5%, p.a. | Roth | ||
| Natriumhydroxid pellets | Roth | ||
| Ni-NTA Agarose, PerfectPro | 5 Prime | ||
| Nucleopore Track-Etch Membrane | Avanti | ||
| PH meter 766 calimatic | Knick | ||
| Phenylmethylsulfonylflourid (PMSF) | Roth | ||
| Polypropylene Columns (1 mL) | Qiagen | ||
| PowerPac basic | BioRad | ||
| Propanol-2-ol | Emplura | ||
| Protein ladder 10-250 kDa | NEB | ||
| Recirculating cooler F12 | Julabo | ||
| Reinforcement rings | Herma | ||
| SacI HF | NEB | ||
| SDS Pellets | Roth | ||
| Sodiumdihydrogen phosphate dihydrate, NaH2PO4 | VWR | ||
| Sterile syringe filter 0.2 mm Cellulose Acetate | VWR | ||
| T4 DNA Ligase | NEB | ||
| TEMED | Roth | ||
| TexasRed Dextran-Conjugate | MolecularProbes | ||
| Thermomix comfort | Eppendorf | ||
| THF, >99.5% p.a. | Acros | ||
| Triton X 100 | Roth | ||
| Trypton/Pepton from Casein | Roth | ||
| Ultrasonic cleaner | VWR | ||
| Urea p.a. | Roth | ||
| Vacuum pump 2.5 | Vacuubrand | ||
| XbaI | NEB | ||
| XhoI | NEB | ||
| ZelluTrans regenerated cellulose tubular membrane (12.0 S/ 3.5 S/ 1.0 V) | Roth |
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Citar este artigo
Schreiber, A., Stühn, L. G., Geissinger, S. E., Huber, M. C., Schiller, S. M. Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro. J. Vis. Exp. (158), e60935, doi:10.3791/60935 (2020).