在肽膜前体的维西图洛合成中,用于自主囊泡生长
Summary
这里介绍了创建能够生长的基于肽的小型单片杆菌囊泡的协议。为了便于膜肽的囊泡生产,这些囊泡配备了转录翻译系统和肽编码质粒。
Abstract
生化反应的分块化是合成细胞的核心方面。为此,肽反应室是脂体或脂肪酸基囊泡的诱人替代品。在体囊外部或内部,肽可以很容易地表达并简化膜前体的合成。此处提供了一种基于两栖弹性蛋白样多肽 (ELP) 利用玻璃珠脱水补液创建直径为 ±200 nm 的囊泡的协议。还介绍了细菌ELP表达和通过逆温循环纯化的协议,以及它们与荧光染料的共价功能化。此外,本报告还描述了一种使RNA aptamer dBroccoli在ELP囊泡内转录的协议,作为生化反应的一个不太复杂的例子。最后,提供了一个方案,允许荧光蛋白和膜肽的囊泡表达,而后者的合成导致囊泡生长。
Introduction
合成活细胞系统的创建通常从两个不同的方向进行。在自上而下的方法中,细菌的基因组被简化为其基本成分,最终导致最小的细胞。在自下而上的方法中,人工细胞从分子组分或细胞子系统中组装,需要功能上集成到一致的细胞样系统中。
在de novo方法中,必要的生化成分的分门别类通常是使用磷脂或脂肪酸1、2、3、4制成的膜实现的。这是因为”现代”细胞膜主要由磷脂组成,而脂肪酸被认为是益生菌膜外壳5、6的可信候选体。为了形成新膜或促进膜生长,必须从外部7提供两栖积木,或者最好使用相应的合成代谢工艺在膜腔内生产4 ,8.
脂质合成是一个相对复杂的代谢过程,而肽可以很容易地使用无细胞基因表达反应9,10。因此,由两栖肽形成的肽膜是脂质膜的有趣替代品,作为能够生长11的人工细胞模拟的外壳。
嗜血弹性蛋白样二块共聚物(ELPs)是一种有吸引力的肽类,它可以作为这种膜12的构建基块。ELPs的基本氨基酸序列图案是(GaGVP)n,其中”a”可以是除proline以外的任何氨基酸,”n”是主题重复的数量13,14,15,16,17.ELPs已经创建与主要含有苯丙氨酸a和亲水性块主要由谷氨酸11组成的疏水块。根据和溶液参数(如pH和盐浓度),ELPs在温度Tt下表现出所谓的逆温过渡,其中肽经历了从亲水性到疏水性完全可逆的相变状态。使用”TX-TL”细菌细胞提取物11、18、19、20、21,在囊泡内轻松实现肽的合成,这提供了耦合转录和翻译反应所需的所有组件。
TX-TL系统封装在一起,DNA模板利用玻璃珠的脱水补液将ELP编码到ELP囊泡中作为固体支撑。囊泡的形成是通过从珠表面11的干肽的补液而形成的。可以使用其他方法22用于囊泡形成,这可能显示较低的多分散度和较大的囊泡尺寸(例如,电形成、乳液相转移或基于微流体的方法)。为了测试封装方法的可行性,荧光能的转录法dBroccoli2可以交替使用11,这比TX-TL系统的基因表达要复杂。
由于囊泡中膜积块的表达及其随后的加入膜,囊泡开始生长11。ELP的膜结合可以通过FRET测定进行演示。为此,用于形成初始囊泡总体的 ELPs 与构成 FRET 对的相等份额中的荧光染料结合使用。当在囊泡中表达未标记的ELP并将其并融入膜中时,膜中标记的ELP被稀释,因此FRET信号减小11。铜催化阿齐德-烷基环增法是一种通用且常见的结合方法。使用稳定配体,如三s(苯基苯甲酰乙-胺),反应可以在生理pH的水溶液中进行,无需水解反应物11,这适用于结合反应涉及肽。
以下协议详细介绍了生长基于ELP的肽类的制备情况。介绍了使用玻璃珠法的肽和囊泡形成表达式。此外,还介绍了如何实现氟化德布罗图阿图默的转录和ELP囊泡内蛋白质表达的转录-翻译反应。最后,提供了一个ELPs与荧光荧光的结合程序,它可以用来通过FRET测定11来证明囊泡的生长。
Protocol
1. 类似艾莱他一样的多肽的表达 第1天:为肽表达准备启动培养和供应 制备和高压灭菌表达培养瓶(4 x 2.5 L)和3升LB介质。对于 1 L LB 介质,将 25 g LB 粉末添加到 1 L 的超纯水中。 使用 100 mL LB 培养基、50 μL 无菌过滤 (0.22 μm 过滤器) 氯霉素溶液(EtOH 中为 25 mg/mL)和 50 μL 无菌过滤 (0.22 μm 过滤器) 卡比霉素溶液(50% EtOH 和 50% 超纯水)制备起动机培养基。 从含有…
Representative Results
囊泡生产图1显示了用不同膨胀溶液和玻璃珠法制备的囊泡的透射电子显微镜(TEM)图像(另见Vogele等人11)。对于图1A中的样本,只有PBS用作膨胀溶液,以证明囊泡的形成并确定其大小。当TX-TL被用作膨胀溶液时(图1B),囊泡也形成。进行了动态光散射(DLS)测量,表明玻璃珠法对囊泡形成有影响。<strong class="xfig…
Discussion
薄膜补液是创建小型单片囊泡的常见程序。故障的主要来源是对程序中使用的材料处理错误。
最初,ELPs由大肠杆菌细胞产生。ELP 纯化后的产量可能有很大差异,具体取决于协议在关键步骤中执行的谨慎程度。这些是逆温循环 (ITC) 步骤和降水后 ELP 的重新悬浮。为了纯化,我们通过添加磷酸触发了肽的疏水性崩溃,磷酸将pH?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢通过DFG TRR 235(生命出现,项目P15)、欧洲研究理事会(赠款协议694410 AEDNA)和TUM国际科学与工程研究生院IGSSE(项目9.05)提供的财政支持。.我们感谢E.Falgenhauer在样品制备方面的帮助。我们感谢A.杜平和M.施瓦茨-席林在TX-TL系统和有益的讨论方面所做的帮助。我们感谢荷兰进行了有益的讨论。
Materials
| 2xYT | MP biomedicals | 3012-032 | |
| 3-PGA | Sigma-Aldrich | P8877 | |
| 5PRIME Phase Lock GelTM tube | VWR | 733-2478 | |
| alkine-conjugated Cy3 | Sigma-Aldrich | 777331 | |
| alkine-conjugated Cy5 | Sigma-Aldrich | 777358 | |
| ATP | Sigma-Aldrich | A8937 | |
| benzamidin | Carl Roth | CN38.2 | |
| BL21 Rosetta 2 E. coli strain | Novagen | 71402 | |
