洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体
Summary
生物膜融合由专用融合蛋白催化。通过脂质混合测定,可以测量蛋白质的富源性。我们提出了一种纯化重组果蝇蛋白的方法,这是一种蛋白质,可以调节ER的同质融合,将其重组为预制脂质体,并测试融合能力。
Abstract
膜融合是真核细胞的关键过程。特殊蛋白质是催化融合所必需的。阿普拉斯是内质视网膜 (ER) 居民蛋白,与 ER 的同质融合有关。在这里,我们详细介绍了通过两轮亲和色谱法来纯化谷胱甘肽 S-转移酶 (GST) 和多组氨酸标记果蝇蛋白的方法。研究体外融合反应需要将纯化融合蛋白插入脂质双层。脂质体是理想的模型膜,因为脂质成分和大小可以调整。为此,我们描述了一种重组方法,通过去除果蝇在骨细胞素到预先成型的脂体。虽然有几种重组方法可用,但通过洗涤剂去除进行重组有几个优点,使其适用于蛋白条和其他类似蛋白质。该方法的优点包括高重组产量和重组蛋白的正确方向。该方法可扩展到其他膜蛋白和其他需要蛋白体的其他应用。此外,我们描述了用于测量膜融合的蛋白石体体基于FRET的脂质混合测定。
Introduction
膜融合是许多生物反应的关键过程。在生物条件下,膜融合不是自发的,需要专门的融合蛋白来催化这种反应1。ER同质膜融合在动物中由与发电机相关GTPase在lastin2中介导。阿克拉斯丁在同质融合中的作用对于外围ER中的三向结至关重要,它构成了一个贯穿整个细胞的大型互联小管网络。阿克拉斯林有一个保守的域形态,包括一个大的GTPase,一个三螺旋束中间域,一个疏水膜锚,和一个短的细胞质C端尾部3。体外研究用重组果蝇对骨素表明,当重组为脂质体时,它保持其富源性。其他的lastin,包括人类同源物,未能在体外重述融合。我们在这里描述一种方法,用于纯化GST和多肽标记重组果蝇对lastin,将其重组为脂体,并分析融合。
研究体外膜融合是一个挑战,因为富源蛋白通常具有膜锚。为了研究它们,有必要将它们重新组成模型脂质双层。大型单片囊泡(LUV)是研究脂质蛋白相互作用的有用工具。我们在这里提出了一个系统,使不同脂质成分的LUV蛋白质重组和融合测定。通过多种方法将整体蛋白质重组到LUV中,可以实现,包括有机溶剂介导的重组、机械机制或洗涤剂辅助重组4。我们这里提出了一种通过洗涤剂去除将果蝇在骨素中重组成预制脂体的方法。这种重组方法的优点包括高再生产量和脂质双层中甲苯的适正定向。此外,通过这种方法,蛋白质不干燥或暴露于有机溶剂,从而保持结构和功能。其缺点之一是,洗涤剂的存在可能并不适用于所有蛋白质,而最终的蛋白酶体在脂质双层中可能有一些结合的洗涤剂。进一步透析可用于消除更多的洗涤剂。然而,透析可能需要很长时间,因此可能导致蛋白质活性的丧失。
评估lastin的聚变活性可以通过脂质混合测定,如前所述2。在这里,我们划定了一种通过N-(7-硝基苯-2-oxa-1,3-二氮-4-yl)/利萨明罗达明-B磺胺(罗达明)标记脂质测量lastin介导聚变的方法。这种测定需要融合供体(标记)蛋白酶体和受体(未标记)蛋白酶体。FRET释放可以在反应过程中测量为从”标记”脂质体稀释的供体受体对到”未标记”脂质体,这是膜融合过程中脂质混合的结果(图1)5。虽然这种测定是膜融合的代理,但它在区分膜融合和血合液方面受到限制,这种状态只有外层传单混合。为了解决这个问题,另一种选择是用二硝基石对NBD进行外部包装。遵循与NBD/罗达明脂质混合测定相同的方法,在淬火外单子时,任何NBD FRET通过融合释放的,都将是由于内部传单混合8。
替代融合测定由内部水含量混合地址完全融合只有5。这方面的例子有二聚氰胺(Tb)/二恶酸(DPA)测定和氨基苯三硫酸(ANTS)/p-二甲苯二(二甲苯)溴化物(DPX)测定。在 Tb/DPA 测定中,封装 Tb 的脂体池与封装 DPA 的脂体混合并融合在一起;融合后,荧光通过内部能量从DPA转移到Tb在[Tb(DPA)3+3-结合复合物6内增加。相比之下,对于ANTS/DPX测定,ANTS荧光由DPX7淬火。虽然这些系统解决了内部内容混合问题,但需要更深入地制备脂体,以去除非封装试剂,以及荧光荧光的意外相互作用。
Protocol
Representative Results
Discussion
本文的方法为重组lastin的净化、重组和测量融合活性提供了一种有效的方法。为了确保高产量的功能lastlastin一些关键步骤必须考虑。在低温(16°C)下必须表达阿列酮,以避免聚集,并且一种应针对0.4~1.5mg/mL的最终浓度。非常稀蛋白不会以1:400蛋白质与脂质比进行最佳重组。通过此处描述的浮点测定法(图 2),可以有选择地分析重组效率。脂质体浮化测定的优点之一是,可以扩展分析?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢迈克尔·斯特恩博士和他的实验室对阿克拉松相关项目的见解和反馈。这项工作得到了国家普通医学研究所[R01GM101377]和国家神经疾病和中风研究所[R01NS102676]的支持。
Materials
| 10 mL poly-prep chromatography columns | Biored | 731-1550 | |
| 10 x 75 mm Flint glass tubes | VWR | 608225-402 | |
| 47 mm diameter, 0.45um pore whatman sterile membrane filters | Whatman | 7141 104 | |
| 96 well white plate | NUNC | 437796 | |
| Anapoe X-100 | Anatrace | 9002-931-1 | |
| Cell disrupter | Avestin | Avestin Emulsiflex C3 | |
| DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)) | Avanti | 840035P-10mg | DOPS |