| Bradford BSA Protein Assay Kit | Bio-rad | 500-0201 | |
| cAMP | Sigma-Aldrich | A9501 | |
| carbenicillin | Carl Roth | 6344.2 | |
| Chloramphenicol | Sigma-Aldrich | C1919 | |
| chloramphenicol | Carl Roth | 3886.3 | |
| chloroform | Carl Roth | 4432.1 | |
| CoA | Sigma-Aldrich | C4282 | |
| CTP | USB | 14121 | |
| CuSO4 | Carl Roth | P024.1 | |
| DFHBI | Lucerna Technologies | 410 | |
| DMSO | Carl Roth | A994.1 | |
| DNase I | NEB | M0303S | |
| DTT | Sigma-Aldrich | D0632 | |
| Ethanol | Carl Roth | 9065.2 | |
| Folinic acid | Sigma-Aldrich | F7878 | |
| Glass beads, acid-washed | Sigma-Aldrich | G1277 | |
| GTP | USB | 16800 | |
| HEPES | Sigma-Aldrich | H6147 | |
| IPTG (β-isopropyl thiogalactoside ) | Sigma-Aldrich | I6758 | |
| KCl | Carl Roth | P017.1 | |
| K-glutamate | Sigma-Aldrich | G1149 | |
| LB Broth | Carl Roth | X968.2 | |
| lysozyme | Sigma-Aldrich | L6876 | |
| methanol | Carl Roth | 82.2 | |
| MgCl2 | Carl Roth | KK36.3 | |
| Mg-glutamate | Sigma-Aldrich | 49605 | |
| Micro Bio-Spin Chromatography Columns | Bio-Rad | 732-6204 | |
| NAD | Sigma-Aldrich | N6522 | |
| NHS-azide linker (y-azidobutyric acid oxysuccinimide ester) | Baseclick | BCL-033-5 | |
| PEG-8000 | Carl Roth | 263.2 | |
| pH stripes | Carl Roth | 549.2 | |
| phenylmethylsulfonyl fluoride | Carl Roth | 6367.2 | |
| phosphate-buffered saline | VWR | 76180-684 | |
| phosphoric acid | Sigma-Aldrich | W290017 | |
| polyethyleneimine | Sigma-Aldrich | 408727 | |
| Potassium phosphate dibasic solution | Sigma-Aldrich | P8584 | |
| Potassium phosphate monobasic solution | Sigma-Aldrich | P8709 | |
| Qiagen Miniprep Kit | Qiagen | 27106 | |
| RNAPol reaction buffer | NEB | B9012 | |
| RNase inhibitor murine | NEB | M0314S | |
| RNaseZap Wipes | ThermoFisher | AM9788 | |
| rNTP | NEB | N0466S | |
| Roti-Phenol/Chloroform/Isoamyl alcohol | Carlroth | A156.1 | |
| RTS Amino Acid Sampler | 5 Prime | 2401530 | |
| Slide-A-Lyzer Dialysis Cassettes, 10k MWCO (Kit) | Thermo-Scientific | 66382 | |
| sodium chloride | Carl Roth | 9265.1 | |
| sodium hydroxide | Carl Roth | 8655.1 | |
| Spermidine | Sigma-Aldrich | 85558 | |
| sterile-filtered (0.22 µm filter) | Carl Roth | XH76.1 | |
| T7 polymerase | NEB | M0251S | |
| TBTA (tris(benzyltriazolylmethyl)amine) | Sigma-Aldrich | 678937 | |
| TCEP (tris(2-carboxyethyl)-phosphine hydrochloride) | Sigma-Aldrich | C4706 | |
| Tris base | Fischer | BP1521 | |
| tRNA (from E. coli) | Roche Applied Science | MRE600 | |
| UTP | USB | 23160 |
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Cite This Article
Vogele, K., Frank, T., Gasser, L., Goetzfried, M. A., Hackl, M. W., Sieber, S. A., Simmel, F. C., Pirzer, T. In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth. J. Vis. Exp. (148), e59831, doi:10.3791/59831 (2019).