| EDTA | Research organics inc. | 6381-92-6 | Ethylenediaminetetraacetic acid |
| EDTA-free protease inhibitor cocktail | Roche | 11873580001 | Complete protease inhibitor |
| Extruder | Sigma Aldrich | Z373400 | Liposofast Basic Extruder |
| GE Akta Prime liquid chromatography system | GE Pharmacia | 8149-30-0006 | |
| Glutathione agarose beads | Sigma aldrich | G4510-50ml | |
| Glycerol | EMD | GX0185-5 | |
| GTP | Sigma Aldrich | 36051-31-7 | Guanosine 5' triphosphate sodium salt hydrate |
| HEPES, acid free | Omnipur | 5330 | |
| Imidazole | fluka | 5670 | |
| Immobilized metal affinity chromatography (IMAC) resin column | GE Healthcare | 17040801 | 1 mL HiTrap Chelating HP immobilized metal affinity chromatography columns |
| Iohexol | Accurate chemical and scientific corporation | AN 7050 BLK | Accudenz/Nycodenz |
| IPTG | Research products international corp. | I56000-100.0 | IPTG, dioxane free |
| L-Glutathione reduced | Sigma-Aldrich | G4251-5g | |
| Magnesium chloride | Fisher | 7791-18-6 | |
| Methanol | Omnisolv | MX0488-1 | |
| NBD-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt)) | Avanti | 236495 | NBD-DPPE |
| n-Dodecyl β-D-maltoside | Chem-Impex International | 21950 | |
| Nonpolar polystyrene adsorbent beads | BioRad | 152-3920 | SM2 Biobeads |
| Nuclepore track-etch polycarbonate 19 mm 0.1 um pore membrane | Whatman | 800309 | |
| Optima LE80K Ultra centrifuge | Beckman Coulter | ||
| Phosphatidylcholine, L-α-dipalmitoyl [choline methyl-3H] | ARC | ART0284 | Titriated lipids |
| Plate reader | TECAN | TECAN infinite M200 plate reader | |
| POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine) | Avanti | 850457C-25mg | POPC |
| Potassium chloride | MP | 151944 | |
| Rh-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)) | Avanti | 236495 | Rh-DPPE |
| Scintillation Cocktail | National Diagnostics | LS-272 | Ecoscint XR Scintillation solution for aqueous or non-aqueous samples |
| Scintillation vials | Beckman | 592928 | Fast turn cap Mini Poly-Q Vial |
| Thrombin | Sigma | T1063-1kU | Thrombin from human plasma |
| Triton X-100 | Fisher | BP151-500 | |
| Ultra-clear centrifuge tubes 5 x 41 mm | Beckman | 344090 | |
| Vortex 9 to 13mm Tube Holder | VWR | 58816-138 | Insert for vortexing flint glass tubes |
| Vortex Insert Retainer | VWR | 58816-132 | Retainer needed for vortex tube holder |
| Vortexer | VWR | 2.235074 | Vortex Genie 2 model G560 |
| β-mercaptoethanol molecular biology grade | Calbiochem | 444203 |
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